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Therapeutic Inhibition of Reproduction in Lizards
Helminski, Gabrielle and DeNardo. Dale F.
Abstract As reptiles become increasingly popular as pets, owners have become concerned with the ability to temporarily inhibit their reproduction and the associated, unwanted behavior. Tamoxifen, an estrogen binding inhibitor, and indomethacin, a prostaglandin synthesis inhibitor, were tested as possible therapeutic treatments for temporary reproductive inhibition. Three phases were used in this experiment. In the first phase, 12 female leopard geckos (Eublepharis macularius) were implanted intracoelomically with sixty-day time-release pellets of either hormone at the onset of the reproductive season. All treated females failed to show any follicular yolk deposition for the duration of the sixty-day life-span of the pellets, whereas 15 of the 18 control females showed reproductive activity. Indomethacin treated females were completely inhibited for the entire reproductive season, but 50% of the females developed a generalized edema. Fifty percent of tamoxifen treated females showed some follicular vitellogenesis after the expiration of the pellet, but no female successfully laid a viable clutch. In phase two, unmanipulated females already displaying active vitellogenesis were treated with tamoxifen pellets. The tamoxifen was ineffective at inhibiting further follicular growth or ovulation. In phase three, all tamoxifen treated females were observed in the following breeding season. Eight of nine successfully reproduced and their reproductive output (i.e., number of total clutches, number of fertile clutches) was similar to that of control females. These results suggest that tamoxifen may be a viable treatment to induce temporary reproductive inhibition in reptiles, and further study is warranted. Introduction
Reptilian reproduction can be associated with such physiological and behavioral changes as extended inappetence, weight loss, muscle atrophy, and increased activity. Most pet owners would prefer not to endure such changes in their pets. Furthermore, because the captive environment usually does not duplicate the complexity of the natural nesting environment, gravid females can experience dystocia as a result of this unwanted and unproductive reproductive activity. For most reptiles, reproduction can be avoided by not co-housing males and females. However, in some circumstances, it is neither feasible nor desirable to keep animals individually. Additionally, in some species, such as the commonly kept green iguana (Iguana iguana), females will become reproductively active even in the absence of a male.
Chemical Inhibitors While not the only hormones involved, both estrogens and prostaglandins have substantial roles in regulating female reproductive activity. Estrogens are primarily responsible for the production of yolk in the liver. Prostaglandins appear to play a role in follicle development and ovulation. The goal for the study was to find chemicals that would be able to temporarily interfere with the function of these hormones and test the chemicals' feasibility for clinical use. Both tamoxifen and indomethacin were used in the study. Tamoxifen is an estrogen binding inhibitor and has been shown to inhibit estrogen-induced vitellogenesis in birds (Gschwendt et al,1982), amphibians (Riegel et al, 1986), and fish (Le Menn et al, 1980). Indomethacin is a prostanglandin synthesis inhibitor shown to be effective in mammals (Espy et al, 1982), birds (Shimada et al, 1986), fish (Goetz, 1983) and reptiles (Guillete et al, 1990). In lizards, indomethacin delays both parturition (Guillette et al, 1991) and follicle stimulating hormone-induced ovulation (Jones et al,1990), but its effect on follicular growth is unknown. Materials and Methods Twelve male and 30 female leopard geckos, Eublepharis macularius, were used for this study. The experiment was divided into three phases Four days after implantation, males were introduced into the female cages. Females were exposed to at least one male every week for the duration of the reproductive season. Females were weighed and visually inspected for reproductive activity weekly. When follicles were present, their diameters were measured using calipers. Gravid females were checked daily for the presence of eggs. Upon oviposition, date of oviposition, female mass, egg number, egg mass, and egg quality were recorded. Data was compared between groups using unpaired T-test (StatView, SAS Institute, Cary, NC). Experimental design Phase 1: inhibition of the onset of reproductive activity The goal of the first phase of the experiment was to determine the effectiveness of the two chemicals at inhibiting the onset of reproductive activity. Ten days after emergence from the cooling period, six female geckos were randomly assigned to each of two treatment groups, while the remaining 18 female geckos served as controls. Treatment females were implanted with a 60-day time-release pellet (Innovative Research of America, Sarasota, FL) containing either 5mg tamoxifen or 5mg indomethacin. Control animals underwent surgery but did not receive a pellet. See Figure 1 for results. Phase 2: inhibition of active vitellogenesis The goal of the second phase of the experiment was to determine the effectiveness of the therapeutic agents at interrupting active vitellogenesis. After 120 days post-treatment (two times the projected life span of the pellets), control females with follicles were alternately subjected to either implantation with a pellet similar to those used in phase 1 or a second control surgery. See Figure 2 for results. Phase 3: reversibility of reproductive inhibition The goal of the third phase of the experiment was to determine whether any reproductive inhibition caused by the implanted pellets was reversible (e.g., would females that were reproductively inhibited one year successfully reproduce the next year). After year one, females were similarly cooled the following January to again stimulate reproductive activity. Upon emergence, females were not manipulated, but simply maintained as in the previous year. Data collection and analysis followed that of the previous phases of the experiment. See Figure 3 for results. Discussion Indomethacin - While indomethacin was effective at completely inhibiting follicular yolk deposition during the entire reproductive season, the generalized edema which occurred in 50% of the females indicate that it would not be clinically valuable as an inhibitor of reproduction. Tamoxifen - Tamoxifen appears to be effective in inhibiting reproduction when it is introduced at the beginning of the reproductive season. Additionally, females treated with tamoxifen were able to reproduce normally the following season. These results suggest that tamoxifen may be an option for clinical use for the temporary inhibition of reproduction in reptiles. Due to its ineffectiveness at halting late stage follicular development, tamoxifen may only be effective when given during or before early follicular growth. The timing for implantation can easily be determined in species that respond to seasonal changes such as reduced ambient temperatures. In species where the onset of reproduction is variable, tamoxifen may not be of value. Further Studies Tamoxifen appears to be a promising therapeutic option for the temporary inhibition of reproduction. This study was only preliminary and further studies are warranted. Reptiles popularly chosen as household pets (e.g., Iguana iguana and tortoises) should be used in a similar study to verify the clinical value of tamoxifen. Acknowledgements This work was funded in part by the Department of Biology, Arizona State University, and grants provided by the Howard Hughes Medical Institute and the Association of Reptilian and Amphibian Veterinarians Conservation and Research.
References
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