## 4 Discussion  
The present study aimed to set the basis for implementing a nano-drug delivery system (i.e., GnRH-loading chitosan nanoparticles; GnRH-ChNPs) for improving procedures and yields of artificial insemination (AI) in rabbits by reducing hormone dose and facilitating animal handling during AI. Achievement of these aims depends to a far extent on the physicochemical properties of the formula. The size of NPs is one of the most important determinants for passing barriers of mucosal tissues of and for the intracellular uptake [17]. The size range of most of the nanoparticles applied for the drug delivery system is between 50-250 nm. This size allows the particles to pass efficiently through different barriers and cell pores, and thus improves the cellular uptake [18; 19]. The biological efficiency of NPs greatly depends on the potential of the nano-carrier to conjugate with loaded molecules. Increasing loading efficiency of active molecules within carriers improves its lifetime in the bloodstream due to the protection from enzymatic degradation. In the present study, chitosan (Ch):TPP with a 2:1 ratio resulted in 90% loading efficiency for GnRH (Table 1). In context, [PERSON] and [PERSON] [11] obtained 91.2% LE when the same Ch:TPP ratio was used for GnRH (gonadorelin) encapsulation. On the other hand, [PERSON] et al. [19] obtained 69% LE for LH-RH when using the same Ch:TPP ratio in female fish. ; [PERSON] et al. [10] also obtained only 43% LE when they used a Ch:dextran ratio of 4:1 for GnRH encapsulation. These results confirm the appropriateness of selected preparation conditions and the formula (Ch concentration 1 mg/mL and Ch:TPP ratio 2:1) to conjugate most of the loaded hormone used in the present study. In the present study, the addition of GnRH to ChNPs increased the particle size from 95.19 \(\pm\) 1.9 nm to 212 \(\pm\) 2.69 nm and decreased the zeta potential value from +34.0 to +8.0 mV (Table 1). Similar results have been obtained in previous studies by [PERSON] et al. [20], who found that the addition of trypsin increased the size of ChNPs from 147 nm to \(\approx\)220 nm. On the other hand, [PERSON] and [PERSON] [11] obtained a lower GnRH-ChNPs size after loading the 93.91 \(\pm\) 0.85 nm compound to non-loaded ChNPs of 125.9 \(\pm\) 3.06 nm. The variation in the size of nanoparticles after loading (increase or decrease) could be related to many factors including the type of loaded molecules, type of carrier molecules, the preparation conditions, and the surface charge (zeta potential). [PERSON] et al. [20] noted that the increased diameter of ChNPs after trypsin loading might be due to the size and the molecular weight of the enzyme during the loading process and adsorption on the  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Variables**} & \multicolumn{3}{c}{**Treatments (\(n=14\) Does/Group)**} \\ \cline{2-4}  & **C** & **HM** & **HV** \\ \hline \multicolumn{4}{c}{Fertility evaluation parameters1  \\ \hline \multicolumn{4}{c}{Fertility evaluation parameters1  } \\ \hline \multicolumn{4}{c}{Pertility evaluation parameters1  } \\ \hline \multicolumn{4}{c}{Pertruition rate (\%)} & 78.5 2  (11/14) & 71.5 2  (10/14) & 50 2  (7/14) & 0.019 \\ \multicolumn{4}{c}{Parturition rate (\%)} & 78.5 2  (11/14) & 64.2  (9/14) & 35.7 2  (5/14) & 0.050 \\ \multicolumn{4}{c}{Abortion rate (\%)} & 0.0 (0/11) & 10.0 (1/10) & 28.5 (2/7) & 0.136 \\ \multicolumn{4}{c}{Pregnancy outcomes parameters (mean \(\pm\) S.E.M. )2  } \\ \multicolumn{4}{c}{Litter size at birth} & 6.08 \(\pm\) 0.70  & 5.47 \(\pm\) 0.59  & 2.50 \(\pm\) 0.54  & 0.001 \\ \multicolumn{4}{c}{No. of live litter} & 6.08 \(\pm\) 0.70  & 5.47 \(\pm\) 0.59  & 1.92 \(\pm\) 0.63  & 0.001 \\ \multicolumn{4}{c}{No. of dead litter} & 0.00 \(\pm\) 0.00  & 0.00 \(\pm\) 0.00 [FOOTNOChNP surface. However, in our study, the increase in GnRH-ChNP size could be ascribed to the occurrence of aggregation between GnRH-ChNPs.