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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Recipient maternal genotypes improved the litter size components of a paternal line involved in a MOET programme in rabbits

[EN] An essential factor in the success of multiple ovulation and embryo transfer programmes (MOET) in any species is the selection of the recipient females. In rabbit there is a notable lack of studies on the effects of recipient genotype on postnatal growth. The aim of this study was to evaluate the effects of recipient maternal genotypes on litter size components within a MOET programme applied to a commercial paternal line that appears to have exhausted its selection programme after 37 generations. The experiment was designed using 13 nulliparous donors from the R line (paternal line) to produce 453 embryos, which were transferred to two recipient maternal genotypes (A and V lines) and the own donor paternal line (R line). Litter size components and pre-and postnatal body mass of kits were evaluated. The differences between the genetic groups of recipients were estimated using a general linear model applying Bayesian analysis. The results showed that maternal lines have a high capacity to implant the embryos, maintain the pregnancy and present a favourable environment for embryo development compared to the R line. Specifically, A line dams showed the highest prenatal survival, total born and number born alive without effects on growth traits. The present study demonstrated the applicability of a MOET programme based on maternal ability recipients to improve the number of kits per cycle. Therefore, to allow the genetic improvement programme of meat rabbits to continue using the R line, we recommended applying for a MOET programme as a routine procedure.This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Interministerial Committee on Science and TechnologyRagab, M.; Valdés-Hernández, J.; Vicente Antón, JS.; Marco-Jiménez, F. (2022). Recipient maternal genotypes improved the litter size components of a paternal line involved in a MOET programme in rabbits. Italian Journal of Animal Science. 21(1):1584-1592. https://doi.org/10.1080/1828051X.2022.21421671584159221

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

[EN] 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 g 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 ug of buserelin acetate administered intramuscularly. The entrapment efficiencyof 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 ug to 4 ug.This research was funded through grant PID2021-127867OB-100 by MCIN/AEI/10.13039/ 501100011033 and by ERDF, A way of making Europe , and through grant 52201K by GVA-IVIA and the FEDER Operational Program of the Valencian Community, 2021 2027.Viudes-De-Castro, MP.; Marco-Jiménez, F.; Vicente Antón, JS. (2023). Reproductive Performance of Female Rabbits Inseminated with Extenders Supplemented with GnRH Analogue Entrapped in Chitosan-Based Nanoparticles. Animals. 13(10). https://doi.org/10.3390/ani13101628131

Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in msy2 gene expression

[EN] Young rabbit females selected for growth rate may have nutritional needs, which may not be met with the common practice of feed restriction during rearing in commercial rabbit production. The aim of this study was to analyse whether two different feeding programmes: ad libitum or restricted (130 g/day) feeding, applied in young rabbit females for 1 month at the end of rearing, could modulate the origin of ovulation process and the quality of the oocytes. At 16 weeks of age, 34 females were randomly assigned to restricted or ad libitum feeding, maintaining these conditions for a month. Then, in an initial experiment, transcriptional profiling of hypothalamus-hypophysis tissue was performed to assess failure to ovulate. In the second experiment, the gene expression analysis of some candidate genes related to oocytes quality was performed. Our results demonstrated that neither of the two feeding programmes modified the transcription of hypothalamus-hypophysis tissue, while the only differences in MSYR expression were found in in vivo mature oocytes ready for successful fertilization. Specifically, MSYR was over-expressed in oocytes from females fed ad libitum. MSYR is one of the most abundant proteins in the oocyte and has proven to be a key regulator of maternal RNA transcription and translation. This finding suggests that MSYR gene is a promising gene in our understanding of the relationship between high growth rate and reproductive performance decline.This work was supported by the Spanish Research Projects AGL2014-53405-C2-P and AGL2011-30170-C02-01 (CICYT). Carmen Naturil was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296).Naturil Alfonso, C.; Peñaranda, D.; Vicente Antón, JS.; Marco-Jiménez, F. (2017). 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Reproduction in Domestic Animals, 51(1), 114-122. doi:10.1111/rda.12653Naturil-Alfonso, C., Marco-Jiménez, F., Jiménez-Trigos, E., Saenz-de-Juano, M., Viudes-de-Castro, M., Lavara, R., & Vicente, J. (2015). Role of Embryonic and Maternal Genotype on Prenatal Survival and Foetal Growth in Rabbit. Reproduction in Domestic Animals, 50(2), 312-320. doi:10.1111/rda.12493O’Callaghan, D., Yaakub, H., Hyttel, P., Spicer, L., & Boland, M. (2000). Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes. Reproduction, 118(2), 303-313. doi:10.1530/jrf.0.1180303Papadopoulos, S., Lonergan, P., Gath, V., Quinn, K. M., Evans, A. C. O., O’Callaghan, D., & Boland, M. P. (2001). Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep. Theriogenology, 55(5), 1059-1069. doi:10.1016/s0093-691x(01)00466-6Pascual, J. J., Castella, F., Cervera, C., Blas, E., & Fernández-Carmona, J. (2000). The use of ultrasound measurement of perirenal fat thickness to estimate changes in body condition of young female rabbits. Animal Science, 70(3), 435-442. doi:10.1017/s135772980005178xRomanatto, T., Cesquini, M., Amaral, M. E., Roman, É. A., Moraes, J. C., Torsoni, M. A., … Velloso, L. A. (2007). TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient—Effects on leptin and insulin signaling pathways. Peptides, 28(5), 1050-1058. doi:10.1016/j.peptides.2007.03.006Rommers, J. M., Meijerhof, R., Noordhuizen, J. P. T. M., & Kemp, B. (2004). Effect of feeding program during rearing and age at first insemination on performances during subsequent reproduction in young rabbit does. Reproduction Nutrition Development, 44(4), 321-332. doi:10.1051/rnd:2004037Smith, J. T., Acohido, B. V., Clifton, D. K., & Steiner, R. A. (2006). KiSS-1 Neurones Are Direct Targets for Leptin in the ob/ob Mouse. 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Expression of MSY2 in Mouse Oocytes and Preimplantation Embryos1. Biology of Reproduction, 65(4), 1260-1270. doi:10.1095/biolreprod65.4.126

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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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

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

Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification

[EN] Parthenote embryos offer multiple opportunities in biotechnological research, so it is important to analyse the possibilities for their cryopreservation in order to establish a biobank. The aim of this experiment was to determine the effect of culture conditions and vitrification on rabbit parthenogenetic embryos. Parthenotes were cultured under in vivo and in vitro conditions until day 3 (late morula/early blastocyst), when they were vitrified. Immediately after warming, they were newly cultured under in vivo and in vitro conditions till day 6 (blastocyst stage). Both culture conditions showed similar late morula/early blastocyst (0.39±0.056 vs. 0.46±0.043, for in vivo and in vitro, respectively) and blastocyst rates (0.12±0.068 vs. 0.13±0.070, for in vivo and in vitro, respectively). However, no parthenote was recovered when a combination of culture conditions was performed. To our best knowledge, this is the first demonstration of the ability of rabbit parthenogenetic embryos to develop after vitrification, with similar embryo development after in vivo or in vitro culture. Nevertheless, our results highlight the importance of culture conditions on the morphology of parthenote embryos. Therefore, we have described that special attention should be paid on culture conditions to generate parthenote embryos, with a view to their subsequent use, for example in embryonic stem cell production.Statement of funding: This work was supported by funds from the Generalitat Valenciana Research Programme (Prometeoll 2014/036).Naturil Alfonso, C.; Jiménez Trigos, ME.; Vicente Antón, JS.; Marco Jiménez, F. (2015). Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification. Cryobiology. 71(1):91-96. doi:10.1016/j.cryobiol.2015.05.003S919671

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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Developmental plasticity and its relevance to assisted human reproduction. Human Reproduction, 33(4), 546-552. doi:10.1093/humrep/dey034Vrooman, 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.2016.07.015Ng, 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/dmx028Zacchini, F., Sampino, S., Stankiewicz, A. M., Haaf, T., & Ptak, G. E. (2019). Assessing the epigenetic risks of assisted reproductive technologies: a way forward. The International Journal of Developmental Biology, 63(3-4-5), 217-222. doi:10.1387/ijdb.180402gpDuranthon, V., & Chavatte-Palmer, P. (2018). Long term effects of ART: What do animals tell us? 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