Session: Veterinary Sciences

Added: 2026-05-15

[00:00:51]Yeah, man.

[00:01:08]Okay.

[00:01:29]Okay, we're good. Thank you for your patience.

[00:01:33]You're all still here.

[00:01:36]Okay, thank you, Po. Sorry for the we are a bit late this morning because of a technical issues concerning the the zoom presentation but now I think we are all set. So good morning everyone. um very glad to start this veterary session this morning and I'd like to u to thank the the CSG um for inviting me to to speak today about uh the crocoline heart which is probably the most um sophisticated cardiovascular system you will find in any vertebbrate.

[00:02:13]I know what you think and you're right.

[00:02:16]Reptile cardiology is a bit challenging, but you will see it's a bit easier when it comes to crocodilians since the heart is um less different from that of mammals than other reptiles.

[00:02:32]Okay, so first back to basics. I'm sure you all know that the reptilian hearts can be loosely classified as being nonprocalin hearts that is to say in squamates and chelonians with a single ventricle even if a sort of incomplete ventricular septation can be observed in certain groups of reptiles such as bowits, pythonids, varits and sea turtles. And on the other hand you have the crocodile hearts that is to say as you know in crocodiles alligators gariel and caymans with two ventricle which are physically divided by a muscular septum allowing for separate pulmonary and systemic circulation.

[00:03:33]Okay.

[00:03:37]Okay. As you can see, both have three epherent arteries emerging from the base of the heart called together the outflow tract, one pulmonary trunk and two aotic arches. And this is absolutely unique in the animal kingdom. That's the first point.

[00:03:57]All right. Now, let's zoom on crocs. On the left, you have the heart's position nearly in the middle of the cellum nestled between the two liver loes. In the center, this is what the heart looks like in an American alligator during necropsy. And on the right, you have a shimatic illustration of the ventral view of the heart seen from the outside in a saltwater crocodile. As you can see, the two aotas and the pulmonary artery are bundled together.

[00:04:32]Does it work?

[00:04:36]What's that?

[00:04:38]Okay. Seems to stay. Okay. So, I said they bundled together in a thick fibrous sheath as you can see here. And you won't find this in other reptiles.

[00:04:50]At first glance, the heart looks pretty similar to what you would see in mammals and birds, except for the presence, as we said, of a left and a right along with the pulmonary artery, which quickly splits into left and right pulmonary arteries. Okay, that's the the second point.

[00:05:14]Now let's carry on this guided tour with the internal structures and outflow tract. As we said, crocodilians possess a complete intervententricular septum.

[00:05:26]And as you can see here, the right ventricular wall is nearly as thick as the left one unlike in mammals and birds where it is typically much thinner. The two atria are separated for the from the ventricles by the atrio ventricular valves which are biker speed valves. The right aotic arch arises from the left ventricle and gives birth to the right subclavian artery, the common caroted artery and the right aorta forming together the brachioipalic trunk.

[00:06:03]The pulmonary artery emerged from the right ventricle and shortly after its emergence it branches into the right and left pulmonary arteries.

[00:06:15]At the base of each apherent artery there is a based similar valve as you can see. So there are four specificities in crocodilian when compared to mammals and birds. First the additional aot the left one that arises from the right ventricle along with the pulmonary trunk. Second, there is an arterial anesmosis between the two aotas in the celamic cavity cordally to the liver and beyond this point the right aot continues as the dorsal aorta while the left one becomes the celiac artery which supplies the gastrointestinal tract.

[00:07:00]Third, as you can see at the base of the heart, immediately outside the ventricle, the two aotas are connected via a small opening and you all know that it is called the formen of panita. This structure allows for mixing blood between the two aotas. And fourth and lastly at the base of the pulmonary trunk, crocodilians possess a unique structure which is a cocktail like valve composed of connective tissue which is actively controlled and involved in closing the base of the pulmonary artery.

[00:07:40]Now let's take a look at blood flow in non-shunting conditions which predominates in resting procodilians.

[00:07:48]During early diastol the biker speed valves at the base of both aorta and the pulmonary trunk close. At this time blood flows from the right aorta into the left one through the fmen of panida helping to equalize pressure between the two vessels. At the same time, both atrio ventricular valves open and oxygenated blood from the left atrium flows into the left ventricle and deoxxygenated blood from the right atrium flows into the right ventricle like in mammals and birds.

[00:08:24]During systol, oxygenated blood is ejected from the left ventricle into the right aorta. And at big cytol when the valve is fully open the medial cusp of the right aortic valve completely oludes the forammen of panza. You can see it here.

[00:08:43]Okay. The famemen of panza is um then uh absolutely uh closed. Then during systol as you can guess no blood can flow from the right aorta into the left one through this opening.

[00:09:01]At the same time, de oxygenated blood from the right ventricle is ejected into the pulmonary artery and not into the left aorta because the blood pressure in the left which is equal to the right one is higher than that in the right ventricle. And this this low pressure in the right ventricle is not sufficient to open the valves at the base of the left el.

[00:09:28]Now if we look cordally during cy blood from the right elota flows into the celiac artery and consequently into the splantnic circulation via the dorsal anesmosis.

[00:09:41]So to sum up during normal breathing blood is distributed into the left through two communication points first the forammen of panza during diastol and second the dorsal anesmosis during systol. This is how it works most of the time and I said most of the time in resting crocodilians.

[00:10:05]Now what happens during shunting and as a reminder by the way what is a shunt?

[00:10:11]As you know it is a bypass congenital or acquired. It is the result of a movement of fluid from one part of the body to another. It can be cardiac cerebral porttoymic. It is defined by its direction and in cardiology a right to left shunt is a bypass of the pulmonary circulation and the left to right shunt is a bypass of the systemic circulation.

[00:10:37]Shents are known to be detrimental in mammals and birds but they are supposed to have many benefits in reptiles such as allowing perfusion of vital organs during diving or bumation for example.

[00:10:53]So now what happens in shunting conditions for example during diving the pulmonary bypass is initiated by the [ __ ] teeth like valve at the base of a pulmonary artery. It gets closed in response to increased veagal tone. It increases pulmonary outflow resistance and thus increases right ventricular pressure. So this time because of the closure of this valve blood is not ejected into the pulmonary artery but is redirected into the left arch instead.

[00:11:28]At the same time oxygenated blood from the left ventricle is ejected into the right aorta but this flow is reduced due to the decreased pulmonary venous return. And then as pressure in the left aotic arch exceeds that one of the right aotic arch blood flows through the forammen of panida into the right aota helping to maintain blood pressure in the right aorta and ensuring perusion of the vital organs. This phenomenon is called reversed feramin outflow.

[00:12:03]It supplies blood to the brain and to the heart itself. It's one of the key mechanisms that allows crocodiles to remain submerged for hours without needing to resurface to breathe. But it also supplies blood to the gut with a H+ and CO2 rich blood helping gastric acid secretion. And finally blood goes into the right aorta via the anesmosis to balance the lowered output in this. So like in squamates and chelonians this is a right to left shent. But of course because of the presence of the intervententricular septum unlike in other reptiles only a right to left shent is possible in these guys.

[00:12:55]So diving or digesting induces a decrease of adrenaline in the bloodstream, increased parasympathetic tone, closure of the valve at the base of the pulmonary artery, increased pulmonary outflow resistance, and then blood shifts to the left aorta, then to the right one to create what we call a right to left shunt.

[00:13:22]Okay.

[00:13:24]And finally you understood that four key mechanisms make right to left shanting possible in crocodilians. First the left aorta allowing the oxygenated blood to enter systemic circulation.

[00:13:38]Second the forammen of panida that enables redistribution of blood between the two systemic outflows. Third, the gear shaped valve at the base of the pulmonary artery which actively regulates pulmonary outflow. And fourth, the dorsal anesmosis that links the two vessels dorsally and codally.

[00:14:03]This um incredible mechanism is supposed to allow crocodilians to extend dive times to conserve energy to optimize oxygen oxygen use to facilitate oxygen uptake from the lungs to facilitate digestion and to recover quickly from metabolic acidosis.

[00:14:28]But to be honest, this cardiacction still remains a mystery to the scientific community, particularly when it comes to its real functions and benefits. We know that right to left shant allows for selective perusion of the head region. The framan of panita as you saw plays a major role supplying b blood to the coronary circulation and to the brain during diving.

[00:14:56]Reintroducing this hypercapnic blood into the systemic circulation might optimize the oxygen dissociation curve.

[00:15:04]Surprisingly, it's been demonstrated that shunting also occurs in resting animals even where air is readily available.

[00:15:14]Right to left shunt helps to retain hydrogen ions and CO2 which helps gastrointestinal processes and digestion. But what remains unclear is that in experiment in experimental studies eliminating right to left shunt by surgically removing the left aotine engators led to remodeling of the right ventricle but this did not alter diving ability. Eliminating the right to left shin did not significantly impair growth and even if it resulted in chronic ventricular enlargement.

[00:15:50]Maybe right to left shant benefits crocodilians when they exceed their aerobic dive limit. And we don't know if this mechanism allows the lungs to serve as a temporary oxygen store during extended dives. So finally, we still don't know if right to left is really essential to their diving physiology.

[00:16:15]And this brings us to a final open-ended question. Are these complex anatomical feature merily evolutionary leftovers from ancestors that were more aquatic than modern crocodilians? And finally, do cardiactions persist today simply because they haven't been selected against that's the question.

[00:16:37]I think that's all the time I had. So, thank you for your attention.

[00:16:50]complex.

[00:16:53]So the our next speaker is Sally. I think you all know Sally. I won't introduce her.

[00:17:00]And she'll be talking to us about a system for visual body score. Uh we talked about the difficulty of uh coming up with a fair idea of the metabolic state of an animal and that you have to combine body score and uh other parameters. the body score being the first thing you can do. And there's been a number of attempts of coming up with scoring. And Sally is going to present hers.

[00:17:45]Good morning everybody. Um, in my real job, um, I do a lot of work with, uh, crocodile farms and tourist facilities and zoos. And what I've found through the, you know, hundreds of thousands of animals that I see every year is that I need a really quick and effective way to be able to communicate with everyone u around what their animals body conditions are. So, I found this uh, quite useful and I thought I'd just present on it. I did write a a CSG newsletter article on this, but I thought I'd just give a quick presentation on this and and you know, just invoke some discussion. So, I've called it CORC BSI and it's short for CROC body score index. Um, you know, how to what it is, it's simple. It's on a scale of 1 to five, which we all heard on sun uh Monday that Paulo hates. He hates the odd number. But to me, I actually like it for the reverse uh uh reason, which is, you know, we're aiming for the middle. You know, PA doesn't like that, but I do.

[00:18:46]Yeah. That that's uh you know, integr good good scientific discussion. Um but look, the other features are it's visual. It it uses really observable uh key features. It's rapid. It's on the spot. Uh there's no complex calculations like some of the other methods around Fulton's uh correlations and stuff like that. Most importantly, it's hands-off.

[00:19:06]It's not invasive. I do not need to touch an animal, increase corticost, lactate levels or anything like that to be able to get the determination that I need. The benefits include anyone can use it. Anyone can use it. Um I train uh you know staff with this. I train people with uh you know low uh English level uh you know uh level communication ability, but we can still get it across. I talk I train my board members in this. So it becomes a very easy mechanism for just everyone to be able to use. Um that means it is easily communicated between stakeholders, right? So I can talk to you, you know, a big variation. Um it can be determined remotely. I can just, you know, if someone isn't confident in using the methodology, we can FaceTime.

[00:19:55]We can use a photo. Um and in fact, we're going to have an interactive session um as soon as I get through the boring medial part of this. Um it can be done at herd level. So I can look in a pen of animals and quickly assess all of the animals within the pen. Um and there's some really clear action plan um you know outcomes um as and if required that can be applied.

[00:20:18]So how does it work? Well, it's basically uh looking at the three fat storage areas of the animal. So we've got the gel just behind the head. We've got the like a belly girth or a belly width. are generally look just in front of the back legs. And a lot of that's determined by how um how the amount of time since the crocodile was last fed.

[00:20:39]Uh it also determines uh how much uh like how big the fat body is. And you can see a bit of a variation there between some uh harvestiz animals that were all in a body score of of three and as well as how much messenteric and abdominal fat is in that area. The final area is that we look at the tail and croc uh crocodiles don't have intramuscular fat. So not like marbled uh beef for in you know meat in beef.

[00:21:07]They uh store all of their fat in between the muscles. And as you can see here in the um the tail, right? So that's all the fat storage in there. So that so as an animal gets fatter, those those fat uh fat areas increase.

[00:21:27]Sorry, I've just realized I'm looking at that.

[00:21:30]Okay. Um it's based on just like this is like the formal definition of the five different uh body score conditions. Um and we'll work through each of those.

[00:21:42]Not each of them. We'll work through the the um middle one and the extreme, should I say. So, we'll start with um an animal in a body score of three. Um there's a slight bulge, you know, um around the gels. Um outwardly, his tail's a little bit bowed, so we know that there's a nice fat uh store in there. Um and he's obviously been eating, right? There's there's, you know, he's he's clearly got a nice belly width there. Um so, there's no action there. Obviously, we want to continue to monitor animals because we don't want to see them decline or increase, but that's ultimately what we're aiming for.

[00:22:16]The other extreme is obese. So here we've got obviously an animal with excessive fat stores in the jel and even the tail region. Um by this stage the animal starting to look rectangular because the abdominal uh sorry the the fat starting to to accumulate around the thoracic area. Um the intercala areas are starting to pull out. So it's very obvious. Um the actions here would be obviously to restrict food not necessarily by decreasing the num the frequency of the food that's being fed but by reducing the portion size although frequency can also be decre decreased. Um but obviously encouraging exercise through environmental enrichment and and training cues. Um and then the other thing is to offer alternative low nutritional food items as well. At the other extreme, we have animals in a body score of one which are basically emaciated. So, you've got um that no fat stores behind the neck. You can often see the tendons um uh coming down the back end, not always. There's no bulge in the tail. You can actually sort of almost see the tail starting to wrink, the skin starting to wrinkle um because, you know, the the the the lack of fill of the tail. And then there's uh you know, obviously they haven't been eating.

[00:23:34]Um, one really important rule, use whole numbers. You know, as scientists, we start talking about, oh, I think it's a 3.125.

[00:23:45]What does that mean? All right, the animal is clearly in a category. Assign it a category. If you think, ah, but it's a three, but it's not quite what I want it to be. Talk about it as being a lower three because it's clearly not a two yet, which which triggers other action items. So even if it's in a lower two, you can go, okay, I'm going to increase that animal's food, but it doesn't mean that it's outside of the ideal body condition at that time. So I said this was going to be interactive.

[00:24:14]Let's have a bit of a look. So this is a wild Australian freshwater crocodile in the Kimbley region of WA. What does everyone think this should be assigned?

[00:24:26]>> Josh, three, I should have a I should have like chocolates to throw. Sorry, my first time doing this.

[00:24:34]All right, this is a Tistima. Um I'm not exactly sure where. We'll obviously um ignore the fact that he's got some snout problems going on there. Anyone want to call out? Yep. Five. That's right. My goodness. So, what would we do there? We would start to restrict the size uh of the food portions. We'd review the diet and we'd encourage more exercise. Um I should just say there as well, reviewing diet is actually really important. Um sometimes um fat and protein reserves can actually be um accumulated sort of uh not proportionally because there's a lacking of minerals and vitamins in the diet as well. And I think that's an area that is seriously lacking in a lot of captive crocodilians particularly. Um hopefully this video will play.

[00:25:20]Um so this is a post that um Rira, one of our CSG members, put up the other day. This is a wild saltwater crocodile in the Northern Territory.

[00:25:29]Right. This is just, you know, so again, this is on Facebook. What does everyone think that body score is?

[00:25:37]Who said two? Well done. Right. So, yeah. Again, like I mean, it's a wild crocodile. I don't know that the animal's uh story or history. What can we do about that in the wild context?

[00:25:48]That raises a whole lot of other questions. But again, immediately we can assign something to it and go, well, that is not what we actually want it to be ideally. Looking at some different species here. Again, I do not know the story behind this Gariel. Um, Gariel do have a slightly different body shape compared to some of the crocodils and the alligators in in this regard just based on head shape and stuff. But, you know, what does someone what do you what do you think about this one?

[00:26:14]>> You're thinking four. I I looked I reviewed this this morning and went I've called it a five, but maybe I want to put it a two. Either way, we know that it's overweight, right? So, that's okay.

[00:26:24]Um and just to set just to show you what a like an ideal um uh gariel would look like. This is another example of an animal in a body score of three.

[00:26:36]>> Yep.

[00:26:40]>> Exactly. And that's why using the three criteria is really important because they can't suck in their tail or their behind their neck, but they can suck in their in their um their their um in front of their their belly, that kind of thing. So yeah, again, like you you know, that's why using the three areas and putting it in context does help. But um this is a wild West African slender snouted croc that we caught uh two years ago now in Thai National Park with Matt Shirley. What does everyone think about this one?

[00:27:13]>> Three.

[00:27:13]>> Yeah. Three. Um, these animals seem to have a little bit more of a like less um, you know, around their jaws and all the rest of it. So, again, we need to make a bit of a, you know, adjustments there for a species, but clearly the animals, um, got a nice uh, bow here in his tail and he's clearly been eating.

[00:27:30]Um, I said that we can do this collectively. Um, so this is a group of captive saltwater crocodiles. What are we assigning these?

[00:27:42]Three. Right. So again, you can just quickly glance cast your eye over all of them. Um yes. So all three. So um I've got a few pamphlets around that I've left outside if you want to take one. Um the as I said, I wrote a newsletter article last year. So it's in the CSG newsletter. You're welcome to download it from that QR code. We've uh this is actually now implemented when the federal government finally releases the new updated code of practice for Australian um uh wild and farm saltwater croc or crocodiles should I say. Um this has actually been included in that code.

[00:28:20]So you know it's starting to get a little bit of traction and yeah thank you very much for listening.

[00:28:39]We can move on to our next presentation.

[00:28:41]That's a recording sent by Arena.

[00:28:45]>> Hola is at the >> That's it. Sorry.

[00:28:50]>> That's all right.

[00:28:52]>> Sorry.

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[00:37:32]Gracias, Serena. That is a topic that many of us are trying to work on. So that's really useful. I think we'll move on back to Sally. If I can open your presentation.

[00:37:47]This one. Right. So S is going to share some uh treatment of one of the most common cause of death of hatchlings with antibiotic that's from the last century. So that's good because it's accessible and cheap. Very very good.

[00:38:03]Thanks.

[00:38:05]>> Thanks U Paulo. Um yeah, as Paulo just said, um you know, pharmaccoinetic data is really lacking in all crocodilian species. Um but particularly within, um you know, the the captive uh you know, farming context as well, like individual crocodiles uh getting uh you know, some some works done in regards to individual animals with injections and all the rest of it, but on a broad you how how that applies in a broad context has been largely overlooked.

[00:38:33]So um providencia reeri in hatchling saltwater crocodiles when septasemia has occurred that seems to be the organism that comes about um the majority of the um uh deaths occur between 2 and 8 months of age. Um and they're sporadic and really unpredictable.

[00:38:52]Now a lot of the literature around the providencia rearizmas sort of implies that it's it's a bit of mismanagement. you know it all, you know, it's inappropriate temperatures blah blah blah blah blah which is true.

[00:39:05]It does corres uh correspond to the onset of the dry season. What's been largely overlooked at this point though is that it's also at the period of the what we call the window of susceptibility and that is when the egg yolk which is where the maternal antibodies are stored starts to get fully absorbed and therefore the animal needs to start standing on its own two feet in regards to its immunity. And from a study that I did with a colleague back in two or lots of colleagues back in 2015, we did a um uh like it's an invivo test uh called a called a bacterial killing assay and when we challenged uh providencia regum.

[00:39:46]Um the animals at 3 months of age had an innate ability to kill the providencia at around uh 10% like sorry that was a meat. So that this is a box plot. Uh so this is a median. the dark lines are median not mean. Um they had an ability to to kill about 10% of the providencia.

[00:40:04]Now you can see over time that increase.

[00:40:06]So at three at 6 months of age they had a 50% ability to to kill providencia and at 9 months of age they had an 80% uh chance of killing providencia which means that they're that you know that this is all innate immunity not acquired but that that innate ability is actually increasing over time. And what you can also see here is some animals don't acquire um immunity at all even innate immunity at all. So providencia regari has just found a niche part in these farming systems. Um you know Kathy Shortton and I have been discussing this and debating it for many years. Uh providencia is a gram negative bacteria.

[00:40:44]It's ubiquitous. So uh there are a whole heap of other gram negative ubiquitous organisms that could could also equally um you know have this impact. But by far and doubt the providence erect has just found a niche. Um husbandry protocols uh you know the refinement of husbandry protocols have definitely decreased the number of mortalities over the years. Um and particularly the big step forward was actually the technological stability around environmental controls. So heating controls and all the rest of it.

[00:41:16]Um, interestingly, the deaths only occur in the the better growing crocodiles, which obviously uh devastates the farmers even more. So, uh, Kathy Shortton and Sesh Benedict published uh, some this paper on 139 historical um, isolates that they had in their laboratory at Bervet lab. And what this basically shows what this graph basically shows is that 44% of the isolates were sensitive to sulfur furol tetracyc and the uh sulfur trimeth combination. Um I won't go into those those statistics are pretty obvious. Um so what has become common place is that a combination uh sulfur trimeth uh antibiotic known as sulprum commercially known as sulprum is just used as a standard rule. Um but there lacks any sort of pharmaccoinetic phocodnamic information. So we set about doing a preliminary trial around this.

[00:42:18]So we what we did was we just modeled the effectiveness of a single dose of sul sorry a very important point that I've overlooked is that all antibiotics in Australia used on crocodilians and I believe it's sort of around the world are what they call off label use. So there's no antibiotics registered specifically for crocodiles. Um the other thing is we're doing this in the farming context. So it's impractical to pick up and inject 15,000 animals a day or whatever. So this food has to be in oral format. Um so that does limit our choices of what we can use as well. So in the trial we uh used the com like the rate that they use on farm which is 2 gram of sulprum uh per kilogram of food fed. Um we used 48 crocodiles in total but we did it in two phases. So the first phase we had 16 crocodiles and our last data collection point was at 144 hours but we still detected antibbody uh antibiotics in the blood at that point.

[00:43:37]Thank you. Um, okay. Where was I at? Um, so yeah, so in our second second phase, and that's why we did it in two phases as well, we added 168 hour data point as well. Um, we used a rotational blood sampling uh strategy so that the animals weren't bled mult they were bled multiple times, but a maximum of three times. Um, and this was all in the refinement part of our animal ethics permit.

[00:44:02]I'm not going to pretend to understand the pharmaccoinetic stuff. That's why I have collaborators that are much smarter than me. Um, so I'm not even going to explain these graphs other than than to say that the halflife, which is apparently really important when you're calculating this stuff, uh, for the sulfur uh, sulfur dimmidine was 43 hours. For trimethoperm, it was 10 hours. So obviously the clearance times in the blood of the sulfur antibiotic is four times that of the trimeth.

[00:44:34]Now from the literature uh to have appropriate bacterial effects you're aiming for a ratio of about 1 to 15 to 1 to 22 of trimeth to sulfur drugs. Now this graph's a little bit complicated but bear with me. Okay. So the the light green the light gray line here is the the amount of sulfur antibiotic in the blood over time. Uh the the uh black is the trimeth which remember we just said then has a quicker clearance time. So of course it's being excreted and it also goes in at a lower rate to begin with.

[00:45:08]The important thing to look at here is this dark blue line. Right? So this is the ratio of the the trimeth to to sulfur drug and this uh uh blue shaded area is that 1:15 to 1:22 ratio that's optimal. So when we feed just one dose of sulprum at that 2 g per kilogram food, you can see how out of whack that is, right? So that's the line there that we want it to be sitting in. But because of the huge amount of sulfur going in and the longer clearance time as opposed to the lower amount of trimethan, the clearer sulfur line that that the ratios all screwed up. So from the modeling what we've that's so funny like look what it's done to my icon. Anyway, sorry small things.

[00:45:57]Um so what we've done as a solution there is whil still feeding one dose of sulprum on day one. Um if we add 150 mg of trimeth that actually brings up that ratio. And this again this is our our blue shaded area here which is our ideal bacterial cidal effect. Um and then if we also continue to feed trimeth at 150 mgs on days 2 3 and four we can somewhat maintain that ratio within the blue area that we're that we consider to be ideal.

[00:46:31]So the recommendation um for the farms moving forward and they've started to implement this is that on day one they they continue with their 2 g per kilogram of sulpram um as well as administering 100 mgs per kilogram of food of additional trimeth. Um then three additional doses of trimeth are provided on days 2, three and four. And this dosage regime is going to promote better uh antimicrobial uh stewardship by reducing the um the risk of antibi microbial resistance to the providencia itself as well as to other environmental organisms but also most importantly achieves the desired result of treating uh treating the animals or minimizing the crocodile septasemic deaths because here's the caveat. Um this I reported this in Darwin when I talked about antibiot uh antibiotic resistant genes.

[00:47:30]Whenever a crocodile is submitted to the lab with providencia septtomia the stomach's always empty. That therefore means that it's not eating it's not going to eat the antibiotics that's in the food. So what we're actually doing by putting antibiotics in the food is we're pro essentially prophylactically treating any animal that is you know acutely affected or hasn't yet been affected for whatever those triggers are. So we still need to be asking the greater research objectives to try and avoid antibiotic use on the overall scale of you know what are the infection dynamics of providence erecta things like virulence and and all questions that I can't even ask because I'm not a microbiologist um but also other ways to promote to assess and promote uh hatchling immunity so very interested to talk to anyone who can help me answer either of those questions otherwise thank you very Thank you, Sally. Now we have to do the difficult part.

[00:48:38]>> Oh, no.

[00:48:40]Okay, I should be able to open the zoom here.

[00:48:47]All right. Can you hear me, Brinky?

[00:48:59]Brinky. Brinky, can you hear me?

[00:49:08]>> Um, hi. Uh, is my voice audible?

[00:49:17]Yeah, just a second. We're moving your presentation to the correct screen.

[00:49:21]>> Can Hi, >> can you hear me?

[00:49:44]Hello.

[00:49:46]>> Can you hear me?

[00:49:47]>> Am I audible?

[00:50:02]>> Okay. Uh, is my screen screen visible?

[00:50:06]Uh it is visible but uh it's on the wrong screen. So just give us a minute.

[00:50:10]We'll transfer it to the other screen.

[00:50:16]>> Shall I start?

[00:50:17]>> Okay. Now you can start.

[00:50:20]>> Okay. Okay. Hi everyone. Good morning.

[00:50:22]This is Binkit Desai from India. Uh today I'm presenting a part of my PhD work which is understanding behavior and endocrine profiles of muggle crocodile during reproductive period.

[00:50:33]Uh the first question that comes is why study reproductive behavior and physiology? Well um studying reproductive behavior and physiology is very fundamental to understanding how a species uh reproduce and thrive in its environment. Moreover, it gives us uh insights into different type of reproductive behaviors like portraitar, nesting behaviors and parental care. uh by studying these aspects of reproduction we can actually understand what reproductive strategies uh they use to adapt and survive in their environment. Now at the same time it is also very important to understand the physiology as it helps us in uh understanding the underlying mechanism that governs different reproductive behaviors and that we can understand using um endocrine profiles uh which is through hormones. Initially a lot of uh studies used to focus at species level but the more uh research people did they tried to study population level as well as uh individual levels.

[00:51:41]Uh now the hormones that I'm looking at uh is uh testosterone. Uh in a study on mineral uh color formation on face and uh face and male fight was associated with elevated testosterone level. At the same time in birds uh during malemale competition the testosterone levels are elevated. Now coming to the females even female shows high level of testosterone during uh reproductive season which is associated with female female competition for male. uh in lemurs it has been uh associated with caring for the young one and in turtles it has been associated with multiple uh clutches of egg laying which is uh actually a physical demanding uh activity.

[00:52:29]The next are uh estrogen and progesterone.

[00:52:34]Studies have shown that female breeding cycles begin with an estrogen peak driving mating behaviors and receptivity which is then followed by elevated levels of progesterone during nesting across uh se uh several species and texa. Progesterone and estrogen has been associated with several reproductive behaviors like nesting, egg laying, caring for young ones, territoriality, uh protecting and defending eggs and young ones. And very few studies which are lab- based have also shown the association of progesterone in males which has been associated with uh breeding coloration uh during breeding season and caring for young ones.

[00:53:18]Now uh coming to the criteria of hormone behavior interaction. There are three main criteria which is very important.

[00:53:25]Uh I've taken this example of a study of green sea turtle where you can actually see that there are three different estrog uh three different progesterone.

[00:53:37]Well this has been associated with three different uh clutches of egg that were laid by an individual female. Uh so uh as you can see here that different hormones has been associated with different activities which were behaviorally observed and at the same times uh their hormones were uh studied to associate the pattern. A recent work on American alligators also showed that bellow's uh individual male uh alligators are different and individuals are able to identified uh uh each other based on their uh bellow sounds.

[00:54:20]uh studies have you uh hormone uh studies have used uh hormones to understand the reproductive patterns which are uh done by collecting blood, scat, scales or hairs and to study behavior. Long-term tracking of individual is done either by satellite tag, call collars, cattle tacks and the more recent studies have started using uh non-invasive techniques which is either uh by uh using injury marks but injury marks fade over time and is proven to be uh unrealistic when uh it comes through long-term studies. And the other method is biometry method where uh the body pattern is body pattern or coloration is used to identify individual animals. To sum up uh my slide, I just want to say that to order in order to understand hormone behavior interaction, simultaneous measurements of both reproductive behaviors and endocrine correlates of focal individuals is needed over a long period of time. Uh coming to my model organism which is mugger crocodile. Uh the reason for choosing uh mugger crocodile uh one is that a lot of studies for reproductive behavior and hor physiology has been focused on mammals and birds.

[00:55:36]Coming to reptilian and amphibian taxa uh a lot of species are data deficient.

[00:55:44]Um huh. Muggers are ectothermic animal. Uh and they live uh muggers are ectothermic animal meaning they live in very diverse habitats. When take an example of mugger crocodile uh they live across 50 small pockets in India and subcontinent and they live in diverse habitat from agricultural landscape to urban cities uh living in gutters and uh even in semi-arid desert habitat which means they constantly have to adapt to different environment behaviorally as well as physiologically in order to reproduce in those environments. Hence they serve as a very good model organism to understand adaptation.

[00:56:28]However there is no systematic data on behavior as well as endocrine profile for mugger crocodiles.

[00:56:35]So uh my objectives was to first u uh uh identify all the reproductive behaviors in muggle crocodiles develop and identify the sequence of behavioral repertoire in context of reproduction.

[00:56:50]validation of fecal based measurements for progesterone and tessterone in mother crocodiles and to generate endocrine coralates of the observed reproductive behaviors in mother crocodiles and this was specifically focused on female crocodiles.

[00:57:04]Uh so first I selected breeding population and data collection. So Madras I selected population from Madras crocodile bank uh trust. Reason being they had the reproductive and health assessment history of their population for the past 30 years. uh they were naturally breeding. Uh by the term naturally uh I mean that there was no human intervention uh of any kind uh to uh trigger or uh help them breed. Uh and they produced viable eggs. Uh one of the caveat of my study was we had 20 females and only two males uh in the study out of which one was a larger male and one was a smaller whale which never came out of the water. So we only have uh behavioral data and scats from 1 mill.

[00:57:52]The study was conducted uh from November to June covering one breeding season. We use CCTV camera, DSLR, voice notes uh to understand the behavior and uh we divided uh whatever data we got, we divided our breeding season into three different phases. One was meeting phase where only meeting incidents took place. One was mixed phase where both uh where along with mating there was nest digging, egg laying and nest guarding and then there was the non-breeding phase.

[00:58:30]Uh sorry I forgot to take water with me.

[00:58:37]13 behaviors were identified. uh where here you can see that there are some behaviors in blue box which were only uh displayed by male pink ones were only displayed by females and black ones were the behaviors that were displayed by both male and female I don't know if you'll be able to hear the um voice in the video uh but this is the head out ta posture by male this is a vocalization Oh, sorry. This is jaw clap. Uh, this is vocalization.

[00:59:33]So a loud vocalization is followed by a low vocalization uh which is then followed by bubbles.

[00:59:45]Then there is clasping by females.

[01:00:01]Snout lift.

[01:00:13]So we uh recorded diadic interaction uh which is an interaction between a male and a female and uh uh on on a single day we used to get more than one diad.

[01:00:25]Uh we took each diet and made a transition matrix. So uh we in total we record 65 diads and 165 transition matrix were prepared.

[01:00:38]Once we had all the uh all the data uh 165 uh datic uh interaction which were the matrix were then added and in order to calculate the frequency of each transition uh the each cell was divided by its row total to get uh absolute to frequency number.

[01:01:02]Now these frequency was then used to plot a mat uh marov chain analysis matrix. Uh to um make this easy for you to understand uh the blue lines that occurred consistently ac across the 165 diic interactions and the yellow lines are the weaker transition that did not occur consistently across the diic interaction. The other thing I wanted to know if these uh transition across the repertoire are happening randomly or are they not random. So we did a k square test which confirmed that these uh transitions are not random and they are uh occurring in a repertoire.

[01:01:47]We also did a residual plot which basically shows that the darker and the blue blue darker large circles are the transitions that are happening consistently and that are contributing more to the entire repertoire than the other transitions which matched with uh what we also got with the marov chain analysis. So basically the uh repertoire starts with head out, tail lock, posture followed by jocklap and vocalization by male which is then followed by uh uh various behaviors performed by both male and female including bubble, staying still, submerge, snout touch approach which leads to uh snout lift by females and uh mounted condition by male.

[01:02:36]uh to summarize my behavior data uh we were able to identify different reproductive behaviors and we also found that the reporter that we identified follows a fixed pattern. Now coming to uh generating the endocrine coralates of the observed reproductive behaviors in mugger crocile. This was mainly focused on females due to low male in the in our sample size.

[01:03:02]uh scat collection and storage was done at the MCBT and sex identification of scats was done using playback of CCTV videos. So all the muggle crocodiles in the pen uh all the females were tagged and all the males uh the two males were untagged. So we were able to identify difference between a male and a female but the tags had worn out numbers. Hence we were not able to identify individual females. So the data that we have analyzed is the pool of all females for different reproductive phases. Um standardized protocol was followed to do the essay to uh identify hormone levels uh which was done by drying and siding the scat samples followed by methanol extraction and performing enzyme linked imunos essay. We do we did two validation. One was biological validation where we pulled all the phases for FPM and FTM. Uh FPM is progesterone and FTM is testosterone. So we saw that testosterone was twofold higher in male than in females.

[01:04:09]And uh progesterone was 63fold higher in females than in males. Again since we only had one male uh sample, we did not perform any statistical analysis for biological validation. Analytical validation was performed by taking standard and one sample from mating phase and one sample from mixed phase to actually see if the standard uh the slope of the regression line for the standard curve and the serially diluted samples were significant or not. And as you can see uh in both the graphs that they uh did they did not have any significant difference meaning the samples uh the kits that we used were uh appropriate.

[01:04:55]Uh now coming to the results for testosterone we did not see any significance difference for uh testosterone in female crocodiles across the three different reproductive phases.

[01:05:07]Now tessterone has been associated with physically demanding activities during reproductive phase. Now mugger crocodiles in this particular enclosure at MCBT are ectotheromic animals and they may have to compete u uh since there are 20 females and one male they have to compete for male during mating phases. uh during mix phase they were constantly defending their eggs and even during non-breeding phases they had to constantly fight for basking space.

[01:05:39]Hence uh we are assuming that this is the baseline testosterone level for the croc population that we studied at NCBT and maybe due to this they may maintain this basal level irrespective of what breeding phase was going on and similar observation was also found in Cuban crocodiles uh which also could be an artifact of captivity.

[01:06:06]Future studies on freeranging population with varying levels of resources can help us build robust conclusion for the hormone behavior interaction as in the wild the resources are different and they may also follow different reproductive strategies which would be different from what we saw at MCVT.

[01:06:24]Coming to progesterron we saw that there was a significant difference uh where uh progesterone was significantly higher during mixed phase compared to mating and non-breeding phase. Progesterone helps maintain gravity in reptilian taxa and as board taxa and it also helps with development of egg and it has been associated with activities like territory defense, incubation, egg laying and nest guarding which was also happening at with the mugger population at MCBT and high level of progesterone during nesting is also documented in Cuban crocodile as well as uh for my study on muggers possibly suggesting the role of hormone known in nesting activities. However, since uh we did not had individual identification and this was pulled sample for from several females, we can't pinpoint the exact uh activity where the progesterone gets triggered or progesterone takes a peak.

[01:07:29]Uh this is a collated profile of behavior and hormones which b basically shows that progesterone takes a peak during different nesting activities and uh this was similar to what was observed in Cuban crocodiles as well as uh study on American alligators and Nile crocodiles where they studied patterns using blood samples but more or less uh every study that progesterone does take peak during uh nesting and egg laying activities.

[01:08:05]We also did uh study estrogen patterns and we did not find any significant difference. Estrogen may remain relable across reproductive phase supporting the baseline reproductive function. We know that they help with follicular maintenance and vitilogenesis in uh different crocodilian species. But uh there could be different environmental factors that are overriding the clear hormonal variation here at MCBT. And uh maybe when we study the these kind of uh uh when we do similar kind of studies in a wild population, they may show a different result.

[01:08:46]This is the overall schematic of my hormone behavior interaction which basically shows that progesterone takes a peak uh during mixed phase which is associated with different kind of nesting uh act uh nesting activities nest guarding egg laying and nest digging activities.

[01:09:06]Uh acknowledgement to everyone who contributed to this project.

[01:09:12]>> Thank you.

[01:09:13]>> Thank you Rinky. Thank you.

[01:09:20]Okay, as uh always we're a bit late.

[01:09:25]Uh the next speaker is Ryan Johnson, who looks looks a lot better than last time I saw him. Thank you, Sally, for filling the gaps again.

[01:09:57]Oh, what's happening? What's happening?

[01:10:11]Wonderful. Thank you. Okay. As um Paulo said, I'm actually presenting this on behalf of Ryan Johnson and the my collaborators at the University of Queensland. Uh Ryan is actually an SR student, but I guess the fact that I'm um presenting on his behalf discounts him as being part of the student prizes, Charlie, does it?

[01:10:37]Anyway, so um I'm presenting today on we're doing some work with around West Nile virus in saltwater crocodiles and uh developing a field based test to be able to detect antibodies in the field clearly.

[01:10:52]So a bit of background um west no vir this is a world map obviously and the red areas here indicate where westn no virus has been found in human cases uh so far um there is a bit of caution here in regards to the gray areas may actually be a lack of reporting not necessarily a lack of um antibodies but we won't go there today. Um it's a bit of an interesting story the West Nile virus in the crocodile context though.

[01:11:18]Um uh to to sort of begin Westnile virus was first discovered in Uganda in 1939 in the West Nile uh district hence its name. Um but it first entered the US in sort of you know in the in the crocodile context. This is where it becomes significant. It entered the US in 1999.

[01:11:37]Now, I won't go into how that happened, but it basically there was 62 confirmed cases and there were seven deaths even though um epidemiological modeling actually uh suggested there was more like 10,000 cases. It then spread all over the US, but uh more specifically for our context, it traveled down the east coast and in 2001 2002 um the first alligator mortalities were reported on uh uh farms in west in southern uh Georgia um as well as into Louisiana. There was also some antibodies found in some wild crocodiles.

[01:12:16]Um just to you know after that there was a series of of studies done. Antibodies were found in farmed crocodiles in Israel um wild and farmed uh crocodiles in Australia uh wild and farmed moral crocodiles in Mexico as well as farmed Nile crocodiles in Zambia and Zimbabwe.

[01:12:36]There might be other studies that I'm unaware of but that's they were the sort of progression of it. Now, what's interesting here is that for Nile and saltwater crocodiles, they've actually had West Nile virus endemic through their range. So, they've they've sort of evolved with it and they don't appear to have any adverse health effects.

[01:12:54]Interestingly, neither does Morott's crocodile from what we know of as well.

[01:12:58]So, even though West Nile virus didn't enter Mexico until 2002, um that there was no reported like mortality events or anything like that.

[01:13:09]This is in contrast to American alligators which until that 1999 incursion of Westnile virus into the US um they were naive to it and it did cause significant mortalities on farms.

[01:13:22]At the same time there was an increase in the number of skin grades that started to occur and that that the the name of these skin downgrade of the defects was called picss.

[01:13:34]Now, in my very first CSG meeting back in 2002 in Gainesville, Paul Cardiac actually uh presented a paper on this with uh for his colleagues whereby he noted that these picss were being uh called that because they appeared to look like little ice picks in the skin and it was causing sub uh substantial down downgrades by up to 50%.

[01:13:57]What's interesting is that he also said that these number of de uh downgrades had occurred since November 1999. same time that West Nile virus incurred in New York. So you know there was a bit of sort of dis discourse to begin with but in 19 sorry 2008 Harvey and Navarez and colleagues sort of drew all the information together. So for those of you who don't uh haven't seen pics on well on this is this is the lesions on the the live animal. I'll show you in the next slide what it looks like on the tan skin. Um so Harvier was able to show that the alligators with the lesions were also serositive for Westnile virus.

[01:14:37]They had antibodies. Um he also confirmed that the Westnile virus in the skin lesions and in the brain and liver tissue were also Westnile vir contained West Nile virus through genetic PCRs.

[01:14:51]And I think this ends up being like the define like the definitive photo around Westnile virus lesions in the skin though. So this is this is a hisystologology picture of the skin. So this is the epidermis and this is a large lymphocitic mass uh within the dermal um dermal uh layers and that coined the term lympio lymphohysteioitic proliferative syndrome which I'm going to continue to call picss because clearly that's a mouthful. Um but why is that important to the to the farm's uh crocodile industry? Um the this is a raw skin on the light table. So, a light shining up below it. This is the tanned product. These here are ISOs, so they're pretty st they're standard on every crocodilian scale. So, we're going to ignore them. Um, but this is the lesion here that we're interested in. All right. So, what happens? You saw just before on that hisytologology picture where there's a large lymphoysteocitic aggregate. Um, that is like just an immune response to the disease. But what it does is it displaces the collagen or the dermal dermal structure and it leaves a void and over time the animal starts to replace that the the uh lymphosytes with collagen but it's spindly and it's not the same. So upon tanning what happens is that area collapses. So it actually forms a divot and then because the collagen is like spindly and not not normal it doesn't uptake the dye at the same rate as the rest of the skin. So hence you know it's it's very obvious on the final product.

[01:16:24]So hence the downgrades.

[01:16:27]So when we started working with um West Nile virus uh you know Westnile virus in the literature is really like well documented for human cases and it's very well recognized that you know it's it's sort of a bird mosquito transfer system whereby a vymic bird that's an animal undergoing a current infection um you know is bitten by a mosquito that mosqu that mosquito is now carrying the live virus it goes and bites a a another bird or a crocodilian or indeed a human or a horse or something like that and that animal is now infected.

[01:17:03]But from a um infection trial that we did, we in purposefully infected some animals with Westnile virus to see if we could invoke the lesions to confirm cautious postulates. Um but what we also noted was that that non animals that hadn't been infected within that pen also developed antibodies. So what uh what we also confirmed was that there was fecal shedding from the infected animals and then that was obviously contaminating the water and the animals are drinking the water and so that the the non-infected pon specifics also became infected. So that then started to show that you know any sort of you know any feces from an infected bird going into the water any any infected uh feces from infected crocodiles. So, so it just it became bigger and I think that made a bit more sense to me because when I was looking around a crocodile pen, I'm like, "Yeah, okay. Crocs can mosquitoes can bite crocodiles, but they do spend a lot of their time underwater." Um, you know, the mosquitoes have to bite the sensitive parts. It just the the level of of skin downgrades didn't make sense.

[01:18:10]But when we started looking at it at this level, it start, you know, the context was a bit more um obvious.

[01:18:17]There's potentially another transmission um pathway as well. um mosquitoes actually pass viruses and bacteria onto their eggs and subsequently they're the larae. So whilst it hasn't been proven yet um you know crocod mosquitoes breed in crocodile pens the crocodile larae hatch after 3 days uh you know in the in the whole thing around environmental uh uh social responsibility we try and uh use as less water as possible which potentially means that the animals are also ingesting um larae through through just drinking and and swallowing exercises and that could also be another possible transmission route. That's a long-winded way of saying get rid of your birds and your mosquitoes and you reduce the probability of having Westnile virus. So um uh yeah sorry yeah bird of mosquito control. Um so what do we know? Uh in saltwater crocodiles the majority of animals hatch with maternal antibodies and those maternal antibodies as I said before um you know it takes you know up to four months generally for animals to absorb all their egg yolk.

[01:19:26]Oh, egg yolk. Um, you distracted me now.

[01:19:31]Okay. And so what that then means is that after that time they become uh vulnerable to wild infection. So um you know if there's if there's if they do get infected they are then vymic for 17 days after which they start to develop antibodies. Um after which they develop antibodies.

[01:19:51]Working with vaccination strategies. If we vaccinate the animals at 4 months and again at 30 days after that as a booster that by that's that by the 30-day mark they're already starting to produce antibodies and we're currently doing a longitudinal trial to um uh ascertain how long that antibbody response goes for. So the long-winded part of me getting here is you know how do we then like know where our animals are up to like do they have the virus? Do they antibodies? Um the two ways to detect antibodies are VNTs and Elizers, but they are costly and they take time to process through the lab and that makes it really untimely to make commercial decisions on. So what Ryan has done is develop a linear flow assay. It's similar to a COVID test. So we all know that. Um he there's a each each strip has a control band to make sure that the test worked. And then uh this is just a a a series of samples here showing that there is actually a gradient in the um uh concentration of antibodies that he can detect and more importantly the negative. We're really after positive and negative. We're not looking after after the quantification of that at the moment. Now, I'm not sure if that video has played through all the way yet, but the point of it was the point of it was that you should have been able to see the bl uh the lines, but in the interest of time, I'll keep going. Um, I want to thank the uh uh funders of this. Again, the CSG has supported uh Ryan in his SRA. And I just thought I'd point out what those marks look like on the crocodile skin just um uh in the background on this on this slide. Thank you.

[01:21:41]Thank you, Sally.

[01:21:54]>> All right.

[01:21:56]Yeah. Thank you very much. Apologies for already being late uh from the first session. Uh we can now uh have the tea break and uh we will not have a question session. There are a few speakers. You can find them outside and ask them your questions.

[01:22:13]Thank you.

[01:22:16]>> Thank you everyone. Just a few housekeeping things. Um if you haven't dropped off your auction items, please do. Uh can I also see Joanna Batala and uh Curtain Colette? I haven't spotted them yet, but I have a message for them if they're here.