## 1 Introduction  
The occurrence of external stimulation during mating (neurohormonal reflex) is required to evoke the gonadotropin-releasing hormone (GnRH)-dependent luteinizing hormone (LH) surge, and consequently ovulation, in non-spontaneously ovulating species like the rabbit [1]. This biological event plays a crucial limiting role for the successful application of assisted reproductive techniques such as artificial insemination (AI) because, in the absence of the male, ovulation needs to be induced by artificial hormonal stimulation. Hence, the induction of ovulation in routine AI protocols is usually achieved by the administration of either GnRH or LH or other gonadotrophins with LH activity (i.e., equine chorionic gonadotrophin, eCG, or human chorionic gonadotrophin, hCG). The GnRH analogues (such as gonadorelin, lecirelin, tritptorelin, or buserelin) are the most recommended compounds because they have additional biological advantages and the possibility of repeated treatments without developing specific antibodies [2]. Currently, GnRH is included in semen extenders to be directly administered to the doe via the seminal dose, which improves animal management and welfare [2]. Certainly, the successful implementation of the intravaginal route in protocols for ovulation induction may minimize animal distress and staff workload when compared to traditional protocols via intramuscular doses. However, the enzymatic proteolytic activity of seminal plasma and vaginal fluids and the biological barriers for mucosal permeation may limit the efficiency of such protocols. In addition, GnRH has a short half-life time in blood circulation, 2-4 min, because it is rapidly degraded by peptidases and cleared by glomerular filtration [3], which limits both its biological activity and sustained action. Consequently, a first approach for improving the efficiency of the treatment is to increase the dose or the activity of the GnRH analogue. However, doses more than ten to fifteen-fold higher are needed (about 8 and 16 \(\upmu\)g buserelin/seminal dose), which constitutes a potential health risk for workers and significantly increases the cost-efficiency of the protocols [4]. A promising alternative may be based on the use of nano-drug delivery systems. Nano-drug delivery systems have been optimized to extend the half-life time of the compound to improve its passage across endothelial or epithelial barriers into blood or lymph circulation and to sustain its delivery to the target sites. Consequently, overcoming these biological barriers can improve cellular uptake and therefore lower doses of drugs/hormones may be used [5; 6; 7; 8; 9]. Hence, nano-drug systems for hormonal treatments (e.g., GnRH for ovulation induction) may benefit many biotechnological assisted reproductive techniques in the livestock field (e.g., AI [5]). However, there is a scarcity of data regarding both hormone bioactivity and animal performance when GnRH is used in nano-formula, although previous results are promising. To date, chitosan-dextran sulfate GnRH (buserelin acetate) nanoparticles have been added to seminal doses of rabbits [10], allowing the GnRH dose to be reduced to half the conventional dose without affecting the fertility of the does. Moreover, the results of a trial performed with the administration of chitosan-tripolyphosphate (TPP)-conjugated GnRH nanoparticles in goats indicate that the GnRH dose may be even reduced three- to four-fold without affecting fertility and proliferative [11]. The practical application of such protocols makes necessary extensive studies on technical and physiological features that may limit the efficiency and final outcomes of treatments involving GnRH-loaded nanoparticles (fabrication, route of administration, bioavailability and hormonal balance). Therefore, the present study aimed to set the basis and evaluate the feasibility and efficiency of GnRH nanoparticles for inducing ovulation in rabbits.