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Alpha-2 agonists and stressed turtles   The table below illustrates that the alpha-2 agonist Domitor (medetomidine), when added to oxytocin + Lutalyse (PGF-2), seemed to be less effective with healthy RES than oxytocin + Lutalyse alone.  However, that poor outcome was caused by the sequence of injection.   Because we gave the Domitor after the mix of oxytocin and PGF-2 the laying process was disturbed.  We would learn later that we should have given the Domitor first. Years later, when working with softshells and snapping turtles, and injecting the alpha-2 agonists first, the alpha-2 agonists proved to be critical to achieving a successful outcome.  We will detail those result later in section 15 under the heading “The drugs we tested on SSS (spiny softshell turtles).” Shortly after we first worked with Domitor it was removed from the market and replaced by the isomer Dexdomitor (dexmedetomidine) which was about twice as potent as Domitor.  After that change we experimented with Dexdomitor and two other alpha-2 agonists: xylazine and Sedivet (romifidine).  The first challenge was to determine the appropriate dosage range for our trials.  We did that by reviewing the recommended doses for various small animals and then testing a range around those…

Possible injection sites We had learned from earlier work that intraperitoneal injections were difficult to do. They were fraught with potential errors like injecting the drug into a solid organ, bowel or egg follicle. Drug absorption from the peritoneal cavity is very rapid because of the large surface area and, for some drugs like prostaglandins, that is undesirable. Intramuscular injections were fine for larger turtles but with smaller animals there was the risk of nerve damage if a large volume was injected into a small muscle (Villarejo 1993). Intramuscular injections are also absorbed faster then subcutaneous (subq) ones so are most suited to drugs where rapid absorption is desired. Intradermal injections were only suitable for larger turtles and small volumes. Since we wanted a location where prostaglandins would be absorbed slowly we needed an injection site where we could give a subcutaneous injection that was safe for the turtle and the operator. To find a suitable site we did a dissection of a RES that had died after a prolonged illness. We selected a location near the base of the tail which you can see in the illustration below. It is labeled “empty space.” The injection site is labeled “empty…

Repeating an induction after the initial induction failed Previous work had suggested we would get the best results with a re-induction if we held an animal for 3 days after the initial, failed induction. This was difficult because there was only limited space to hold the turtles, especially the SSS which had to be held separately (because of aggression) and required a very large tank. Once we started using Lutalyse with RES there was only a 6% failure rate with the first induction so turtles to be re-injected were hard to come by. However, the failure rate with Lutalyse alone was 43% in 2016 with RES stressed by malnutrition so we had plenty of turtles to include in a follow-up study that year. When these RES were re-injected with Lutalyse and Sedivet three days after the initial failure we had a success rate of 81% as shown below. In 2013 we were inducing SSS with a combination of Lutalyse and oxytocin plus Dexdomitor. We re-injected three of these. Two were re-injected after two days and one after three days. They were re-injected with the same doses of Lutalyse, oxytocin, and Dexdomitor as were used for the previous failed induction. All…

Side effects of drugs used with RES and SSS Oxytocin used on its own has two significant side effects.  One side effect is that it interferes with the next ovulation in species that lay multiple clutches.  That means there is often only one clutch per season where there might have been three or four.  It also causes false nesting;  a behavior where the female attempts to nest after a successful oxytocin induction even though she’s carrying no eggs.  This is a big danger for wild turtles who must face the threats of automobiles and predators when they emerge from the water to lay their eggs. We addressed both these issues. We were able to test Lutalyse in several ways to see if it interfered with the following ovulation.  One method was to follow up labeled RES on the Concordia Turtle Farm.  We applied tree paint to each RES with a code that referred back to the day they were induced.  The problem with this method was that the labels deteriorated rapidly.  This happened despite each animal’s carapace being washed with water and then with rubbing alcohol before being labeled. After three weeks (the average period between clutches)  the label was…

The drugs we tested on Spiny softshells We started out by using oxytocin alone to induce the SSS. We tried it at two dosage levels, after different holding periods, with a low success rate in all cases, as shown below. After trying oxytocin alone we ran a trial of a mix of oxytocin and Lutalyse with both drugs being injected at the same time.  Forty four animals were involved.  It took nine working  days to collect and process this many animals so numbers were low in every category.  As you can see from the four tables below the success rate seemed higher on the stat day than on days 1, 2, 3 and 4. Even when all the results were pooled without specifying the day of induction, as shown below, none of the combinations of oxytocin and Lutalyse were very effective. Cytotec is misoprostol or prostaglandin E1. This is a drug that can induce abortion in mammals. The details of how we used it are described previously under the heading “The drugs we used on their own.”  The table below illustrates that it was not very effective.  It was also time consuming to prepare and dangerous to instil so we abandoned…

Working with spiny softshell turtles   Spiny softshells (SSS) are large and aggressive. They are also fast and hard to catch; even when using drift fences on a turtle farm! SSS are much too large to palpate for eggs so we had to x-ray each animal to determine how many eggs they were carrying.  Before any injections were given each turtle had to be wrapped in towels to protect the turtle and ourselves. An examination of the anatomy of the SSS showed two mechanisms that enabled them to retain their eggs.  One is a powerful sphincter at the end of the oviduct much like the cervix in humans.  This is illustrated below. The other mechanism that enables egg retention is a weaker sphincter at the distal end of the everted cloaca as shown in the figure below.  Both sphincters must be relaxed for oviposition to occur. In addition to finding the right drug to relax these sphincters, we had to determine the dose and the the sequence of injection; should we give drug #1 first, drug #2 first, or should we give them together?.  If we gave the drugs in sequence how long should we wait between injections?  There were…

Methods to determine the number of eggs in a clutch During our work with RES we depended on palpation to determine if the females were carrying eggs and how successful an induction had been. An induction was only classified as successful if no eggs were palpable afterwards. In 2009 we did a study and palpated 46 RES after induction.  There were three examiners.  Results were checked by ultra-sound. The outcomes in the table below showed an accuracy rate around 93%.  That is, 93% of the time the examiner would be correct when checking a turtle for eggs post-induction. When we decided to work with spiny soft shells (SSS) palpation was not possible because of their large size. We tried ultra-sound but that could only tell us if there were eggs, not how many eggs there were. So we turned to x-ray as our method to determine the number of eggs in these much larger turtles.  Our funds were limited but we managed to secure the loan of a handheld, portable, Nomad dental x-ray machine.  This machine had a low output so it took 3-4 seconds to get a good film. Each spiny soft shell turtle had to be washed before…

Less common species we worked with on the turtle farm We had limited opportunities to work with small numbers of other North American turtle species at the Concordia Turtle Farm.  Usually we were only able to induce a few of each species each year so the methods we used evolved over time and the numbers of animals we induced of any one species were low except for the spiny softshells. From 2008-2019 we injected over a hundred turtles of various other species besides RES (razor back musks, snapping turtles, and a variety of cooters, sliders, painted and map turtles) with variable outcomes. Because of the limitations of low numbers there were few observations of value.  The table above deals with the yellow bellied and cumberland sliders.  These species seemed as responsive as RES to 1.5 mg/kg of Lutalyse. The table below details the results with 21 snapping turtles.  Note that the eight turtles that received an alpha-2 agonist laid all their eggs. Sabine map turtles (Graptemys sabinensis), as shown below, appeared around the farm during rain storms. The barriers around the ponds were porous and these small turtles could slip through and wander freely. Because of their small size sabine…

Some ideas that did not work Since the biochemical complexities of reptilian reproduction are poorly understood we decided to experiment with a few unlikely drugs to induce egg laying because it is known that different species of lizards use similar hormones/prostaglandins in different ways (Guillette et al. 1992) and that might be true for turtles as well. Methylergometrine is an ergot alkaloid that causes the contraction of uterine smooth muscle and is used as a treatment for postpartum bleeding in women. We thought it was possible it would also trigger functional contractions of the oviduct. We tried a wide range of doses as shown below but it had no effect on oviposition. Ovacyst (gonadorelin diacetate tetrahydrate) is a hypothalamic releasing factor that causes the secretion of FSH and LH from the anterior pituitary. It is known that PGF-2 is involved in the secretion of FSH and LH so we thought it was possible these hormones might have something to do with oviposition. We tried a wide range of doses as shown in the table below; none had any effect. Mifepristone, also known as RU 486, is an oral drug used to produce abortion early in mammalian pregnancy. It is a…

Discovering the importance of prostaglandin F2 alpha (PGF-2) Carbetocin is a long acting analogue of oxytocin. It was difficult to obtain at the time we used it but it was worth the effort because we had an expectation that it would be much more effective than oxytocin. As the table below shows that was not the case. It had almost no effect at all! But, when we combined the carbetocin with Lutalyse, the results were dramatic. The table below illustrates this. When carbetocin was combined with 1.5 mg/kg of Lutalyse the success rate rose to 100% in 26 cases! We were puzzled by this and then “the light came on” and we realized that it was the Lutalyse alone that was so effective. Consequently we began to trial Lutalyse given alone by subq injection at the base of the tail. The next table shows how successful this was; a 94% success rate with a dose of 1.5 mg/kg. The figure below converts that data to graph form. Since we could only induce 20 RES per day these results reflect close to 15 days of work over several years. The inductions were spread over several years to eliminate the possibility the…