Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits

[EN] This study was conducted to demonstrate how embryo manipulation techniques incur phenotypic changes throughout life. This study reports the first evidence demonstrating that the vitrification device used is not a trivial decision, providing valuable information about how the cooling-warming rates during vitrification can be partly responsible of the postnatal phenotypic variations. In this study, we evaluated the effect of embryo vitrification using two different devices on adulthood phenotype in rabbits. In vitro development, prenatal embryo survival, body weight, growth performance, haematological and biochemical peripheral blood analysis, reproductive performance, and lactation performance traits were compared between the experimental groups. They derived from naturally-conceived embryos (NC), fresh-transferred embryos (FT), vitrified-transferred embryos using mini-straw (VTs), or vitrified-transferred embryos using Cryotop (VTc). Straw-vitrified embryos exhibited lower in vitro developmental rates and in vivo survival rates following embryo transfer compared to its Cryotop-vitrified counterparts. Moreover, the VTs group exhibited higher foetal losses than VTc, FT, and NC groups. Independently of the vitrification device, vitrified-transferred (VT) offspring showed a skewed sex ratio in favour of males, and an increased birth bodyweight. In contrast, postnatal daily growth was diminished in all ART (i.e., FT and VT) animals. In adulthood, significant differences in body weight between all groups was founded-all ART progenies weighed less than NC animals and, within ART, VT animals weighed less than FT. For VT groups, weight at adulthood was higher for the VTs group compared with the VTc group. Peripheral blood parameters ranged between common values. Moreover, no differences were found in the fertility rates between experimental groups. Furthermore, similar pregnancy rates, litter sizes, and the number of liveborns were observed, regardless of the experimental group. However, decreased milk yield occurred for VTc and FT animals compared to VTs and NC animals. A similar trend was observed for the milk composition of dry matter and fat. Concordantly, reduced body weight was found for suckling kits in the VTc and FT groups compared to VTs and NC animals. Our findings reveal that developmental changes after the embryo vitrification procedure could be associated with an exhibition of the embryonic developmental plasticity. Moreover, to our best knowledge, this study reports the first evidence demonstrating that the vitrification device used is not a trivial decision, providing valuable information about how the cooling-warming rates during vitrification can be partly responsible of the postnatal phenotypic variations.Funding from the Ministry of Economy, Industry and Competitiveness (research project: AGL2014-53405-C2-1-P and AGL2017-85162-C2-1-R) is acknowledged. X.G.-D. was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429). English text version was revised by N. Macowan English Language Service.Garcia-Dominguez, X.; Vicente Antón, JS.; Marco-Jiménez, F. (2020). Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits. Animals. 10(5):1-17. https://doi.org/10.3390/ani10050804S117105Ng, K. Y. B., Mingels, R., Morgan, H., Macklon, N., & Cheong, Y. (2017). 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Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model

[EN] This study was conducted to demonstrate how a vitrified embryo transfer procedure incurs phenotypic and molecular changes throughout life. This study reports the first evidence describing that embryonic manipulation during a vitrified embryo transfer cycle induced molecular modifications, concerning oxidative phosphorylation and dysregulations in zinc and lipid metabolism in liver tissue, which has been reported as responsible for postnatal variations of the phenotype. Nowadays, assisted reproductive technologies (ARTs) are considered valuable contributors to our past, but a future without their use is inconceivable. However, in recent years, several studies have evidenced a potential impact of ART on long-term development in mammal species. To date, the long-term follow-up data are still limited. So far, studies have mainly focused on in vitro fertilization or in vitro culture, with information from gametes/embryos cryopreservation field being practically missing. Herein, we report an approach to determine whether a vitrified embryo transfer procedure would have long-term consequences on the offspring. Using the rabbit as a model, we compared animals derived from vitrified-transferred embryos versus those naturally conceived, studying the growth performance, plus the weight throughout life, and the internal organs/tissues phenotype. The healthy status was assessed over the hematological and biochemical parameters in peripheral blood. Additionally, a comparative proteomic analysis was conducted in the liver tissue to investigate molecular cues related to vitrified embryo transfer in an adult tissue. After vitrified embryo transfer, birth weight was increased, and the growth performance was diminished in a sex-specific manner. In addition, vitrified-transferred animals showed significantly lower body, liver and heart weights in adulthood. Molecular analyses revealed that vitrified embryo transfer triggers reprogramming of the liver proteome. Functional analysis of the differentially expressed proteins showed changes in relation to oxidative phosphorylation and dysregulations in the zinc and lipid metabolism, which has been reported as possible causes of a disturbed growth pattern. Therefore, we conclude that vitrified embryo transfer is not a neutral procedure, and it incurs long-term effects in the offspring both at phenotypic and molecular levels. These results described a striking example of the developmental plasticity exhibited by the mammalian embryo.Funding from the Ministry of Economy, Industry and Competitiveness (Research project: AGL2017-85162-C2-1-R and AGL2014-53405-C2-1-P) is acknowledged. X.G.D. was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429).Garcia-Dominguez, X.; Marco-Jiménez, F.; Peñaranda, D.; Vicente Antón, JS. (2020). Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model. Animals. 10(6):1-16. https://doi.org/10.3390/ani10061043S116106Crawford, G., & Ledger, W. (2018). In vitro fertilisation/intracytoplasmic sperm injection beyond 2020. BJOG: An International Journal of Obstetrics & Gynaecology, 126(2), 237-243. doi:10.1111/1471-0528.15526Findlay, J. K., Holland, M. K., & Wong, B. B. M. (2019). Reproductive science and the future of the planet. Reproduction, 158(3), R91-R96. doi:10.1530/rep-18-0640Vrooman, 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.2016.07.015Roseboom, 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-xFeuer, 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/healthcare4030051Feuer, 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/s204017441700023xDuranthon, 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.22970Ramos‐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.23119Chen, 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/s2040174417000228Novakovic, 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/s41467-019-11929-9Belva, F., Bonduelle, M., Roelants, M., Michielsen, D., Van Steirteghem, A., Verheyen, G., & Tournaye, H. (2016). Semen quality of young adult ICSI offspring: the first results. Human Reproduction, 31(12), 2811-2820. doi:10.1093/humrep/dew245Calle, A., Fernandez-Gonzalez, R., Ramos-Ibeas, P., Laguna-Barraza, R., Perez-Cerezales, S., Bermejo-Alvarez, P., … Gutierrez-Adan, A. (2012). Long-term and transgenerational effects of in vitro culture on mouse embryos. Theriogenology, 77(4), 785-793. doi:10.1016/j.theriogenology.2011.07.016Feuer, S. K., Liu, X., Donjacour, A., Lin, W., Simbulan, R. K., Giritharan, G., … Rinaudo, P. F. (2014). Use of a Mouse In Vitro Fertilization Model to Understand the Developmental Origins of Health and Disease Hypothesis. Endocrinology, 155(5), 1956-1969. doi:10.1210/en.2013-2081Garcia-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/ani10050804Dulioust, 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.92.2.589Fischer, B., Chavatte-Palmer, P., Viebahn, C., Navarrete Santos, A., & Duranthon, V. (2012). Rabbit as a reproductive model for human health. REPRODUCTION, 144(1), 1-10. doi:10.1530/rep-12-0091Servick, K. (2014). Unsettled questions trail IVF’s success. Science, 345(6198), 744-746. doi:10.1126/science.345.6198.744De 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/hoz038Sparks, 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-0035-1546826Vicente, 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. 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A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species

[EN] Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet® device (n = 175; 25 embryos per device), the open Cryotop® device (n = 175; 10 embryos per device), and the traditional closed French mini-straw device (n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group (n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet® and the other devices. In experiment 2, the CryoEyelet® device showed a higher implantation rate compared with the Cryotop® (6.3% unit of SD, p = 0.87) and French mini-straw® (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet® device was similar to the Cryotop® device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet® showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes-a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet® device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet® device in other polytocous species for the simultaneous vitrification of a large number of embryos.This research was funded by the Funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU /PRTR (PDC2021-120767-I00).Marco-Jiménez, F.; Garcia-Dominguez, X.; García-Valero, L.; Vicente Antón, JS. (2023). A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species. Animals. 13(5). https://doi.org/10.3390/ani1305079113

Impact of embryo technologies on secondary sex ratio in rabbit

[EN] Increasing evidence indicates that assisted reproductive technologies (ARTs) disturb skewed sex-ratio and induce sex-dimorphic postnatal effects. Undoubtedly, the combination of multiple ovulation and embryo transfer (MOET) together with the use of vitrification technique (MOVET) is currently being used in breeding programs. However, since the first case of sex skewing reported in 1991, the accumulative and long-term transmission of skewed sex-ratio to future generations has not been thoroughly evaluated. Here we test as MOVET program induce a skewed sex ratio, and we consider skewed sex ratio transmission to future generations. To this end, we first evaluated the F1 generation, demonstrating that a MOVET program causes a severe imbalance skewed secondary sex ratio (SSR) towards male by 12%. This imbalanced persist after a second MOVET program (F2 generation), with an accumulative skewed SSR towards male by 25%. Finally, using a crossbred generation derived from crossing F1 males derived from a MOVET program with naturally-conceived (NC) females, we show that the imbalance skewed SRR persist. Bodyweight comparison between MOVET animals and NC counterparts revealed significant changes at birth, weaning and adulthood. However, there was a significant interaction between F2 MOVET animals and sex, demonstrating an apparent accumulative sex-dimorphic effect. At adulthood, MOVET derived males presented a lower body weight. In conclusion, we show that the MOVET program causes a direct, accumulative and long-term transmission of skewed SSR.This work was supported by the Ministry of Economy, Industry and Competitiveness (Research project: AGL2017-85162-C2-1-R) is acknowledged. X. Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429). English text version was revised by N. Macowan English Language Service.Garcia-Dominguez, X.; Juarez, JD.; Vicente Antón, JS.; Marco-Jiménez, F. (2020). Impact of embryo technologies on secondary sex ratio in rabbit. Cryobiology. 97:60-65. https://doi.org/10.1016/j.cryobiol.2020.10.008S606597Auroux, M., Cerutti, I., Ducot, B., & Loeuillet, A. (2004). Is embryo-cryopreservation really neutral? Reproductive Toxicology, 18(6), 813-818. doi:10.1016/j.reprotox.2004.04.010Avery, B., Madison, V., & Greve, T. (1991). Sex and development in bovine in-vitro fertilized embryos. Theriogenology, 35(5), 953-963. doi:10.1016/0093-691x(91)90306-xBermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P., & Gutierrez-Adan, A. (2010). Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts. Proceedings of the National Academy of Sciences, 107(8), 3394-3399. doi:10.1073/pnas.0913843107Bermejo-Álvarez, P., Rizos, D., Rath, D., Lonergan, P., & Gutierrez-Adan, A. (2008). 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Bovine Blastocyst Development In Vitro: Timing, Sex, and Viability Following Vitrification1. Biology of Reproduction, 71(5), 1671-1676. doi:10.1095/biolreprod.104.027987Ng, 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/dmx028Roseboom, T. J. (2018). Developmental plasticity and its relevance to assisted human reproduction. Human Reproduction, 33(4), 546-552. doi:10.1093/humrep/dey034Sparks, 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-0035-1546826Supramaniam, P. R., Mittal, M., Ohuma, E. O., Lim, L. N., McVeigh, E., Granne, I., & Becker, C. M. (2019). Secondary sex ratio in assisted reproduction: an analysis of 1 376 454 treatment cycles performed in the UK. Human Reproduction Open, 2019(4). doi:10.1093/hropen/hoz020Tan, K., An, L., Miao, K., Ren, L., Hou, Z., Tao, L., … Tian, J. (2016). Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization. Proceedings of the National Academy of Sciences, 113(12), 3197-3202. doi:10.1073/pnas.1523538113Tan, K., Wang, Z., Zhang, Z., An, L., & Tian, J. (2016). IVF affects embryonic development in a sex-biased manner in mice. REPRODUCTION, 151(4), 443-453. doi:10.1530/rep-15-0588Tarín, J. J., García-Pérez, M. A., Hermenegildo, C., & Cano, A. (2014). Changes in sex ratio from fertilization to birth in assisted-reproductive-treatment cycles. Reproductive Biology and Endocrinology, 12(1), 56. doi:10.1186/1477-7827-12-56Torner, E., Bussalleu, E., Briz, M. D., Yeste, M., & Bonet, S. (2014). Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium. Reproduction, Fertility and Development, 26(4), 570. doi:10.1071/rd13004Valdivia, R. P. A., Kunieda, T., Azuma, S., & Toyoda, Y. (1993). PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro. Molecular Reproduction and Development, 35(2), 121-126. doi:10.1002/mrd.1080350204Ventura-Juncá, P., Irarrázaval, I., Rolle, A. J., Gutiérrez, J. I., Moreno, R. D., & Santos, M. J. (2015). In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations. Scientific and bioethical implications for IVF in humans. Biological Research, 48(1). doi:10.1186/s40659-015-0059-yVicente, 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. 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Using the available indicators of potential biodiversity damage for Life Cycle Assessment on soybean crop according to Brazilian ecoregions

The purpose of the study was to evaluate the use of and suggest possible adjustments to indicators of biodiversity loss for LCA proposed by Chaudhary and Books (2018). For this, we analyzed soybean production in Brazil. Potential damage to biodiversity has been determined for all Brazilian ecoregions: the Amazon; the Atlantic forest; Caatinga; Cerrado; Pampas; and the Pantanal. Two dimensions of assessment were considered – global and regional – in addition to Average-country. An adjustment was proposed for the vulnerability coefficient to the indicators Average-country Brazil: the Regional Species Fragility Index (FI). Two inventories were created using two different functional units: area of production of soybean by ecoregion (year m2); and area corresponding to production of 1 kg of soybean (year m2). Thus, we observed that when the indicators of aggregate values were adopted, the Atlantic Forest was the ecoregion most affected by the crop. Regarding the assessments of the Potential Biodiversity Damage (BD) Global and Regional indicators, the Atlantic Forest and the Amazon were the ecoregions that suffered the highest impacts, mainly on plants, birds and amphibians taxa. Besides, the impacts at the global level were always more expressive than the regional ones. Due to this, we noticed that the results were influenced by the Vulnerability Score (VS). The suitability of the VS for FI is relevant and the adjustment in the equation can be suggested for other regions. Considering the results found here, to prevent regional impacts, technical measures such as extensive farming and crop rotation should be prioritized as impact mitigation actions. However, political measures tend to be more effective at geographic levels when addressing more than one ecoregion, due to the standardization of preservation procedures. Thus, from the results reported here, we conclude that the FI is relevant to diagnose measures at the administrative geographic levels of the ecoregions present in a single country, and the applied indicators reinforce that the Atlantic Forest ecoregion is the most vulnerable due to the replacement of wild forest for cultivated areas, which includes soybean crops.info:eu-repo/semantics/publishedVersio

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer

[EN] Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo's developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 +/- 0.02, 0.78 +/- 0.05 and 0.63 +/- 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 +/- 0.49 mm, 2.15 +/- 0.51 mm, 1.76 +/- 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.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).Garcia-Dominguez, X.; Diretto, G.; Frusciante, S.; Vicente Antón, JS.; Marco-Jiménez, F. (2020). Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer. International Journal of Molecular Sciences. 21(19):1-14. https://doi.org/10.3390/ijms21197116S1142119Rizos, D., Maillo, V., Sánchez-Calabuig, M.-J., & Lonergan, P. (2017). The Consequences of Maternal-Embryonic Cross Talk During the Periconception Period on Subsequent Embryonic Development. 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Measures to evaluate quality of care in head and neck cancer: Results of a Delphi study.

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Successful development of vitrified embryonic kidney after laparoscopy transplantation into non-immunosuppressed hosts

[EN] Transplantation from living or deceased donors has been limited by donor availability that is opposed to the increasing demand and by the risk of allograft loss rejection and immunosuppressive therapy toxicity. In recent years, xenotransplantation of metanephroi has offered a novel solution for the unlimited supply of human donor organs. However, even if in a most favourable and idyllic situation the organ availability and its demand could be balanced using transplantation of animal embryonic organs, the future of this treatment would still be compromised without proper long-term storage procedure. Thus, based on the ongoing long-term storage necessities, this study was designed to investigate the effect of two specific time window of the metanephroi development (15 days-old and 16 days-old) on the in vivo developmental capacity and the developed morphologically normal glomeruli of vitrified metanephroi in non-immunosuppressive rabbits. Metanephroi originating from 15 and 16 days old rabbit embryos were vitrified using M22 solution and Cryotop® as a device. After three months of storage in liquid nitrogen, metanephroi were transplanted into non-immunosuppressed adult hosts by laparoscopy surgery. Twenty-one days after allotransplantation, 6 (32%) and 7 (35%) new kidneys were recovered. All the new kidneys recovered exhibited significant growth and mature glomeruli. However, histomorphometry analysis revealed that new kidneys developed from 16 days-old metanephroi exhibit a greater degree of maturity compared with 15 days-old metanephroi. Results obtained in the present study point out that, in rabbit model, vitrified 16 days-old metanephroi could be stored in liquid nitrogen, achieving good in vivo developmental capacity and the developed morphologically normal glomeruli after laparoscopy transplantation into non-immunosuppressed hosts.This work was supported by funds from the Generalitat Valenciana Research Programme (PrometeoII 2014/036), ALCER-TURIA foundation and PRECIPITA CROWDFUNDING. English text version was revised by N. Macowan English Language Service.Garcia-Dominguez, X.; Vicente Antón, JS.; Vera Donoso, CD.; Marco-Jiménez, F. (2017). Successful development of vitrified embryonic kidney after laparoscopy transplantation into non-immunosuppressed hosts. Transplantation open. 2(2):1-5. https://doi.org/10.15761/JTO.1000125S152

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