STANDARDIZATION OF MINIMALLY INVASIVE SURGICAL AND PERI-SURGICAL PROCEDURES IN POND SLIDERS (TRACHEMYS SCRIPTA)

North American pond sliders (Trachemys scripta) are invasive alien species that, following the release of pet specimens in the wild over the decades, have established breeding populations outside their native habitat, with detrimental effects on local biodiversity. Their commerce and breeding have been recently banned in the European Union, and national programs are being introduced by Union Members to eradicate and control their presence. Aims of the present PhD project were to elaborate on this species’ minimally invasive ovariectomy regarding surgical, anesthesiologic and analgesic aspects, and to dwell on the concept of population control, possibly providing means alternative to surgical gonadectomy. The first study included in the present thesis is a comparison between dorsal and lateral recumbency during endoscope assisted oophorectomy. Ovariectomy via the prefemoral fossa, with or without endoscopic assistance, is a well-described technique for elective and therapeutic sterilization in chelonians. The choice between lateral and dorsal recumbency is generally left to the surgeon’s preference, with no data supporting an objective superiority of one over the other. Twenty-four sexually mature female pond sliders were enrolled for this study, and were randomly divided in two groups: 12 animals were placed in right lateral recumbency with a left fossa approach (Group A), and 12 in dorsal recumbency with a right fossa approach (Group B). Scoring systems were applied to assess the ease of access to the coelomic cavity, and the ease of identification of the ovary opposite to the surgical incision; total surgery times and necessity to perform bilateral access were recorded. A negative correlation was found between the body weight of the animals and the ease of access to the coelom (p = 0.013), making the access easier in smaller animals. No significant difference was found between the two groups in terms of ease of access to the coelomic cavity, first ovary removal time, ease of identification of the second ovary, second ovary removal time, or total surgery time. It can be concluded that endoscope-assisted prefemoral ovariectomy in mature pond sliders can be indifferently performed in lateral or dorsal recumbency, depending on the preference of the surgeon, on the animal’s pathophysiologic status, or on the necessity to perform concurrent procedures. The second study is the description and comparison of two totally intramuscular anesthetic protocols. Chelonian anesthesia is a challenge for reptile practitioners: the capability of these animals to hold their breath for a considerable amount of time, and the ability of the chelonian heart to perform right-to-left shunts, make inhalant maintenance less predictable and reliable. Intravenous administration of anesthetic drugs can pose problems as well, since the difficulty of obtaining and maintaining IV catheters in non-complying patients. Anesthetic induction and maintenance by the intramuscular (IM) route circumvent these problems, but the number of multimodal protocols available for this purpose is limited. Twenty-six sexually mature female pond sliders undergoing elective prefemoral ovariectomy were enrolled for this study, and were randomly divided in two groups: Group A received a IM combination of ketamine 3 mg/kg + dexmedetomidine 0.1 mg/kg + midazolam 0.5 mg/kg + alfaxalone 8.5 mg/kg; Group B was premedicated with morphine 1 mg/kg IM 1 hour prior to induction, which was achieved via ketamine 3 mg/kg + dexmedetomidine 0.1 mg/kg + midazolam 0.5 mg/kg + alfaxalone 5 mg/kg IM. Time of loss of and recurrence of spontaneous movement, muscle and cloacal tone, and limb withdrawal latency, time to intubation, heart rate (HR), need for isoflurane administration, recovery time and total anesthesia time were recorded and compared between the two groups. Results indicate that IM administration of dexmedetomidine/ketamine/midazolam/alfaxalone alone (Group A) or in combination with morphine (Group B) provided an adequate anesthetic depth for surgical purpose respectively in 76.9% and 100% of animals, witnessed by the lack of reaction to skin incision and organs manipulation and resection. No marked cardiovascular effects were recorded but, if compared to basal values, Group B displayed a significantly lower HR in the first 15 minutes of surgery, likely determined by the cardiorespiratory effects of morphine. Protocol B showed quicker loss of measured parameters, shorter mean induction time (11 ± 5 vs 22 ±5 minutes, p = 0.00001), quicker recurrence of measured parameters and shorter recovery time (21 ±12 vs 36 ±22 minutes, p = 0.04). Both protocols were safe and provided quick induction and recovery time; premedication with morphine allowed to lower the dosage of alfaxalone, and the resulting protocol provided surgical anesthesia suitable for the whole length of the procedure without requiring inhalant maintenance. The third study evaluated the role of differentially expressed plasmatic micro RNAs (miRNAs) as possible biomarkers for surgical pain in pond sliders. Plasma of female turtles that underwent surgical gonadectomy were collected 24 hours pre-surgery, and 2.5 hours and 36 hours post-surgery; the expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves, and the pointed out that, out of 14 differentially expressed miRNAs, miR-499-3p and miR-203-5p were effectively modulated. MiR-203-5p was fair (AUC: 0.7934) in discriminating pre-and 36h post-surgery samples and poor for other time points, while miR-499-3p was excellent (AUC: 0.944) in discriminating pre-surgery and 2.5h post-surgery samples, and fair in discriminating pre-surgery and 36h post-surgery (AUC:0.7292) and 2.5h and 36h post-surgery (AUC:0.7569) samples. In conclusion, it was demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers. The last study investigated the efficacy of single and double IM 4.7 deslorelin acetate implant in suppressing fertility in female pond sliders. The use of long-acting GnRH-agonists has been poorly investigated in reptiles, and the few available studies show inconsistent results. Twenty sexually mature turtles were divided into three groups: one single-implant groups (6 animals), one double-implant group (6 animals) and one control group (no implant). During one reproductive season (March to October), plasmatic concentration of sexual hormones (estradiol, progesterone and testosterone) and ovarian morphometric activity via computed tomography were monitored about every 30 days. A significative (p = 0.011) decrease in the number of phase-II ovarian follicles was detected in the double-implant group when compared with the control group, but no significant difference was noted regarding the number of phase-III and phase-IV follicles, egg production, and sexual hormones plasmatic concentration. Results show that neither a single or double deslorelin acetate implant can successfully inhibit reproduction in female pond sliders during the ongoing season, but the lower number of phase-II follicles in the double-implant group can possibly be associated to a reduced fertility in following seasons. The present PhD project confirms the poor feasibility of non-surgical sterilization in chelonians and improves the knowledge on endoscopic-assisted ovariectomy, providing useful insight regarding not only the surgical procedure, but also regarding anesthesiologic concerns and pain management in these animals. Obtained results confirm this surgery as a highly specialistic procedures, and its massive application in the context of population control programs is questionable.