Uncategorized

Executive summary The tables and figures in this section summarize our key findings. The recommendations for induction based on this data can be found under the web-site heading “P.I.T. instructional.” P.I.T. instructional consists of a PDF that outlines our suggestions for a successful induction and a series of videos that details the practical techniques.   Large numbers of red-eared sliders (RES) were involved in multiple trials over several years to determine the most effective dose of Lutalyse in healthy RES. The most effective dose was 1.5 mg/kg given subcutaneously. ———————————————- The figure above is a graphical representation of the relative effectiveness of various doses of subcutaneous Lutalyse used to induce oviposition. ———————————————– This figure illustrates the dramatic improvement of effectiveness when Lutalyse is used when compared to oxytocin alone. Large numbers of RES were involved in these trials which were done over several years. Note the slight improvement when Lutalyse is used alone compared to various combinations. ————————————————— Smaller numbers of RES were involved in this trial to determine the best induction method for RES stressed by underfeeding. Sedivet 0.8 mg/kg followed immediately by Lutalyse 1.5 mg/kg was the most effective treatment. ———————————————————– As you can see in the table…

The background of our research During a visit to John Legler’s lab in 1977 we met a young graduate student named Jim Berry. I explained to Jim that we liked to keep hatchling turtles for a year or so, release them, and then replace them with new hatchlings. When I complained about how difficult it was to catch the hatchlings he suggested I learn how to “make them.” Jim then showed me how they had been experimenting with injecting oxytocin intraperitoneally to induce turtles to lay their eggs (Ewert and Legler 1978). I didn’t realize it then, but that simple lesson was to change our lives. From 1978 to 2005 we tried to induce about 250 turtles (see web-site section labeled “2007 article”). Our first obstacle was to find the gravid turtles. I made it a habit to take my vacation time in June and we would go out most evenings and mornings to collect turtles that were looking for a nesting site in our area of Pennsylvania, USA.  We sought aquatic turtles that were emerging from the water to lay their eggs and eastern box turtles (Terrapene carolina) which we found in known nesting areas. All the turtles were palpated…

Establishing a pattern for working with red eared sliders In the spring of 2008 we found ourselves at the Concordia Turtle Farm in Wildsville, Louisiana. Concordia is the largest turtle farm in the United States. It’s owned by the Evans family. They were very supportive of our goals and gave us access to a pond that had 5,000 red eared sliders (Trachemys scripta elegans) in it to use for our research.   They also gave us a shed to work in with power and water, and an ATV to move the animals between the pond and the shed. In later years we became concerned that the reproductive physiology of all turtles was not the same. To deal with that problem the Evan’s family also gave us access to a spiny softshell turtle (Apolone spinifera) pond so we could test the new methods we were developing on a species that was very different from red eared sliders (RES). The first challenge was to be uniform in our approach. For our results to be of any use we would need to treat every turtle the same. First we defined our goal as having every turtle lay all its eggs after the first…

Determining the techniques we would use Before we could begin to experiment with different drugs that would induce oviposition we had to design a standard method of giving injections, a standard site for those injections and a consistent environment to contain the turtles. Environmental concerns included the possible need for noise control, how to blind the turtles to potentially frightening movements around them, preferred temperatures and deciding if the outcome was better if the turtle was kept dry or was kept in water. We also had to adopt a method to label the turtles so we could identify them days or weeks later for follow-up. During these initial tests we used 10 units of oxytocin per kilogram as our standard dose because that was found to be most effective by John Tucker when he was working with wild RES in Illinois in 2007 and by us in the 1990s (see the “2007 article” section of this web-site). We also adopted the use of a grid as Tucker did in 2007 because it was the easiest method to keep the oviposited eggs safe from damage. When we were faced with a choice between two methods that had similar outcomes we always…

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 the drugs would be absorbed slowly we needed an injection site where we could give a subq 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. The site is labeled “empty space” but, in a healthy…

Designing the best environment for oviposition   We had learned in previous years how important it was to shield the turtles from being frightened by movements around them. We used to suspend them over a wet towel in a closed closet to achieve this goal. This worked fine for painted turtles (Chrysemys picta picta) and box turtles but such a procedure was time consuming and some species became agitated if suspended.   Placing them in deep water also worked but it required frequent observation to remove the eggs and it was also time consuming. We eventually decided on using a wide spaced grid that the eggs could fall through onto a moist surface below. Constructing the grid took about twenty minutes but it could be reused hundreds of times and was safe for the turtles and the eggs. We wondered if noise affected the laying process. To test this we put the plastic tubs outside the lab in an alleyway that diesel powered 4×4 vehicles drove through about every ten minutes. To our surprise the noise had no effect at all. We did this the first time using oxytocin alone.   The results are shown in the chart below. Years later…

The drugs we used on their own   The table above shows how poor the results were using oxytocin alone (Remember that “success” for us means that the turtle laid all its eggs after one injection).  We pooled the intraperitoneal and subq results because they were essentially identical as the table below shows. Because of these poor results with oxytocin, a success rate of better than 40% became our target when we experimented with other agents. The table below shows a number of agents that we had no success with on their own. We used normal saline as a control and, of course, it had no effect.   One striking failure was arachidonic acid, the precursor to the prostaglandins. It is known to induce oviposition in birds (Wechsung and Houvenaghel 1981), and lizards (Guillette et al.1992). We tested a wide range of doses. Since arachidonic acid is the precursor for many prostaglandins we thought it might be rapidly metabolized to the prostaglandins involved in oviposition. Unfortunately we were wrong; it had no effect. A gel made with misoprostol (prostaglandin E1) by crushing Cytotec tablets and suspending it in a gel was introduced into the cloaca with a 20 ml syringe…

Combinations of drugs we used The dramatic improvement in success shown in the table below (when we combined oxytocin and Lutalyse) led to a long series of experiments to determine the best proportions of each drug.   A common side effect of Lutalyse is diarrhea. Because the incidence of diarrhea rose 231% when the dose of Lutalyse was increased from 1.0 mg/kg to 3.0 mg/kg, and the success rate dropped, we did not pursue dose combinations using 3.0 mg/kg or more of Lutalyse. The table above shows the various combinations of prostaglandins and oxytocin that we tried initially. Oxytocin (10u/kg) + Lutalyse (1.5mg/kg) had a 78% success rate compared to a 32% success rate with oxytocin alone; a dramatic improvement! In the early studies the oxytocin and Lutalyse were given by separate injections but later on, when the drugs were combined into one injection, the success rate was even higher (Instead of giving separate injections of oxytocin and Lutalyse ten minutes apart we decided to combine them into one injection). The combination was at least as effective and a lot less time consuming as shown below.   The manufacturer of Lutalyse suggests that it be stored at room temperature. Oxytocin…

Discovering the importance of prostaglandin F2 alpha 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…

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…