scholarly journals Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260645
Yasuyuki Kikuchi ◽  
Sayaka Wakayama ◽  
Daiyu Ito ◽  
Masatoshi Ooga ◽  
Teruhiko Wakayama

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%–5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

2019 ◽  
Vol 11 (1) ◽  
Audrey J. Kindsfather ◽  
Megan A. Czekalski ◽  
Catherine A. Pressimone ◽  
Margaret P. Erisman ◽  
Mellissa R. W. Mann

Abstract Background Over the last several decades, the average age of first-time mothers has risen steadily. With increasing maternal age comes a decrease in fertility, which in turn has led to an increase in the use of assisted reproductive technologies by these women. Assisted reproductive technologies (ARTs), including superovulation and embryo culture, have been shown separately to alter imprinted DNA methylation maintenance in blastocysts. However, there has been little investigation on the effects of advanced maternal age, with or without ARTs, on genomic imprinting. We hypothesized that ARTs and advanced maternal age, separately and together, alter imprinted methylation in mouse preimplantation embryos. For this study, we examined imprinted methylation at three genes, Snrpn, Kcnq1ot1, and H19, which in humans are linked to ART-associated methylation errors that lead to imprinting disorders. Results Our data showed that imprinted methylation acquisition in oocytes was unaffected by increasing maternal age. Furthermore, imprinted methylation was normally acquired when advanced maternal age was combined with superovulation. Analysis of blastocyst-stage embryos revealed that imprinted methylation maintenance was also not affected by increasing maternal age. In a comparison of ARTs, we observed that the frequency of blastocysts with imprinted methylation loss was similar between the superovulation only and the embryo culture only groups, while the combination of superovulation and embryo culture resulted in a higher frequency of mouse blastocysts with maternal imprinted methylation perturbations than superovulation alone. Finally, the combination of increasing maternal age with ARTs had no additional effect on the frequency of imprinted methylation errors. Conclusion Collectively, increasing maternal age with or without superovulation had no effect of imprinted methylation acquisition at Snrpn, Kcnq1ot1, and H19 in oocytes. Furthermore, during preimplantation development, while ARTs generated perturbations in imprinted methylation maintenance in blastocysts, advanced maternal age did not increase the burden of imprinted methylation errors at Snrpn, Kcnq1ot1, and H19 when combined with ARTs. These results provide cautious optimism that advanced maternal age is not a contributing factor to imprinted methylation errors in embryos produced in the clinic. Furthermore, our data on the effects of ARTs strengthen the need to advance clinical methods to reduce imprinted methylation errors in in vitro-produced embryos.

2021 ◽  
Vol 2 (2) ◽  
pp. 325-334
Neda Javadi ◽  
Hamed Khodadadi Tirkolaei ◽  
Nasser Hamdan ◽  
Edward Kavazanjian

The stability (longevity of activity) of three crude urease extracts was evaluated in a laboratory study as part of an effort to reduce the cost of urease for applications that do not require high purity enzyme. A low-cost, stable source of urease will greatly facilitate engineering applications of urease such as biocementation of soil. Inexpensive crude extracts of urease have been shown to be effective at hydrolyzing urea for carbonate precipitation. However, some studies have suggested that the activity of a crude extract may decrease with time, limiting the potential for its mass production for commercial applications. The stability of crude urease extracts shown to be effective for biocementation was studied. The crude extracts were obtained from jack beans via a simple extraction process, stored at room temperature and at 4 ℃, and periodically tested to evaluate their stability. To facilitate storage and transportation of the extracted enzyme, the longevity of the enzyme following freeze drying (lyophilization) to reduce the crude extract to a powder and subsequent re-hydration into an aqueous solution was evaluated. In an attempt to improve the shelf life of the lyophilized extract, dextran and sucrose were added during lyophilization. The stability of purified commercial urease following rehydration was also investigated. Results of the laboratory tests showed that the lyophilized crude extract maintained its activity during storage more effectively than either the crude extract solution or the rehydrated commercial urease. While incorporating 2% dextran (w/v) prior to lyophilization of the crude extract increased the overall enzymatic activity, it did not enhance the stability of the urease during storage.

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 187-194 ◽  
Y H Choi ◽  
L B Love ◽  
D D Varner ◽  
K Hinrichs

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

2015 ◽  
Vol 27 (1) ◽  
pp. 119
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P < 0.05) and tended (P < 0.08) to reduce re-expansion rate at 24 and 48 h, respectively (76.5 ± 2.5 v. 70.4 ± 2.5 and 79.5 ± 2.2 v. 76.0 ± 2.2, respectively). Whereas hatching rate at 72 h tended (P < 0.08) to be higher for embryos cultured with LC (67.8 ± 2.5 v. 74.4 ± 2.5), treatment with CLA reduced (P < 0.05) hatching rate at 48 h (62.3 ± 2.6 v. 54.9 ± 2.6). In Exp. 2, to determine lipid content, expanded blastocyst-stage embryos (n = 263) were harvested and stained using Nile Red. Embryos were examined for fluorescence using an epifluorescence microscope, and intensity of fluorescence per unit area was quantified using ImageJ software (NIH, Bethesda, MD, USA). There was a significant interaction (P < 0.01) between LC and CLA affecting embryo lipid content. Whereas addition of CLA during culture increased lipid, treatment with LC and the combination of LC and CLA reduced lipid (22.8 ± 1.1 v. 19.1 ± 1.1 v. 28.4 ± 1.1 v. 19.2 ± 1.2 for no additive, +LC, +CLA, and +LC and CLA, respectively). For Exp. 3, the effect of LC and CLA on the relative abundance of genes involved in lipid metabolism (ELOVL6, SCD1, SQLE, HMGCS1, CYP51A1, FDPS, FDFT1, LDLR, and SC4MOL) was determined. Pools of 5 expanded blastocyst-stage embryos from each treatment were collected across 5 replicates. The RNA was purified and synthesised into cDNA for RT-qPCR analysis. The SDHA, GAPDH, and YWAZ were used as housekeeping genes. Addition of LC during culture reduced (P < 0.05) the abundance of 4 of the 9 genes analysed (SQLE, HMGCS1, CYP51A1, and FDPS) and tended (P < 0.08) to reduce a fifth (FDFT1). In addition, there was a tendency (P < 0.08) for LC to increase the abundance of SCD1. Addition of CLA during culture had minimal effects on transcript abundance. In particular, CLA treatment reduced (P < 0.01) ELOVL6 and tended (P < 0.08) to increase SCD1. In contrast to previous studies, post-thaw cryosurvival was not significantly improved by treatment with LC or CLA. Results indicate that reduced embryo lipid content caused by LC treatment is due, in part, to an alteration in the abundance of genes involved in lipid metabolism. Further research is still necessary to determine whether in vivo survival following transfer of cryopreserved embryos can be enhanced by treatment with LC or CLA.Support was provided by USDA AFRI Grant 2010–85122–20623.

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (, which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

Zygote ◽  
2009 ◽  
Vol 18 (2) ◽  
pp. 121-129 ◽  
A.T. Palasz ◽  
P. Beltrán Breña ◽  
J. De la Fuente ◽  
A. Gutiérrez-Adán

SummaryThe effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration andin vitrodevelopment. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to ≤8-cell stage by day 4 were not affected by lack of BSA in the medium, but increased numbers of blastocysts developed in medium supplemented with BSA from days 1 or 4 with or without HA than in medium that had HA only. Addition of both HA and BSA at day 4 increased mtDNA copy numbers at the blastocyst stage. Data suggest that the addition of BSA and/or HA at 96 hpi increased expression ofRHAMMandHas2genes, but notCD44,Has1orHas3genes. Higher expression levels of Has2 than Has1 and the three isoforms indicate that high- rather than low-molecular-weight HA should be used for preimplantation bovine embryo culture.

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

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