## 2 Materials and Methods
### In vivo Assessment of the Reproductive Response of Does Treated with GnRH-Loaded Nanoparticles
#### 2.3.2 Trial 1. Patterns of LH Secretion, Ovulatory Efficiency, and Early Fertility  
Twenty adult nulliparous rabbit does (5-5.5 months-old and 2.82 \(\pm\) 0.21 kg of body-weight) were treated for estrus synchronization with 25 IU of eCG (Gonaser\({}^{\text{\textregistered}}\), Hipra, Girona, Spain) [14] and 48 h later, does were subjected to one of the following five ovulation induction treatments: a control conventional i.m. protocol (0.8 \(\upmu\)g of GnRH in distilled water; C-treatment) and four protocols using GnRH-ChNPs with half i.m. dose (0.4 \(\upmu\)g. GnRH-ChNPs; HM-treatment), quarter i.m. dose (0.2 \(\upmu\)g i.m. GnRH-ChNPs; QM-treatment), half vaginal dose (4 \(\upmu\)g vs. GnRH-ChNPs; HV-treatment), and quarter vaginal dose (2 \(\upmu\)g vs. GnRH-ChNPs; QV-treatment). Females in C, HM, and HV treatments were immediately inseminated artificially with 0.3 mL diluted semen (30 \(\times\) 10\({}^{6}\) sperm/insemination). Females in HV and QV treatments were inseminated, at the same time, using the same seminal dose, but supplemented with the corresponding GnRH-ChNPs. Semen samples used for AI were collected from five fertile rabbit bucks by using an artificial vagina and a teaser doe. The quality of semen was evaluated, and only semen samples fulfilling quality criteria were used for AI [15]. The doses of GnRH-ChNPs were based on the findings of previous studies, where the dose of 0.8 \(\upmu\)g GnRH is the conventional dose in intramuscular treatment and dose of 8 \(\upmu\)g GnRH is the conventional dose in intravaginal treatment [4; 11]. The schematic diagram of the experiment is presented in Figure 1.  
The assessment of the patterns of LH secretion was performed on blood samples collected from the marginal ear vein of each doe with non-heparinized tubes at 0 (time of insemination) 30, 60, 90, and 120 min post-insemination. The samples were centrifuged at 700\(\times\) g for 20 min to obtain serum, which was stored at \(-\)20 \({}^{\circ}\)C until analysis. Then, the serum LH concentration was determined using an enzyme immunoassay commercial kit (Cusabio Biotech, Hubei, China). The assay sensitivity was 0.35 mIU/mL and intra- and inter-assay coefficients of variation were 8% and 10%, respectively.  
Afterward, all females were sacrificed 48 h post-insemination and the reproductive tracts were immediately obtained. The ovaries were dissected for determining the number  
Figure 1: Experimental planning of trial 1, aiming to determine the effect of gonadotrophin-releasing hormone (GnRH) in different doses and routes of administration on LH secretion (by means of successive blood samples; BS) and presence of oocytes/embryos in the reproductive tract, RT, at day 2 after artificial insemination (AI).  
of total ovarian follicles (clear visible follicles) and the number of ovulatory points (ovulated follicles), as previously described [14]. The oviducts were flushed with 15 mL of phosphate buffer solution (PBS) containing 0.2% of bovine serum albumin (BSA, Sigma-Aldrich, Madrid, Spain) to assess the number of embryos and their development stage and the number of non-fertil oocytes with a binocular microscope (Olympus Optical Co. Ltd., Tokyo, Japan).