## 4 Discussion
This suggestion is confirmed by the decreased values of zeta potential (Table 1) and image taken by transmission electronic microscope (TEM), which clearly showed the aggregation between GnRH-ChNPs (Figure 3B.). Zeta potential refers to the value of surface charge and is considered as an indicator of particle stability. Regardless of the surface charge (+ or \(-\)), high values of zeta potential (>30 mV) refer to high stability and repulsion of the particles in the solution because the high value of the electrical double-layer thickness prevents the aggregation between the particles. Thus, a low value of zeta potential accelerates the process of particle aggregation [21; 22]. However, the increase in zeta potential values improves the stability of nanoparticles, this effect may not be suitable for cell viability. High zeta potential value, particularly the positive charge, stimulates cellular uptake, which affects cell survival [18; 23]. The spectra of functional groups of Ch, ChNPs, and GnRH-ChNPs presented in Figure 4 were recorded in the region between 4000 cm\({}^{-1}\) to 400 cm\({}^{-1}\). The Ch spectra showed peaks at 3458.3 cm\({}^{-1}\), 2924.3 cm\({}^{-1}\), 1645.5 cm\({}^{-1}\), 1419.1 cm\({}^{-1}\), 1314.8 cm\({}^{-1}\), 1572.05 cm\({}^{-1}\), and 1077 cm\({}^{-1}\), which belong to the following functional groups: hydrogen-bond, O-H; C-H bond in pyranose rings; C=O in NHCOCH\({}_{3}\); C-H in CH\({}_{2}\)OH; C-N stretching vibration of type II amine; N-H band; and C-O-C in glucosidic-linkages. The addition of TPP to chitosan solution (ChNPs) resulted in the disappearance of C-H and N-H functional groups, indicating binding of these groups with phosphate groups in TPP. The addition of GnRH to ChNPs resulted in shifts for the remaining functional groups identified in the Ch spectra, which was confirmed in previous studies [19; 24; 25]. The results of the in vitro release study showed an initial surge release of GnRH from GnRH-ChNPs of around 50% during the first 10 h of incubation in phosphate buffer solution (PBS, pH = 7.4). This might be due to the weak bonds between adsorbed GnRH and the surface of ChNPs [26]. The other 50% of conjugated GnRH was released slowly through the incubation time (from 10 h to 30 h), which is due to the degradation of the GnRH-ChNPs' surface and the release of encapsulated GnRH [13]. In general, nanoparticles entrap, adsorb, bind, or encapsulate the active agents in their structure, depending on the preparation method [27]. The main use of the nano-formula innovated in the present study was the induction of ovulation in rabbit does. The preparation method used in our study seems to be suitable for this purpose, since the initial release of GnRH is required to stimulate the LH surge secretion, while the slow release maintains the GnRH concentration over a long period and this sustained the surge of gonadotrophins [28]. The assessment of the potential of the developed GnRH-ChNP formula for inducing the LH-surge and ovulation revealed the relevance of the HM, QM, and HV treatments. In these three treatments, a reduction from 25% to 50% in the conventional GnRH dose was achieved without negative effects on LH surge or ovulation rate. In contrast, the QV treatment failed to induce LH surge or ovulation. These results indicate the ability of the nano-formula to protect GnRH from enzymatic degradation [20], improving the cellular uptake and thus greater bioavailability. It is interesting to note that HV treatment advanced the pre-implanted embryo development, as 25% of the collected embryos at 48 h post-treatment were beyond the blastocyst stage whilst morula and early blastocyst were the common stages in the other groups (C, HM, and QM). In the present study, the loading efficiency of ChNPs for GnRH reached 90%. Thus, intravaginal administration of GnRH-ChNPs may result in the sustained and high release of GnRH into the reproductive tract.