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

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

Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential

[EN] Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected.This research was supported in part by the Valencian regional government research project PROMETEOII/2014/036. The authors would like to thank Neil Macowan Language Services for revising the English version of the article.Viudes De Castro, MP.; Herreros Pomares, A.; Saenz De Juano Ribes, MDLD.; Marco Jiménez, F.; Vicente Antón, JS. (2015). Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential. Theriogenology. 84(3):446-451. https://doi.org/10.1016/j.theriogenology.2015.04.001S44645184

Effect of embryo vitrification on the steroid biosynthesis of liver tissue in rabbit offspring

[EN] Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer.This research was funded by Conselleria d'Educacio, Investigacio, Cultura i Esport, Spain, grant number AICO/2019/272. Ximo Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness of Spain (BES-2015-072429).Marco-Jiménez, F.; Garcia-Dominguez, X.; Domínguez-Martínez, M.; Viudes-De-Castro, MP.; Diretto, G.; Peñaranda, D.; Vicente Antón, JS. (2020). Effect of embryo vitrification on the steroid biosynthesis of liver tissue in rabbit offspring. International Journal of Molecular Sciences. 21(22):1-17. https://doi.org/10.3390/ijms21228642S1172122Novakovic, B., Lewis, S., Halliday, J., Kennedy, J., Burgner, D. P., Czajko, A., … Saffery, R. (2019). Assisted reproductive technologies are associated with limited epigenetic variation at birth that largely resolves by adulthood. Nature Communications, 10(1). doi:10.1038/s41467-019-11929-9Roseboom, T. J. (2018). Developmental plasticity and its relevance to assisted human reproduction. Human Reproduction, 33(4), 546-552. doi:10.1093/humrep/dey034Fleming, T. P., Watkins, A. J., Velazquez, M. A., Mathers, J. C., Prentice, A. M., Stephenson, J., … Godfrey, K. M. (2018). Origins of lifetime health around the time of conception: causes and consequences. The Lancet, 391(10132), 1842-1852. doi:10.1016/s0140-6736(18)30312-xDulioust, E., Toyama, K., Busnel, M. C., Moutier, R., Carlier, M., Marchaland, C., … Auroux, M. (1995). Long-term effects of embryo freezing in mice. Proceedings of the National Academy of Sciences, 92(2), 589-593. doi:10.1073/pnas.92.2.589Auroux, M., Cerutti, I., Ducot, B., & Loeuillet, A. (2004). Is embryo-cryopreservation really neutral? Reproductive Toxicology, 18(6), 813-818. doi:10.1016/j.reprotox.2004.04.010Vicente, J. S., Saenz-de-Juano, M. D., Jiménez-Trigos, E., Viudes-de-Castro, M. P., Peñaranda, D. S., & Marco-Jiménez, F. (2013). Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation. Cryobiology, 67(3), 321-326. doi:10.1016/j.cryobiol.2013.09.165Saenz-de-Juano, M. D., Marco-Jimenez, F., Schmaltz-Panneau, B., Jimenez-Trigos, E., Viudes-de-Castro, M. P., Peñaranda, D. S., … Vicente, J. S. (2014). Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level. REPRODUCTION, 147(6), 789-801. doi:10.1530/rep-14-0019Saenz-de-Juano, M. D., Vicente, J. S., Hollung, K., & Marco-Jiménez, F. (2015). Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy. PLOS ONE, 10(4), e0125157. doi:10.1371/journal.pone.0125157Berntsen, S., & Pinborg, A. (2018). Large for gestational age and macrosomia in singletons born after frozen/thawed embryo transfer (FET) in assisted reproductive technology (ART). Birth Defects Research, 110(8), 630-643. doi:10.1002/bdr2.1219Maheshwari, A., Pandey, S., Amalraj Raja, E., Shetty, A., Hamilton, M., & Bhattacharya, S. (2017). Is frozen embryo transfer better for mothers and babies? Can cumulative meta-analysis provide a definitive answer? Human Reproduction Update, 24(1), 35-58. doi:10.1093/humupd/dmx031Garcia-Dominguez, X., Vicente, J. S., & Marco-Jiménez, F. (2020). Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits. Animals, 10(5), 804. doi:10.3390/ani10050804Kohda, T. (2013). Effects of embryonic manipulation and epigenetics. Journal of Human Genetics, 58(7), 416-420. doi:10.1038/jhg.2013.61Canovas, S., Ross, P. J., Kelsey, G., & Coy, P. (2017). DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies). BioEssays, 39(11), 1700106. doi:10.1002/bies.201700106Canovas, S., Ivanova, E., Romar, R., García-Martínez, S., Soriano-Úbeda, C., García-Vázquez, F. A., … Coy, P. (2017). DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids. eLife, 6. doi:10.7554/elife.23670Ivanova, E., Canovas, S., Garcia-Martínez, S., Romar, R., Lopes, J. S., Rizos, D., … Coy, P. (2020). DNA methylation changes during preimplantation development reveal inter-species differences and reprogramming events at imprinted genes. Clinical Epigenetics, 12(1). doi:10.1186/s13148-020-00857-xGarcía-Martínez, S., Sánchez Hurtado, M. A., Gutiérrez, H., Sánchez Margallo, F. M., Romar, R., Latorre, R., … López Albors, O. (2018). Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig. MHR: Basic science of reproductive medicine, 24(5), 260-270. doi:10.1093/molehr/gay008Ng, K. Y. B., Mingels, R., Morgan, H., Macklon, N., & Cheong, Y. (2017). In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: a systematic review. Human Reproduction Update, 24(1), 15-34. doi:10.1093/humupd/dmx028Marchesi, D., Qiao, J., & Feng, H. (2012). Embryo Manipulation and Imprinting. Seminars in Reproductive Medicine, 30(04), 323-334. doi:10.1055/s-0032-1320013Ramos‐Ibeas, P., Heras, S., Gómez‐Redondo, I., Planells, B., Fernández‐González, R., Pericuesta, E., … Gutiérrez‐Adán, A. (2019). Embryo responses to stress induced by assisted reproductive technologies. Molecular Reproduction and Development, 86(10), 1292-1306. doi:10.1002/mrd.23119Vrooman, L. A., & Bartolomei, M. S. (2017). Can assisted reproductive technologies cause adult-onset disease? Evidence from human and mouse. Reproductive Toxicology, 68, 72-84. doi:10.1016/j.reprotox.2016.07.015Chen, M., & Heilbronn, L. K. (2017). The health outcomes of human offspring conceived by assisted reproductive technologies (ART). Journal of Developmental Origins of Health and Disease, 8(4), 388-402. doi:10.1017/s2040174417000228Duranthon, V., & Chavatte-Palmer, P. (2018). Long term effects of ART: What do animals tell us? Molecular Reproduction and Development, 85(4), 348-368. doi:10.1002/mrd.22970Leibo, S. P., & Sztein, J. M. (2019). Cryopreservation of mammalian embryos: Derivation of a method. Cryobiology, 86, 1-9. doi:10.1016/j.cryobiol.2019.01.007Sparks, A. (2015). Human Embryo Cryopreservation—Methods, Timing, and other Considerations for Optimizing an Embryo Cryopreservation Program. Seminars in Reproductive Medicine, 33(02), 128-144. doi:10.1055/s-0035-1546826De Geyter, C., Calhaz-Jorge, C., Kupka, M. S., Wyns, C., Mocanu, E., Motrenko, T., … Goossens, V. (2020). ART in Europe, 2015: results generated from European registries by ESHRE†. Human Reproduction Open, 2020(1). doi:10.1093/hropen/hoz038Saenz-de-Juano, M., Marco-Jimenez, F., Viudes-de-Castro, M., Lavara, R., & Vicente, J. (2014). Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae. Reproduction in Domestic Animals, 49(3), 505-511. doi:10.1111/rda.12320Garcia-Dominguez, X., Marco-Jiménez, F., Peñaranda, D. S., & Vicente, J. S. (2020). Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model. Animals, 10(6), 1043. doi:10.3390/ani10061043Lavara, R., Baselga, M., Marco-Jiménez, F., & Vicente, J. S. (2015). Embryo vitrification in rabbits: Consequences for progeny growth. Theriogenology, 84(5), 674-680. doi:10.1016/j.theriogenology.2015.04.025Lavara, R., Baselga, M., Marco-Jiménez, F., & Vicente, J. S. (2014). Long-term and transgenerational effects of cryopreservation on rabbit embryos. Theriogenology, 81(7), 988-992. doi:10.1016/j.theriogenology.2014.01.030Garcia-Dominguez, X., Marco-Jiménez, F., Peñaranda, D. S., Diretto, G., García-Carpintero, V., Cañizares, J., & Vicente, J. S. (2020). Long-term and transgenerational phenotypic, transcriptional and metabolic effects in rabbit males born following vitrified embryo transfer. Scientific Reports, 10(1). doi:10.1038/s41598-020-68195-9Feuer, S., & Rinaudo, P. (2016). From Embryos to Adults: A DOHaD Perspective on In Vitro Fertilization and Other Assisted Reproductive Technologies. Healthcare, 4(3), 51. doi:10.3390/healthcare4030051Zandstra, H., Brentjens, L. B. P. M., Spauwen, B., Touwslager, R. N. H., Bons, J. A. P., Mulder, A. L., … Van Montfoort, A. P. A. (2018). Association of culture medium with growth, weight and cardiovascular development of IVF children at the age of 9 years. Human Reproduction, 33(9), 1645-1656. doi:10.1093/humrep/dey246Chen, L., Yang, T., Zheng, Z., Yu, H., Wang, H., & Qin, J. (2018). Birth prevalence of congenital malformations in singleton pregnancies resulting from in vitro fertilization/intracytoplasmic sperm injection worldwide: a systematic review and meta-analysis. Archives of Gynecology and Obstetrics, 297(5), 1115-1130. doi:10.1007/s00404-018-4712-xZhang, W. Y., Selamet Tierney, E. S., Chen, A. C., Ling, A. Y., Fleischmann, R. R., & Baker, V. L. (2019). Vascular Health of Children Conceived via In Vitro Fertilization. The Journal of Pediatrics, 214, 47-53. doi:10.1016/j.jpeds.2019.07.033Guo, X.-Y., Liu, X.-M., Jin, L., Wang, T.-T., Ullah, K., Sheng, J.-Z., & Huang, H.-F. (2017). Cardiovascular and metabolic profiles of offspring conceived by assisted reproductive technologies: a systematic review and meta-analysis. Fertility and Sterility, 107(3), 622-631.e5. doi:10.1016/j.fertnstert.2016.12.007Feuer, S. K., Liu, X., Donjacour, A., Simbulan, R., Maltepe, E., & Rinaudo, P. (2017). Transcriptional signatures throughout development: the effects of mouse embryo manipulation in vitro. Reproduction, 153(1), 107-122. doi:10.1530/rep-16-0473Feuer, S. K., & Rinaudo, P. F. (2017). Physiological, metabolic and transcriptional postnatal phenotypes ofin vitrofertilization (IVF) in the mouse. Journal of Developmental Origins of Health and Disease, 8(4), 403-410. doi:10.1017/s204017441700023xEcker, D. J., Stein, P., Xu, Z., Williams, C. J., Kopf, G. S., Bilker, W. B., … Schultz, R. M. (2004). Long-term effects of culture of preimplantation mouse embryos on behavior. Proceedings of the National Academy of Sciences, 101(6), 1595-1600. doi:10.1073/pnas.0306846101Fauque, P., Mondon, F., Letourneur, F., Ripoche, M.-A., Journot, L., Barbaux, S., … Vaiman, D. (2010). In Vitro Fertilization and Embryo Culture Strongly Impact the Placental Transcriptome in the Mouse Model. PLoS ONE, 5(2), e9218. doi:10.1371/journal.pone.0009218Fernandez-Gonzalez, R., Ramirez, M. A., Pericuesta, E., Calle, A., & Gutierrez-Adan, A. (2010). Histone Modifications at the Blastocyst Axin1Fu Locus Mark the Heritability of In Vitro Culture-Induced Epigenetic Alterations in Mice1. Biology of Reproduction, 83(5), 720-727. doi:10.1095/biolreprod.110.084715Fernandez-Gonzalez, R., Moreira, P., Bilbao, A., Jimenez, A., Perez-Crespo, M., Ramirez, M. A., … Gutierrez-Adan, A. (2004). Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proceedings of the National Academy of Sciences, 101(16), 5880-5885. doi:10.1073/pnas.0308560101Winick, M., & Noble, A. (1965). Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Developmental Biology, 12(3), 451-466. doi:10.1016/0012-1606(65)90009-6Saenz-de-Juano, M. D., Marco-Jiménez, F., & Vicente, J. S. (2016). Embryo transfer manipulation cause gene expression variation in blastocysts that disrupt implantation and offspring rates at birth in rabbit. European Journal of Obstetrics & Gynecology and Reproductive Biology, 207, 50-55. doi:10.1016/j.ejogrb.2016.10.049Delle Piane, L., Lin, W., Liu, X., Donjacour, A., Minasi, P., Revelli, A., … Rinaudo, P. F. (2010). Effect of the method of conception and embryo transfer procedure on mid-gestation placenta and fetal development in an IVF mouse model. Human Reproduction, 25(8), 2039-2046. doi:10.1093/humrep/deq165Wale, P. L., & Gardner, D. K. (2015). The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Human Reproduction Update, 22(1), 2-22. doi:10.1093/humupd/dmv034Charni-Natan, M., Aloni-Grinstein, R., Osher, E., & Rotter, V. (2019). Liver and Steroid Hormones—Can a Touch of p53 Make a Difference? Frontiers in Endocrinology, 10. doi:10.3389/fendo.2019.00374Yakar, S., Liu, J.-L., Stannard, B., Butler, A., Accili, D., Sauer, B., & LeRoith, D. (1999). Normal growth and development in the absence of hepatic insulin-like growth factor I. Proceedings of the National Academy of Sciences, 96(13), 7324-7329. doi:10.1073/pnas.96.13.7324Juul, A. (2003). Serum levels of insulin-like growth factor I and its binding proteins in health and disease. Growth Hormone & IGF Research, 13(4), 113-170. doi:10.1016/s1096-6374(03)00038-8Miles, H. L., Hofman, P. L., Peek, J., Harris, M., Wilson, D., Robinson, E. M., … Cutfield, W. S. (2007). In Vitro Fertilization Improves Childhood Growth and Metabolism. The Journal of Clinical Endocrinology & Metabolism, 92(9), 3441-3445. doi:10.1210/jc.2006-2465Belva, F., Bonduelle, M., Provyn, S., Painter, R. C., Tournaye, H., Roelants, M., & De Schepper, J. (2018). Metabolic Syndrome and Its Components in Young Adults Conceived by ICSI. International Journal of Endocrinology, 2018, 1-8. doi:10.1155/2018/8170518Fournier, N., Atger, V., Paul, J.-L., Sturm, M., Duverger, N., Rothblat, G. H., & Moatti, N. (2000). Human ApoA-IV Overexpression in Transgenic Mice Induces cAMP-Stimulated Cholesterol Efflux From J774 Macrophages to Whole Serum. Arteriosclerosis, Thrombosis, and Vascular Biology, 20(5), 1283-1292. doi:10.1161/01.atv.20.5.1283Liang, Y., Jiang, X.-C., Liu, R., Liang, G., Beyer, T. P., Gao, H., … Cao, G. (2004). Liver X Receptors (LXRs) Regulate Apolipoprotein AIV-Implications of the Antiatherosclerotic Effect of LXR Agonists. Molecular Endocrinology, 18(8), 2000-2010. doi:10.1210/me.2003-0477Lin, Q., Cao, Y., & Gao, J. (2015). Decreased expression of the APOA1–APOC3–APOA4 gene cluster is associated with risk of Alzheimer’s disease. Drug Design, Development and Therapy, 5421. doi:10.2147/dddt.s89279Qin, W., Li, X., Xie, L., Li, S., Liu, J., Jia, L., … Chen, Z. (2016). A long non-coding RNA,APOA4-AS, regulatesAPOA4expression depending on HuR in mice. Nucleic Acids Research, 44(13), 6423-6433. doi:10.1093/nar/gkw341Leese, H. J., Guerif, F., Allgar, V., Brison, D. R., Lundin, K., & Sturmey, R. G. (2016). Biological optimization, the Goldilocks principle, and how much islagomin the preimplantation embryo. Molecular Reproduction and Development, 83(9), 748-754. doi:10.1002/mrd.22684Kohda, T., & Ishino, F. (2013). Embryo manipulation via assisted reproductive technology and epigenetic asymmetry in mammalian early development. Philosophical Transactions of the Royal Society B: Biological Sciences, 368(1609), 20120353. doi:10.1098/rstb.2012.0353Garcia-Dominguez, X., Juarez, J. D., Vicente, J. S., & Marco-Jiménez, F. (2020). Impact of embryo technologies on secondary sex ratio in rabbit. Cryobiology, 97, 60-65. doi:10.1016/j.cryobiol.2020.10.008Viudes-de-Castro, M. P., Marco-Jiménez, F., Cedano-Castro, J. I., & Vicente, J. S. (2017). Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit. Theriogenology, 98, 68-74. doi:10.1016/j.theriogenology.2017.05.005Marco-Jiménez, F., Lavara, R., Jiménez-Trigos, E., & Vicente, J. S. (2013). In vivo development of vitrified rabbit embryos: Effects of vitrification device, recipient genotype, and asynchrony. Theriogenology, 79(7), 1124-1129. doi:10.1016/j.theriogenology.2013.02.008Vicente, J.-S., Viudes-de-Castro, M.-P., & García, M.-L. (1999). In vivo survival rate of rabbit morulae after vitrification in a medium without serum protein. Reproduction Nutrition Development, 39(5-6), 657-662. doi:10.1051/rnd:19990511Garcia-Dominguez, X., Marco-Jimenez, F., Viudes-de-Castro, M. P., & Vicente, J. S. (2019). Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. Journal of Visualized Experiments, (147). doi:10.3791/58055Besenfelder, U., Strouhal, C., & Brem, G. (1998). A Method for Endoscopic Embryo Collection and Transfer in the Rabbit. Journal of Veterinary Medicine Series A, 45(1-10), 577-579. doi:10.1111/j.1439-0442.1998.tb00861.

Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance

[EN] The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, beta nerve growth factor ((beta-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as beta-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and beta-NGF, they could be used to develop new extenders with less hormone concentration. (C) 2017 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 Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by funds from Instituto Valenciano de Investigaciones Agrarias (IVIA) and Ministerio de Empleo y Seguridad Social (Programa de Garantia Juvenil).Casares-Crespo, L.; Fernández-Serrano, P.; Vicente Antón, JS.; Moce Cervera, ET.; Castellini, C.; Stabile, A.; Viudes De Castro, MP. (2018). Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance. Theriogenology. 105:61-65. https://doi.org/10.1016/j.theriogenology.2017.09.009S616510

Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH

[EN] The efficiency of an embryo bank depends on provision of optimal conditions for recovery, cryopreservation and transfer to a breed or strain. In this sense, increasing the number of embryos available using superovulation should improve the cryobank efficiency. However, vagueness of response to conventional protocols to control or increase ovarian response and the quality of oocytes and embryos and their cryotolerance remain a challenge. The aim of our study was to evaluate the effect of corifollitropin alpha (CTP) and a recombinant human FSH (rhFSH), alone or supplemented with rhLH, on embryo cryosurvival by in vitro development and OCT4 and NANOG mRNA abundance at blastocyst stage and offspring rate. In vitro development of vitrified embryos was not significantly affected by superstimulation with or without rhLH supplementation, resulting in similar development rates to those of the control groups (fresh and vitrified embryos from non-superstimulated donor does). Blastocysts developed from vitrified embryos showed higher levels of OCT4 transcript abundance than fresh control, while NANOG transcript abundance was only higher in the blastocysts developed from vitrified embryos after superstimulation treatment in comparison with control groups. The implantation and offspring rates at birth were negatively affected by supplementation with rhLH. Both rhFSH or CTP vitrified embryo groups showed an implantation rate similar to those of the control groups, but an offspring rate lower than control. In conclusion, embryos produced using corifollitropin alpha did not compromise the cryosurvival of vitrified embryos in the rabbit. In addition, this study points out the negative effect of rhLH supplementation in terms of offspring rate on embryo vitrification.This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (CICYT) and Generalitat Valenciana research programme (Prometeo II 2014/036). English text version revised by N. Macowan English Language Service.Vicente Antón, JS.; Viudes De Castro, MP.; Cedano-Castro, JI.; Marco-Jiménez, F. (2018). Cryosurvival of rabbit embryos obtained after superovulation with corifollitropin alfa with or without LH. Animal Reproduction Science. 192:321-327. https://doi.org/10.1016/j.anireprosci.2018.03.034S32132719

Trends in Rabbit Insemination Extenders for Fresh and Frozen Semen. A Review

[EN] Artificial insemination (AI) has become a popular technique in rabbit farms worldwide. This report discusses the progress made on semen extenders used in rabbit AI, setting out the latest innovations. Fresh and frozen semen have different requirements, so the extender composition will vary depending on the type of semen used. We discuss the endocrine supplementation of extenders for ovulation induction, the use of active molecules as an alternative to conventional antibiotics and the extenders developed for rabbit sperm cryopreservation.Funding from MCIN/AEI/10.13039/501100011033 and by ERDF, “A way of making Europe” (grant PID2021-127867OB-100), and from GVA-IVIA and the FEDER Operational Program of the Valencian Community, 2021–2027 (grant 52201K) is acknowledged.Viudes-De-Castro, MP.; Vicente, JS. (2023). Trends in Rabbit Insemination Extenders for Fresh and Frozen Semen. A Review. World Rabbit Science. 31(2):109-116. https://doi.org/10.4995/wrs.2023.1850510911631