The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E-like (113 kDa), haemoglobin subunit zetalike (13 kDa) and β-nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.

The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size.

Males from a paternal line selected for growth traits were used to produce semen doses at insemination centres and farms in a breeding scheme for rabbit meat production. The aim of this study was to assess whether a programme of selection by daily gain in fattening period changed the seminal traits, plasma and sperm proteome and the fertility of semen when used in artificial insemination. Thirty-nine males from a paternal line were obtained by re-derivation from vitrified embryos with a difference of 18 generations (G21V and G39V). Sperm production parameters, morphological traits, sperm motility parameters and viability were evaluated from ejaculates. Seminal plasma and sperm proteome of three pool ejaculates from 10 mature males of each group were analysed and semen doses were used to inseminate 311 females. Only the percentage of abnormal sperm showed significant differences, with G21V presenting fewer abnormal sperm than G39V (10.5±2.63 vs 23.8±1.98). The discriminant analysis (DA-PLS) showed a clear effect of the generation for plasma and sperm proteome. In seminal plasma, 643 proteins were reported and 64 proteins were differentially expressed, of which 56 were overexpressed in G39V (87.5%). Sperm proteome reported 1360 proteins with 132 differentially abundant proteins. Of the total, 89 proteins were overexpressed in G39V (67.4%). From the 64 and 132 differentially abundant proteins of plasma and sperm, 19 and 26 had a FC>1.5, 12 and 13 of them belonging to the Oryctolagus cuniculus taxonomy, Author version of the manuscript 2 respectively. Despite observing differences in important proteins related to capacitation, sperm motility or immunoprotection and consequently to the fertilization process (TMPRSS2, Serpin family, Fam71f1, ATPase H+ transporting accessory protein 2, carbonic anhydrase 2, UDPglucose glycoprotein glucosyltransferase 2), no differences in fertility and prolificacy were detected when commercial seminal doses were used for insemination from both male groups. However, overabundance of KIAA1324 protein can be related to the increase in abnormal sperm after selection by growth rate.

| INTRODUC TI ONSuperovulation protocols are designed to ensure the maximum number of transferable embryos per donor. Most of the embryos produced by superovulation are cryopreserved until they are subsequently used, which allows saving embryos for an unlimited time. The human gonadotropins, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are commonly used for superovulation in humans and animals. The effectiveness of superovulation treatments with gonadotropins is dependent on the maintenance of adequate daily levels of FSH throughout the process. The short elimination half-life and rapid metabolic clearance of the traditional FSH require twice daily treatments, which increases the donor handling and the possibility of errors in giving the treatments. The introduction of corifollitropin alfa (FSH-CTP), a long-acting recombinant FSH, has given an opportunity to simplify superstimulation protocols, reducing the number of injections and consequently improving the overall donor management. On the other hand, results of superovulation treatments vary, and one of the reasons for this may be the variable LH:FSH ratio. Although LH has essential and well-established roles in ovarian steroid synthesis and ovulation (