## 3 Results  
In Vitro Assessment of the Physicochemical Properties and Release Patterns of GnRH-Loaded Nanoparticles  
Physicochemical characteristics and loading efficiency of GnRH-ChNPs are shown in Table 1. The loading efficiency of GnRH by ChNPs was 90% and the average size, PdI and zeta potential of ChNPs and GnRH-ChNPs were 95.19 \(\pm\) 1.9 nm vs. 212 \(\pm\) 2.69 nm, 0.165 vs. 0.295, and +34.0 vs. +8.0 mV, respectively. The FTIR showed that the Ch spectra exerted 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 bond; and C-O-C in glucosidic-linkages. Addition of TPP to chitosan solution resulted in the disappearance of C-H and N-H functional groups, indicating binding of these groups with phosphate groups in TPP. Addition of GnRH to ChNPs resulted in shifts for the remaining functional groups identified in Ch spectra (Figure 3). The images of ChNPs under the transmission electron microscope showed spherical nanoparticles with smooth surfaces and a diameter of about 100 nm and, after GnRH loading, the diameter of GnRH-ChNPs reached 200 nm with high agglomerating appearance (Figure 4). \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Nanoparticles.**} & **Particle Size** & \multirow{2}{*}{**PdI**} & **Zeta Potential** & **Loading** \\  & **(nm)** & & **(mV)** & **Efficiency (\%)** \\ \hline ChNPs & 95.19 \(\pm\) 1.90 & 0.165 & +34.0 & - \\ GnRH–ChNPs & 212 \(\pm\) 2.69 & 0.295 & +8.0 & 90 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Particle size, polydispersity index (PdI), zeta potential, and loading efficiency of chitosan nanoparticles (ChNPs) and GnRH-loaded chitosan nanoparticles (GnRH–ChNPs). Figure 3: Fourier transform infrared (FTIR) spectra of chitosan (Ch), chitosan-TPP nanoparticles (ChNPs). The release profile of GnRH-ChNPs showed that the initial surge release of GnRH was about 50% at the first 10 h of incubation time, followed by a slow release of residual concentration during the subsequent 20 h. The release of the almost loaded hormone (about 99%) from ChNPs was after 30 h and remained steady up to 20 h later (Figure 5). In Vivo Assessment of Patterns of LH Secretion, Orulatory Efficiency, and Early Fertility in Does Treated with GnRH-Loaded Nanoparticles  
Figure 6 depicts the effect of different doses and routes of GnRH-ChNPs on serum LH concentrations at 0, 30, 60, 90, and 120 min. There were no significant effects on overall mean LH concentrations but on timing of the preovulatory LH surge. The groups HM, QM, and HV showed an earlier LH surge (90 min vs. 120 min post-insemination in the treatment C; \(p<0.05\)), but the group QV failed to induce LH surge with LH concentrations remaining stable. Figure 4: Transmission electron microscope image of (**A**) chitosan-TPP nanoparticles and (**B**) GnRH-loaded chitosan nanoparticles at a magnification \(\times\) 20,000 and 25 \({}^{\circ}\)C.  
Figure 5: Mean (\(\pm\) S.E.M.) percentage of over-time release of gonadotropin-releasing hormone (GnRH) from GnRH-loaded chitosan nanoparticles. All groups (HM, QM, HV, and QV) significantly increased the number of total ovarian follicles when compared to group C (\(p\) < 0.05; Table 2). The number of total ovarian follicles was significantly lower in group HM, intermediate in groups QM and QV, and higher in group HV (\(p\) < 0.05). There were no significant differences in the number of ovulation points among groups, except for group QV, which showed no ovulation points. The number of recovered embryos was higher in groups HM and HV than in group C (\(p\) < 0.05), while group QM showed intermediate values and no embryos were collected at group QV.