Reproductive Performance of Female Rabbits Inseminated with Extenders Supplemented with GnRH Analogue Entrapped in Chitosan-Based Nanoparticles

**Background:** Rabbits are reflexively ovulating species, so artificial insemination (AI) requires exogenous GnRH administration to induce ovulation. Intravaginal administration is less stressful than intramuscular injection but suffers from poor bioavailability due to proteolytic enzymes in seminal plasma (especially aminopeptidase B) and limited vaginal mucosa permeability. Consequently, GnRH analogue doses in extenders must be 15–25 times higher than intramuscular doses (15–25 μg vs. 1 μg). Nanoencapsulation using biodegradable polymers may protect the analogue from enzymatic degradation and improve mucosal transport. This study evaluated two chitosan-based nanoparticle systems—chitosan–dextran sulphate (CS–DS) and chitosan–alginate (CS–ALG)—as carriers for buserelin acetate in rabbit AI extenders.

**Methods:** Nanoparticles were prepared via coacervation without chemical cross-linkers. Polymers (0.05%) were dissolved in a TCG extender supplemented with 10 μM bestatin and 20 mM EDTA. Buserelin acetate was dissolved in DS and ALG solutions and added dropwise into chitosan solution (charge ratio 1:4 v/v) to form self-assembled nanoparticles. Entrapment efficiency (EE) was measured spectrophotometrically at 280 nm at time zero and after 5 h incubation at 5 °C and 37 °C. Semen from 12 adult New Zealand White males was pooled, evaluated (minimum 70% motility, 85% normal acrosome, <15% abnormal sperm), and split into three fractions: CS–DS extender (8 μg/mL buserelin acetate), CS–ALG extender (8 μg/mL), and control extender (no GnRH). A total of 356 non-lactating females received 0.5 mL of diluted semen via standard curved cannulas. Females in the CS–DS and CS–ALG groups received 4 μg buserelin acetate intravaginally (0.5 mL × 8 μg/mL); control females received extender without GnRH plus 1 μg buserelin acetate intramuscularly. All females received 20 IU eCG intramuscularly two days before insemination. Fertility rate (does giving birth/inseminated does) and prolificacy (total and liveborn kits per litter) were recorded. Statistical analysis used GLM with group as fixed effect; ANOVA for semen quality, EE, and prolificacy; probit link with binomial error for fertility; Bonferroni's test for post-hoc comparisons (p < 0.05).

**Key Results:** Semen quality was similar across groups (total motility 81.2 ± 7.9%, progressive motility 51.2 ± 9.1%, viability 68.9 ± 4.80%, acrosome integrity 89.1 ± 2.5%). Initial EE exceeded 90% for both systems but was significantly higher for CS–DS than CS–ALG. After 5 h at 5 °C, EE remained similar to initial values. After 5 h at 37 °C, EE decreased by 8–10% in both systems, with CS–DS retaining significantly higher EE than CS–ALG. In vivo, no significant differences in fertility rate or prolificacy were observed between groups. Females inseminated with CS–DS and CS–ALG extenders (4 μg buserelin acetate intravaginally) achieved reproductive performance comparable to the control group (1 μg intramuscularly).

**Clinical Implications:** Both chitosan-based nanoencapsulation systems effectively protected the GnRH analogue from degradation and enabled successful intravaginal ovulation induction at 4 μg per female—a substantial reduction from the typical 15–25 μg used in conventional extenders. This approach eliminates the need for intramuscular injection, reducing stress for animals, decreasing handling time for farmers, and lowering the risk of occupational exposure to hormonal products. The use of oppositely charged polymers without chemical cross-linkers provides a safe, biocompatible encapsulation method. The presence of EDTA in the extender may further enhance vaginal absorption via chelation of epithelial Ca²⁺ ions. These findings represent a refinement in rabbit AI practice that improves animal welfare, biological safety, and cost-efficiency. Further studies are warranted to determine whether even lower GnRH concentrations are feasible and to evaluate nanoparticle stability during extended semen storage (2–36 h).