Effect of restricted feeding under rearing on reproduction, body condition and blood metabolites of rabbit does selected for growth rate

[EN] Young rabbit females selected for growth rate can have nutritional needs which may not be met by the common practice of feed restriction during rearing in commercial rabbit production. The aim of this study was to analyse the effect of two different feeding programmes: restricted and ad libitum feeding, applied in young rabbit females for one month at the end of rearing, on reproductive performance, body condition and circulating metabolic hormones and metabolites in a rabbit line selected by growth rate in 3 consecutive reproductive cycles. Thus, twenty-four 16-week-old does were randomly assigned to a group in which the daily recommended nutrient intakes were satisfied (fed restricted: 130 g/day, n=13) or a group fed to satiety (ad libitum: 235.5 g/day, n=11) during one month. Then, all does were inseminated in 3 consecutive cycles using a 42-day reproductive cycle. Measurements of does’ body weight, perirenal fat thickness and plasma leptin, non-esterified-fatty-acids (NEFA), beta-hydroxybutyrate (BOHB) and fructosamine were performed at artificial insemination (AI), parturition and weaning time in 3 consecutive cycles. Reproductive performance of does was evaluated based on fertility, litter size at parturition, prolificacy and productivity. Differences in body weight were found only in the 1st cycle, ad libitum fed females being heavier than restricted ones. Nevertheless, body weight variances disappeared in later cycles. No differences were found in perirenal fat thickness. Finally, in ad libitum fed females slight differences were found in plasma levels of NEFAs (452 vs. 258 μekv/L and 527 vs. 306 μekv/L for 1st and 2nd cycles) and BOHB (0.26 vs. 0.03 mM for 2nd cycle), but disappeared in the 3rd reproductive cycle. Fertility, prolificacy and productivity was not significantly affected by the feeding programme. Nevertheless, total litter size showed to be higher in ad libitum fed females at second parturition (8.7 vs. 5.9 kits). Therefore, the evaluated feeding programmes until first AI in females selected by growth rate had no effect on their reproductive outcomes, as the global reproductive performance was not affected.This work was supported by the Spanish Research Project AGL2014- 53405-C2-1-P (CICYT). Carmen Naturil was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF ⁄ 2013 ⁄ 296). English text version revised by N. Macowan English Language Service.Naturil-Alfonso, C.; Marco-Jiménez, F.; Pascual, J.; Vicente, J. (2017). Effect of restricted feeding under rearing on reproduction, body condition and blood metabolites of rabbit does selected for growth rate. World Rabbit Science. 25(4):303-312. https://doi.org/10.4995/wrs.2017.6848SWORD30331225

Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. V., Turovets, N. A., Seiler, M. J., Nistor, G., Altun, G., Agapova, L. S., … Keirstead, H. S. (2011). Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells. PLoS ONE, 6(1), e14499. doi:10.1371/journal.pone.0014499Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., … Zhou, Q. (2010). Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. Journal of Assisted Reproduction and Genetics, 27(6), 285-291. doi:10.1007/s10815-010-9408-5Koh, C. J., Delo, D. M., Lee, J. W., Siddiqui, M. M., Lanza, R. P., Soker, S., … Atala, A. (2009). Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications. Methods, 47(2), 90-97. doi:10.1016/j.ymeth.2008.08.002Vrana, K. E., Hipp, J. D., Goss, A. M., McCool, B. A., Riddle, D. R., Walker, S. J., … Cibelli, J. B. (2003). Nonhuman primate parthenogenetic stem cells. Proceedings of the National Academy of Sciences, 100(Supplement 1), 11911-11916. doi:10.1073/pnas.2034195100Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., … Liu, L. (2009). Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells. Stem Cells, 27(9), 2136-2145. doi:10.1002/stem.158Sritanaudomchai, H., Ma, H., Clepper, L., Gokhale, S., Bogan, R., Hennebold, J., … Mitalipov, S. (2010). Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells. Human Reproduction, 25(8), 1927-1941. doi:10.1093/humrep/deq144Fang, Z. F., Gai, H., Huang, Y. Z., Li, S. G., Chen, X. J., Shi, J. J., … Sheng, H. Z. (2006). Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Experimental Cell Research, 312(18), 3669-3682. doi:10.1016/j.yexcr.2006.08.013Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., … Ji, W. (2007). Generation and Characterization of Rabbit Embryonic Stem Cells. Stem Cells, 25(2), 481-489. doi:10.1634/stemcells.2006-0226Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology, 34(5), 879-901. doi:10.1016/0093-691x(90)90559-cNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Besenfelder, 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.xMehaisen, G. M. K., Viudes-de-Castro, M. P., Vicente, J. S., & Lavara, R. (2006). In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does. Theriogenology, 65(7), 1279-1291. doi:10.1016/j.theriogenology.2005.08.007Bilodeau-Goeseels, S., & Schultz, G. A. (1997). Changes in Ribosomal Ribonucleic Acid Content Within in Vitro-produced Bovine Embryos1. Biology of Reproduction, 56(5), 1323-1329. doi:10.1095/biolreprod56.5.1323Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18), 3674-3676. doi:10.1093/bioinformatics/bti610Edgar, R. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research, 30(1), 207-210. doi:10.1093/nar/30.1.207Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.019Llobat, L., Marco-Jiménez, F., Peñaranda, D., Saenz-de-Juano, M., & Vicente, J. (2011). Effect of Embryonic Genotype on Reference Gene Selection for RT-qPCR Normalization. Reproduction in Domestic Animals, 47(4), 629-634. doi:10.1111/j.1439-0531.2011.01934.xLiu, N., Enkemann, S. A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., … Liu, L. (2010). Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis. Journal of Molecular Cell Biology, 2(6), 333-344. doi:10.1093/jmcb/mjq029Abdoon, A. S., Ghanem, N., Kandil, O. M., Gad, A., Schellander, K., & Tesfaye, D. (2012). cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6), 1240-1251. doi:10.1016/j.theriogenology.2011.11.004Labrecque, R., & Sirard, M.-A. (2011). Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation. Reproduction, Fertility and Development, 23(4), 591. doi:10.1071/rd10243Rizos, D., Clemente, M., Bermejo-Alvarez, P., de La Fuente, J., Lonergan, P., & Gutiérrez-Adán, A. (2008). Consequences ofIn VitroCulture Conditions on Embryo Development and Quality. Reproduction in Domestic Animals, 43, 44-50. doi:10.1111/j.1439-0531.2008.01230.xLonergan, P., Rizos, D., Kanka, J., Nemcova, L., Mbaye, A., Kingston, M., … Boland, M. (2003). Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction, 337-346. doi:10.1530/rep.0.1260337Memili, E., & First, N. L. (2000). Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote, 8(1), 87-96. doi:10.1017/s0967199400000861Latham, K. E. (2001). Embryonic genome activation. Frontiers in Bioscience, 6(3), d748-759. doi:10.2741/a639Niemann, H., & Wrenzycki, C. (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology, 53(1), 21-34. doi:10.1016/s0093-691x(99)00237-xCorcoran, D., Fair, T., Park, S., Rizos, D., Patel, O. V., Smith, G. W., … Lonergan, P. (2006). Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction, 131(4), 651-660. doi:10.1530/rep.1.01015Morison, I. M., Ramsay, J. P., & Spencer, H. G. (2005). A census of mammalian imprinting. Trends in Genetics, 21(8), 457-465. doi:10.1016/j.tig.2005.06.008Bischoff, S. R., Tsai, S., Hardison, N., Motsinger-Reif, A. A., Freking, B. A., Nonneman, D., … Piedrahita, J. A. (2009). Characterization of Conserved and Nonconserved Imprinted Genes in Swine1. Biology of Reproduction, 81(5), 906-920. doi:10.1095/biolreprod.109.078139Cruz-Correa, M., Zhao, R., Oveido, M., Bernabe, R. D., Lacourt, M., Cardona, A., … Giardiello, F. M. (2009). Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene. Epigenetics, 4(2), 114-118. doi:10.4161/epi.4.2.7954Park, C.-H., Uh, K.-J., Mulligan, B. P., Jeung, E.-B., Hyun, S.-H., Shin, T., … Lee, C.-K. (2011). Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos. PLoS ONE, 6(7), e22216. doi:10.1371/journal.pone.0022216Thurston, A., Taylor, J., Gardner, J., Sinclair, K. D., & Young, L. E. (2007). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction, 135(1), 29-40. doi:10.1530/rep-07-0211Li, Y., & Sasaki, H. (2011). Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Research, 21(3), 466-473. doi:10.1038/cr.2011.15Mamo, S., Gal, A., Polgar, Z., & Dinnyes, A. (2008). Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Molecular Biology, 9(1), 67. doi:10.1186/1471-2199-9-67Navarrete Santos, A., Tonack, S., Kirstein, M., Pantaleon, M., Kaye, P., & Fischer, B. (2004). Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction, 128(5), 517-526. doi:10.1530/rep.1.0020

Effect of different culture systems on mRNA expression in developing rabbit embryos

[EN] The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit preimplantation embryos.This work was supported by the Spanish Research Projects (CICYT AGL2008-03274) and the Generalitat Valenciana research program (Prometeo 2009/125). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.Saenz De Juano Ribes, MDLD.; Naturil Alfonso, C.; Vicente Antón, JS.; Marco Jiménez, F. (2013). Effect of different culture systems on mRNA expression in developing rabbit embryos. Zygote. 21(1):103-109. https://doi.org/10.1017/S0967199411000414S10310921

Effect of exposure to heatwave during blastocyst formation on reproductive performance of female rabbits

We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14-22°C, n = 29) and does heat stressed in a climatic chamber (maintained between 32-37°C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-γ{phonetic}, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8-16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation.This work was supported by the Spanish Research Projects AGL2008–03274 and AGL2011‐30170‐C02‐01. Estrella Jiménez‐Trigos and Carmen Naturil‐Alfonso were supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262 and ACIF/2013/296, respectively)

Maternal exposure to high temperatures disrupts OCT4 mRNA expression of rabbit pre‐implantation embryos and endometrial tissue

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre‐implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat‐treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat‐treated does, lower litter size (9.7 ± 0.48 and 11.4 ± 0.50) and fewer live born kits (7.2 ± 0.55 and 10.2 ± 0.57) were observed, although similar ovulation rates and numbers of pre‐implantation embryos were noted. In experiment 2, after 3rd partum multiparous non‐lactating does from each experimental group were used to obtain pre‐implantation embryos and endometrial tissue. mRNA transcripts from OCT‐4, VEGF, erbB3, Ifn‐ɣ, HSP70 and HSP90 were analysed by real‐time qPCR. Higher values of OCT‐4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up‐regulate VEGF in embryos and down‐regulate Ifn‐ɣ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre‐implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.This work was supported by the Spanish Research Projects AGL2008–03274 (CICYT). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Estrella Jiménez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).Peer reviewe

Oocyte quality and in vivo embryo survival after ovarian stimulation in nulliparous and multiparous rabbit does

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viabilit

Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos

Parthenote embryos offer multiple possibilities in biotechnological investigation, such as stem cell research. However, there is still a dearth of knowledge of this kind of embryo. In this study, development and ploidy were analysed in parthenotes under in vitro and in vivo culture conditions. Subsequently, using real-time PCR, the expressions of factor OCT-4, Vascular Endothelial Growth Factor, Epidermal Growth Factor Receptor 3 and Transforming Growth Factor ß2 genes were analysed to compare the embryo types at the blastocyst stage. Development and implantation of parthenote embryos were described after transfer at day 10 of pregnancy. Parthenotes showed similar blastocyst development for both culture conditions and most of the parthenotes produced were diploid. However, parthenotes developed under in vivo conditions showed similar mRNA expression of OCT-4, VEGF and TGF-ß2 to 5 and 6 day old blastocysts. In contrast, parthenotes developed under in vitro conditions had altered the expression pattern of these genes, except for erbB3 mRNA. Finally, transferred parthenotes had the ability to implant but showed severe growth retardation and lesser size. This is the first demonstration of the influence of culture conditions on parthenote mRNA expression. Our study highlights the importance of culture conditions in subsequent uses of parthenotes, such as the production of stem cell lines. © 2011 Elsevier B.V.This work was supported by Generalitat Valenciana research program (Prometeo 2009-8260: 125) and by the Spanish Research Projects (CICYT AGL2008-03274). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science. (127):222-228. https://doi.org/10.1016/j.anireprosci.2011.08.005S22222812

Foetal and postnatal exposure to high temperatures alter growth pattern but do not modify reproductive function in male rabbits

Purpose: The foetal origin hypothesis postulates that a number of organ structures and associated functions undergo programming during embryonic and foetal life and the neonatal period, which determines the set point of physiological and metabolic responses that carry into adulthood. We evaluate the relationship between high environmental temperatures and the reproductive function of male offspring to determine whether pregnant mammals and their infants are potentially vulnerable to the effects of climate change. Methods: Rabbit pups were exposed to high temperatures during gestation and lactation. Results: Foetal and postnatal exposure to high temperatures did not alter semen characteristics and was associated with a similar fertility rate and number of pups born. Moreover, males showed reduced rate of maturing and carcass traits at adulthood. Conclusion: Our findings suggest that male exposure during the foetal period to high temperatures did not affect sperm quality but permitted an adaptive phenotypic plasticity of growth in adulthood.This work was supported by the Spanish Research Projects AGL2008-03274 and AGL2011-30170-C02-01 (CICYT) and funding from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez-Trigos and Carmen Naturil-Alfonso were supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262 and ACIF/2013/296, respectively). The English text version was revised by N. Macowan English Language Service. The authors declare they have no actual or potential competing financial interests. The authors alone are responsible for the content and writing of the paper.Marco Jiménez, F.; Naturil Alfonso, C.; Jiménez Trigos, ME.; García Diego, FJ.; Lavara García, R.; Vicente Antón, JS. (2014). Foetal and postnatal exposure to high temperatures alter growth pattern but do not modify reproductive function in male rabbits. International Journal of Hyperthermia. 2(30):86-95. https://doi.org/10.3109/02656736.2013.878042S869523