Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?

[EN] 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 amino peptidase 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. (C) 2016 Elsevier Inc. All rights reserved.This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L Casares-Crespo is supported by a scholarship from Institute Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund.Casares-Crespo, L.; Vicente Antón, JS.; Talavan, A.; Viudes De Castro, MP. (2016). Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?. Theriogenology. 85(5):928-932. doi:10.1016/j.theriogenology.2015.10.044S92893285

Semen evaluation of two selected lines of rabbit bucks

[EN] Twenty rabbit bucks of 9 months of age were used to evaluate semen quality of two lines of New Zealand rabbit bucks selected for litter size at weaning (A line) and growth rate from weaning to slaughter (R line). The morphological semen characteristics indicated that the A line spermatozoa had greater acrosome integrity (+3.6 percentage units; P<0.01) and smaller sperm head size (for example, ¿1.46 ¿m2 for sperm head area) than in the R line. Seminal functional traits were also significantly higher for the A line (+13.4 percentage units for viability, +10.6 percentage units for hypo-osmotic swelling test (HOST) and +3.3 g/L for seminal plasma protein. However, no differences were detected between lines for motility parameters and seminal plasma protein electrophoretic profiles. Both lines had the same twelve bands with the following molecular weights to the nearest 1 kD: 124, 117, 99, 86, 75, 62, 40, 32, 21, 19, 10 and 6 kD. A relationship (r=0.308 for A line and 0.359 for R line; P<0.01) was found between the integrity of the plasmatic membrane (viability rate) and tail membrane (HOST) of the spermatozoa in the A line, but not in the R line, which had greater sperm head size. There was also a significant positive correlation coefficient between sperm concentration and either viability or some kinetic traits (r=0.567 and 0.575 for VCL, r=0.584 and 0.561 for VSL and r=0.588 and 0.588 for VAP, for A and R lines, respectively; P<0.001). We concluded that the A line seems to have better semen characteristics than the R line. We also found an interesting correlation among the seminal morphological, functional and kinetic traits, which could possibly be used to facilitate semen evaluation.Hosam M. Safaa was supported by a grant from ICSC-World Laboratory, Switzerland. This work has been supported by the Spanish Research Project (CICYT AGL2004-02710/GAN). The authors are indebted to F. Marco-Jiménez, Universidad Politécnica de Valencia, Valencia, Spain, and G.M.K. Mehaisen, Department of Animal Production, Faculty of Agricultural, Cairo University, Giza, Egypt, for their excellent cooperation throughout this study and to D.A.A. El-Sayed, Faculty of Agricultural, Cairo University, Giza, Egypt, for her help in preparing the manuscript.Safaa, H.; Vicente, J.; Lavara, R.; Viudes De Castro, M. (2008). Semen evaluation of two selected lines of rabbit bucks. World Rabbit Science. 16(3). doi:10.4995/wrs.2008.62216

Effect of gelatin addition to freezing extender on rabbit semen parameters and reproductive performance

An improvement in reproduction management has been associated to the wide use of artificial insemination in rabbits. The use of frozen semen in this species is greatly limited due to its low fertility rates. Moreover, in politocous species, prolificacy is a very important trait and it is particularly affected when semen is frozen. Since gelatine addition to fresh semen extenders improves and prolongs motility and viability sperm parameters, its use in semen freezing could enhance sperm efficiency after thawing. The aim of this study was to assess the effects of gelatine addition to freezing extender using a commercially available extender (MIII) and a tris-based extender (TGC). Four experimental extenders were used: TGC extender with gelatine, the same without gelatine and MIII extender with and without gelatine. In order to evaluate seminal parameters, motility was assessed using the Computer Assisted Sperm Analysis, while viability rate was calculated using flow cytometry. To evaluate in vivo frozen-thawed semen, 273 females were inseminated. No significant difference was found among the four freezing extenders when motility, viability fertility or prolificacy were considered. Motility rates ranged between 22.4% and 35.1%, while viability varied between 22.5 and 30.9%. Fertility rates oscillated from 80.8% to 86.2% and the total born ranged from 7.9 to 8.5. While in vitro results were lower than those obtained in other studies, in vivo results were quite favourable. In conclusion, gelatine addition did not improve sperm motility and viability after thawing nor fertility and prolificacy after insemination with frozen-thawed semen, using MIII or TCG extender

Immune response to repeated rhFSH superovulation treatment in rabbit does

Some studies have demonstrated that, when superovulation is induced more than once in the same animal, the response to treatment may be reduced. This reduced response may be related to an increase of anti-gonadotrophin antibodies. The aim of this study was to evaluate the effect of repeated recombinant human (rh) FSH superovulation treatments on the ovulation rate and anti-FSH antibodies production. For this purpose, 34 females were treated i.m. with rhFSH (0.6 mg every 24 h, for 3 days) in order to induce superovulation four consecutive times. Control does were injected with the vehicle at the appropriate time. The interval between the first three treatments was one month; and three months between the third and fourth treatment during which the females were inseminated without superovulation treatment. Ovulation rate was checked by laparoscopy and blood samples were collected after each treatment. An indirect ELISA was used to detect sera anti-FSH antibodies. The ovulation rate was significantly higher in does treated with rhFSH than in control group. The ovulation rate was significantly higher in does treated for the first time with rhFSH than in those treated two, three, or four times (8.7±1.42, 19.3±1.36, 13.5±1.26, 13.0±1.28, 14.3±1.31 for control and superovulated females, respectively, P<0.05). On the other hand, results obtained after four consecutives rhFSH treatments indicate that there was a significant difference in immune response of does after the second treatment (P<0.05), none of the treated females presented immune response in the first or second treatment, on the contrary, in the third and forth treatment the 40 to 60% of females presented high antibody levels. The results of the present study clearly demonstrate that repeated rhFSH superovulation treatments in rabbit does induce a immune response and have a negative effect on ovulation rate. Although anti-FSH antibodies induce a decrease in superovulation response, the ovulation rate of females superovulated twice, three and four times was significantly higher than control females. The immune response developed has an important individual variability and may be related with the reproductive response decrease after repeated treatments. Nevertheless, since there were females in which ovulation rate diminished without an increase in sera antibodies, it is clear that reproduction failure after consecutive superovulation treatments can be caused by different reasons, which have to be studied in future

Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit

[EN] There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, pos- ology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or sup- plemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to su- perovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP þ LH showed significantly higher implantation rates and survival at birth among the different ovarian stim- ulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplemen- tation allows us to obtain similar in vivo embryo development results as in the control group.This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (CICYT) and Prometeo II 2014/36 Generalitat Valenciana research program. English text version revised by N. Macowan English Language Service.Viudes De Castro, MP.; Marco-Jiménez, F.; Cedano-Castro, JI.; Vicente Antón, JS. (2017). Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit. Theriogenology. 98:68-74. https://doi.org/10.1016/j.theriogenology.2017.05.005S68749

Antibacterial Activity of Some Molecules Added to Rabbit Semen Extender as Alternative to Antibiotics

[EN] This study was conducted to evaluate the antibacterial activity of two aminopeptidase inhibitors and chitosan-based nanoparticles in liquid-stored rabbit semen. This study reports that the aminopeptidase inhibitors used to prevent bacterial growth could be used in semen extender as a suitable alternative to antibiotics. Although great attention is paid to hygiene during semen collection and processing, bacteria are commonly found in the semen of healthy fertile males of different species. As the storage of extended semen might facilitate bacterial growth, extenders are commonly supplemented with antibiotics. This study aimed to evaluate the antibacterial activity of ethylenediaminetetraacetic acid (EDTA), bestatin and chitosan-based nanoparticles added to rabbit semen extender and their effect on reproductive performance under field conditions. Four different extenders were tested, supplemented with antibiotics (TCG+AB), with EDTA and bestatin (EB), with EDTA, bestatin and chitosan-based nanoparticles (QEB) or without antibiotics (TCG-AB). Extended semen was cooled at 15 degrees C for three days. Cooled samples were examined for bacterial growth and semen quality every 24 h for 3 days. The enterobacteria count increased considerably during storage at 72 h in semen extended with TCG+AB and TCG-AB, while extenders EB and QEB showed a bacteriostatic effect over time. After 24, 48 and 72 h, quality characteristics were retained in all groups, with no significant motility differences, either in acrosome integrity, membrane functionality or the viability of spermatozoa. Additionally, bacterial concentration present in fresh semen did not affect reproductive performance. In conclusion, EDTA and bestatin exerted a potent bacteriostatic effect over time and could be used as an alternative to conventional antibiotics in rabbit semen extenders.Funding from the Ministry of Economy, Industry and Competitiveness (Research project: AGL2017-85162-C2-1-R) is acknowledged.Viudes-De-Castro, MP.; Marco-Jiménez, F.; Vicente Antón, JS.; Marin, C. (2021). Antibacterial Activity of Some Molecules Added to Rabbit Semen Extender as Alternative to Antibiotics. Animals. 11(4):1-10. https://doi.org/10.3390/ani11041178S11011

Minimally invasive embryo transfer and embryo vitrification at the optimal embryo stage in rabbit model

[EN] Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment. The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species. Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time- flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.This work was supported by funds from the Ministry of Economy and Competitiveness of Spain Research Programme (AGL2014-53405-C2-1- P) and Generalitat Valenciana Research Programme (PrometeoII 2014/036). English text version revised by N. Macowan English Language Service.Garcia-Dominguez, X.; Marco-Jiménez, F.; Viudes De Castro, MP.; Vicente Antón, JS. (2019). Minimally invasive embryo transfer and embryo vitrification at the optimal embryo stage in rabbit model. Journal of Visualized Experiments. 147. https://doi.org/10.3791/58055Se5805514

Long-Term Effects Following Fresh/Vitrified Embryo Transfer Are Transmitted by Paternal Germline in a Large Size Rabbit Cohort

[EN] Simple Summary Assisted reproductive technologies (ARTs) involve an extraordinary change in the natural developmental trajectory of the mammalian embryo, incurring potential long-term and inheritable effects in the resulting offspring. The results of this study demonstrate, for the first time, that ex vivo embryo manipulations during fresh and vitrified embryo transfer are associated with paternally inherited bodyweight variation, but seemed not transmissible via the female germline. This asymmetry in the transmission of acquired features following ARTs suggests that embryo paternal and maternal genomes differ in their degree of susceptibility to the lasting effects of ARTs. This study would provide a novel view of developmental plasticity in the early mammalian embryo. The concept of developmental programming suggests that the early life environment influences offspring phenotype in later life, whose effects may also be manifested in further generations. Valuable pieces of evidence come from the fields applying assisted reproductive technologies (ARTs), which deprive embryos of their optimal maternal environment and were thus associated with subsequent developmental deviations. Recently, we demonstrated that the in vitro manipulations during a vitrified embryo transfer procedure incurs a cumulative and transgenerational decline in the growth performance of the resulting offspring. Here, we provide a longitudinal study to investigate whether previous developmental deviations could be indistinctly paternally or maternally transmitted using crossbred mattings. Our findings revealed that early embryo manipulations through fresh and vitrified embryo transfer incurred paternally transmissible effects over the growth pattern and adult body weight, which seemed not inheritable via the female germline. Similar inheritable effects were observed after fresh and vitrified embryo transfer, suggesting that disturbing optimal embryo development through in vitro manipulations was the principal trigger of transmissible effects, rather than embryo cryopreservation per se.This research was funded by Conselleria d'Educacio, Investigacio, Cultura i Esport (Generalitat Valenciana, Spain), grant number AICO/2019/272. X.G.-D. was supported by a research grant from the Spanish Ministry of Economy, Industry, and Competitiveness, grant number BES-2015-072429.Garcia-Dominguez, X.; Vicente Antón, JS.; Viudes-De-Castro, MP.; Marco-Jiménez, F. (2020). Long-Term Effects Following Fresh/Vitrified Embryo Transfer Are Transmitted by Paternal Germline in a Large Size Rabbit Cohort. Animals. 10(8):1-7. https://doi.org/10.3390/ani10081272S1710

Rabbit seminal plasma proteome: The importance of the genetic origin

[EN] The present study was conducted to characterise rabbit seminal plasma proteins (SP proteins) focusing on the influence of the genetic origin and seasonality. In addition, ß-NGF protein quantity in SP was determined. Semen samples were recovered from January to December 2014 using 6 males belonging to genotype A and six from genotype R. For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment. A total of 24 pools (3 for each season and genetic line) were analysed. SP proteins of the two experimental groups were recovered and subjected to in-solution digestion nano LC¿MS/MS and bioinformatics analysis. The resulting library included 402 identified proteins validated with ¿95% Confidence (unused Score¿1.3). These data are available via ProteomeXchange with identifier PXD006308. Only 6 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Twenty-three proteins were differentially expressed between genotypes, 11 over-expressed in genotype A and 12 in genotype R. Regarding the effect of season on rabbit SP proteome, results showed that there is no clear pattern of protein variation throughout the year. Similar ß-NGF relative quantity was observed between seasons and genotypes. In conclusion, this study generates the largest library of SP proteins reported to date in rabbits and provides evidence that genotype is related to a specific abundance of SP proteins.This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by Spanish funds from IVIA and Ministerio de Empleo y Seguridad Social (Youth Guarantee Program). The authors are grateful to M. Luz Valero for her excellent technical assistance.Casares-Crespo, L.; Fernández-Serrano, P.; Vicente Antón, JS.; Marco-Jiménez, F.; Viudes De Castro, MP. (2018). Rabbit seminal plasma proteome: The importance of the genetic origin. Animal Reproduction Science. 189:30-42. https://doi.org/10.1016/j.anireprosci.2017.12.004S304218

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

Rabbit is a reflexively ovulating species. Accordingly, in the practice of artificial insemination (AI) ovulation must be induced via exogenous GnRH (Gonadotropin-Releasing Hormone) administration, which may be performed intramuscularly, subcutaneously, or intravaginally. Unfortunately, the bioavailability of the GnRH analogue when added to the extender is lower due to the proteolytic activity in the seminal plasma and the poor permeability of the vaginal mucosa. The aim of the study was to refine the practice of AI practice in rabbits by replacing parenteral GnRH analogue administration (subcutaneous, intravenous, or intramuscular injection) with intravaginal application, while reducing its concentration in the diluent. Extenders containing the buserelin acetate in chitosan–dextran sulphate and chitosan–alginate nanoparticles were designed and 356 females were inseminated. Reproductive performance of females inseminated with the two experimental extenders, receiving 4 μg of buserelin acetate intravaginally per doe, was compared with that in the control group, the does of which were inseminated with the extender without the GnRH analogue and induced to ovulate with 1 μg of buserelin acetate administered intramuscularly. The entrapment efficiency of the chitosan–dextran sulphate complex was higher than that of chitosan–alginate. However, females inseminated with both systems showed similar reproductive performance. We conclude that both nanoencapsulation systems are an efficient way of intravaginal ovulation induction, allowing a reduction in the level of the GnRH analogue normally used in seminal doses from 15–25 μg to 4 μg