Development of new artificial insemination extenders supplemented with GnRH analogues to induce ovulation and proteomic characterization of rabbit semen

Abstract

The general objectives of this thesis were to develop new artificial insemination (AI) extenders supplemented with a GnRH analogue and to characterise the proteomic profile of rabbit semen. In chapter I, the inclusion of a protease inhibitors cocktail in the insemination extender (IE) to avoid part of the rabbit seminal plasma protease activity was evaluated. Seminal quality and fertility rate were not affected by the cocktail, having similar values between experimental and control groups. However, prolificacy rate was significantly lower in experimental group compared to positive and negative control groups (8.2 ±0.22 vs. 9.3 ±0.23 and 9.2 ±0.26 total born per litter, respectively). From this chapter, it may be concluded that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate and it in the future it would be advisable to test specific aminopeptidase inhibitors (AMIs). Therefore, in chapter II, we supplemented the IE with AMIs (bestatin and EDTA), and we studied their effect on rabbit seminal quality and reproductive performance. Seminal quality was not affected by AMIs. Regarding reproductive performance, the inclusion of AMIs, did not affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery) in comparison with control group. Thus, we concluded that AMIs can be used in rabbit IEs to inhibit part of the seminal plasma aminopeptidase activity. In chapter III, we test new rabbit IEs containing AMIs with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. The following experimental extenders were studied: C4 (4 µg buserelin/doe in control medium (CM): Tris-citric acid-glucose supplemented with AMIs), C5 (5 µg of buserelin/doe in CM), Q4 (4 µg of buserelin/doe into CS-DS nanoparticles in CM) and Q5 group (5 µg of busereline/doe into CS-DS nanoparticles in CM). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 vs. 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied. Thus, CS-DS nanoparticles as carrier for buserelin acetate allow reducing the hormone's concentration in extenders supplemented with AMIs without affecting the fertility and prolificacy of rabbit females. Therefore, nanoencapsulation seems to be a promising system to protect the GnRH analogue in rabbit AI extenders. On the other hand, the aim of the last three chapters was to characterize rabbit semen proteins. In chapters IV and V, rabbit seminal plasma (SP) proteins were studied. Semen samples were recovered using 6 males from each genetic line (A and R). For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment (24 pools in total). In chapter IV, we used a 1D polyacrylamide gel electrophoresis approach. Seven protein bands were significantly different between genetic lines and three protein bands were significantly different between seasons. In chapter V, SP was subjected nano LC-MS/MS and 402 proteins were identified and quantified. Twenty-three proteins were differentially expressed between genotypes. Regarding the effect of season on rabbit SP proteome, results showed that there was no clear pattern of protein variation throughout the year. The results obtained in both chapters evidence that genotype is related to a specific abundance of SP proteins. In chapter VI, rabbit sperm proteins were characterised. Six samples were recovered during two months using 5 males from each genotype. Sperm proteins were subjected to nano LC-MS/MS and 487 proteins were identified and quantified. Forty proteins were differentially expressed between genotypes. In conclusion, rabbit sperm proteins showed that genotype has also a huge impact on their abundance. Finally, with these data, the first publicly accessible database of rabbit semen proteome was created.