Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures

2019 ◽  
Vol 31 (5) ◽  
pp. 837 ◽  
Author(s):  
V. H. Do ◽  
S. Catt ◽  
J. E. Kinder ◽  
S. Walton ◽  
A. W. Taylor-Robinson
Keyword(s):  
Extreme Temperature ◽  
Laboratory Culture ◽  
Freezing Process ◽  
Embryo Viability ◽  
Culture Techniques ◽  
Practical Challenge ◽  
Cryopreservation Method ◽  
Fresh Embryos

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. ‘fresh’) embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

136 BLASTOCOELE COLLAPSE IMPROVES POST-THAW SURVIVAL OF SLOW FROZEN AND VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 181 ◽  
Author(s):  
J. P. Barfield ◽  
G. E. Seidel
Keyword(s):  
Defined Medium ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Intact Control ◽  
Commercial Medium ◽  
Cryopreservation Method ◽  
Standard Techniques ◽  
Better Than

Slow-freeze cryopreservation of in vitro-produced bovine embryos often results in poor post-thaw embryo viability. Results with vitrification tend to be better but do not always produce consistent results for a variety of reasons. Experiments with human and equine embryos have demonstrated improved post-thaw survival when blastocoele fluid is removed before vitrification. We hypothesised that removing blastocoele fluid before vitrification or slow-freeze cryopreservation would improve post-thaw survival of in vitro-produced bovine embryos. Three replicates of embryos were generated from abattoir-derived ovaries using standard techniques. Seven days post-fertilization, embryos were evaluated for quality, and grade 1 or 2 embryos were allocated to 1 of 4 treatments: slow-freeze control (n = 103); slow-freeze collapsed (n = 106); vitrified control (n = 117); vitrified collapsed (n = 99). Blastocoele fluid was removed by aspiration with a pipette advanced to the centre of the blastocoele while the embryos were stabilised with a holding pipette. After collapse, embryos were immediately slow frozen in commercial medium [1.5 M ethylene glycol (EG) + 0.1 M sucrose, Bioniche] in 0.25-mL straws or vitrified in 2 steps: 3 min in 1.5 M EG, 30 s in 7 M EG + 0.6 M galactose + 18% Ficoll in open-pulled straws. After thawing or warming, embryos were cultured in chemically defined medium-2 + 5% FCS in 5% CO2, 5% O2, 90% N2 at 38.5°C. Embryos were evaluated for survival based on re-expansion of the blastocoele after 24 h in culture. Data were analysed by GLIMMIX (SAS Institute Inc., Cary, NC, USA). There were significant replicate effects but there were no significant differences in survival between embryos that were vitrified or slow frozen. Approximately twice as many collapsed embryos survived compared to intact control embryos regardless of whether the embryos were vitrified or slow frozen (collapsed: 50.5 ± 4.1%, control: 26.3 ± 3.5%; P < 0.001). Advanced blastocysts (fully expanded and hatched blastocysts) survived better than early blastocysts (pre-expansion blastocysts and expanding blastocysts, 45.9% ± 3.9 v. 30.0% ± 4.4, respectively; P < 0.01). The only significant interaction (P < 0.05) was that vitrification was superior to freezing for early blastocysts (39.5 ± 5.9% v. 22.1 ± 5.0% survival) but not advanced blastocysts (44.4 ± 5.3% v. 47.4 ± 5.8% survival). Removal of blastocoele fluid before cryopreservation of in vitro-produced bovine embryos may be a useful technique for improving post-thaw survival regardless of cryopreservation method or blastocyst stage.

scholarly journals 102VITRIFICATION OF IN VITRO-PRODUCED BOVINE AND OVINE EMBRYOS USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 172 ◽  
Author(s):  
J.M. Kelly ◽  
D.O. Kleemann ◽  
M. Kuwayama ◽  
S.K. Walker
Keyword(s):  
Sucrose Solution ◽  
Survival Rates ◽  
Minimum Volume ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Embryo Survival ◽  
Mammalian Embryos ◽  
Fresh Embryos ◽  
Hatching Rates

Considerable progress has been achieved in the cryopreservation of mammalian embryos. The use of vitrification minimizes chilling injuries by increasing cooling and warming rates. This study assesses the effect of vitrification using the minimum volume cooling (MVC) method (Kuwayama &amp; Kato 2000 J. Assist. Reprod. Genet. 17, 477) on in vitro-produced bovine and ovine embryos. A total of 1756 ovine and 753 bovine cumulus-oocyte complexes were obtained from the abattoir and matured, fertilized (Day 0) and cultured in vitro (Walker et al., 1996 Biol. Reprod. 55, 703–708, Kelly et al., 1997 Theriogenology 47, 291). Overall cleavage rates were 93.7% and 80.5% respectively. Embryos were vitrified (OPS or MVC method) on Days 5 (morula, compact morula), 6 (expanded blastocyst, blastocyst, compact morula) or 7 (hatched and hatching blastocysts, expanded blastocyst, blastocyst). Embryos were equilibrated with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3min and then exposed to 16.5% EG, 16.5% DMSO, 0.5M sucrose and 20% FCS for 30s. Embryos were loaded onto either an MVC plate (Cryotop, Kitazato Supply Co, Toyko, Japan) or open pulled straw (OPS) and plunged into liquid nitrogen. After 5 days, embryos were thawed directly into 1.25M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Embryo survival was assessed by culture to Day 8 and compared to the development of non-vitrified control embryos (Table 1). Variables were assessed using procedure CATMOD in SAS. The Cryotop method yielded a significantly higher percentage of viable ovine embryos after thawing compared with OPS (P&lt;0.0001); neither day nor treatment x day interaction was significant (P&gt;0.05). A significant interaction between vitrification treatment and day (P&lt;0.007) indicated that the percentage of hatched embryos peaked at Day 6 using the Cryotop method compared with Day 7 for OPS. Hatching rates for fresh and vitrified embryos were similar at Day 7 and were independent of treatment. With the Cryotop method, day of vitrification did not influence the percentage of Days 6 and 7 bovine embryos that hatched after thawing but, on each day, this figure was significantly higher (P&lt;0.003 and P&lt;0.0001, respectively) than that obtained with fresh embryos. To further assess embryo viability, 36 fresh, 52 OPS and 56 Cryotop vitrified Day-6 in vitro-produced ovine embryos were transferred to synchronized recipients. Survival rates to Day 13 were 29/33 (87.9%), 23/36 (63.9%) and 42/51 (82.4%), respectively (P&lt;0.05). This study demonstrates that using the MVC Cryotop method, the viability of vitrified embryos, as assessed at Days 8 and 13, is similar to that obtained with fresh embryos. Table 1

scholarly journals 88 PREGNANCY RATES FOR IN VITRO AND IN VIVO PRODUCED OVINE EMBRYOS VITRIFIED USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 194
Author(s):  
J. Kelly ◽  
D. Kleemann ◽  
M. Kuwayama ◽  
S. Walker
Keyword(s):  
Pregnancy Rate ◽  
Sucrose Solution ◽  
Bovine Embryo ◽  
Minimum Volume ◽  
Embryo Viability ◽  
Embryo Survival ◽  
First Order ◽  
Fresh Embryos

Previously we reported that, using the minimum volume cooling (MVC) cryotop vitrification method, in vitro-produced ovine and bovine embryo survival after thawing was similiar to that of fresh embryos (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172). While survival of vitrified embryos after thawing can be indicative of embryo viability, this assessment does not always correlate with embryo survival after transfer. This study assesses the effect of vitrification using the MVC cryotop method on the survival after transfer of in vitro- and in vivo-produced ovine embryos. Fresh or vitrified Day 6 ovine embryos (expanded blastocysts, blastocysts, compact morulae) were used in this study. Ovine cumulus–oocyte complexes were obtained and matured, fertilized (Day 0), and cultured in vitro (Walker et al. 1996 Biol. Reprod. 55, 703–708). In vitro embryos for vitrification were produced and vitrified (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) 10 days prior to the day of transfer. In vivo embryos were recovered from donor Merino ewes and vitrified 7 days prior to the day of transfer while fresh in vivo embryos were collected and transferred on the same day. Semen used for both in vivo and in vitro embryo production was from the same sire. On the day of transfer, vitrified embryos were thawed directly into 1.25 M sucrose solution, followed by stepwise dilution of the cryoprotectants. Embryos were transferred as singles into synchronized recipient ewes on a randomized basis. Fetal number was detected at Day 50. Variables were assessed using the CATMOD procedure in SAS. Pregnancy rate for in vivo-derived embryos was higher (P < 0.01) than for in vitro-derived embryos. Embryo treatment (fresh vs. vitrified) did not significantly affect pregnancy rate. Pregnancy rate for ewes detected (by vasectomized rams) in estrus within 48 h of progesterone pessary removal was higher (P < 0.05) than for both the 48–68 h and unmarked groups. The latter two groups did not differ significantly. None of the first-order interactions were significant (P > 0.05). This study demonstrates that ovine embryos (in vitro and in vivo) can be vitrified, thawed, and transferred without compromising embryo viability. However, the differences in pregnancy rate between the recipient groups warrant further investigation. The MVC cryotop method is a vitrification technique that can be adapted to routine field use. Table 1. Pregnancy rate of fresh and vitrified in vivo and in vitro ovine embryos after embryo transfer

Fine cryopreservation method of porcine blastocysts produced by in vitro fertilization

2014 ◽  
Author(s):  
Sung-Hun Min ◽  
Jin-Woo Kim ◽  
Yong-Hee Lee ◽  
Jae-Hyun Ahn ◽  
Geon-Yeop Do ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization ◽  
Cryopreservation Method

Dwindling status of Epimedium Elatum (Morren & Decne) and its geographical distribution in Kashmir Himalaya, India

2018 ◽  
pp. 47-52
Keyword(s):  
Medicinal Plant ◽  
Biological Activities ◽  
Conservation Status ◽  
Natural Populations ◽  
Culture Techniques ◽  
Kashmir Himalayas ◽  
Near Future ◽  
First Time ◽  
Tissue Culture Techniques

Epimedium elatum (Morren & Decne) of family Berberidaceace is a rare perennial medicinal plant, endemic to high altitude forests of Northwestern Himalayas in India. Ethnobotanically, it has been used as an ingredient for treatment of bone-joint disorders, impotence and kidney disorders in Kashmir Himalayas. Phytochemically, it is rich in Epimedin ABC and Icariin; all of these have been demonstrated to possess remarkable biological activities like PDE-5 inhibition (treatment of erectile dysfunction), anticancer, antiosteoporosis antioxidant and antiviral properties. The present investigation reports its traditional usage, comprehensive distribution and conservation status from twenty ecogeographical regions in Kashmir Himalayas, India. The species was reported from Gurez valley for the first time. Numerous threats like excessive grazing, deforestration, habitat fragmentation, tourism encroachment, landslides and excessive exploitation have decreased its natural populations in most of the surveyed habitats. Consequently, its existence may become threatened in near future if timely conservation steps are not taken immediately by concerned stakeholders involved in medicinal plant research. Moreover, use of plant tissue culture techniques is recommended for development of its in vitro propagation protocols. Therefore, introduction of this medicinal plant in botanical gardens, protected sites and development of monitoring programmes are needed for its immediate conservation in Northwestern Himalayas, India.

Bioactive Peptides Sensitize Cells to Anticancer Effects of Oxaliplatin in Human Colorectal Cancer Xenografts in Nude Mice

2019 ◽  
Vol 26 (7) ◽  
pp. 512-522
Author(s):  
Xian Li ◽  
Long Xia ◽  
Xiaohui Ouyang ◽  
Qimuge Suyila ◽  
Liya Su ◽  
...  
Keyword(s):  
Quality Of Life ◽  
Colorectal Cancer ◽  
Nude Mice ◽  
Low Dose ◽  
Bioactive Peptides ◽  
Human Colorectal Cancer ◽  
Short Term ◽  
Hct 116

<P>Background: Despite new agent development and short-term benefits in patients with Colorectal Cancer (CRC), metastatic CRC cure rates have not improved due to high rates of oxaliplatin resistance and toxicity. There is an urgent need for effective tools to prevent and treat CRC and reduce morbidity and mortality of CRC patients. Exploring the effects of bioactive peptides on the antitumor to CRC was of vital importance to the clinical application. </P><P> Objective: This study aimed to investigate the therapeutic impact of Anticancer Bioactive Peptides (ACBP) on anticancer effect of oxaliplatin (LOHP) in human colorectal cancer xenografts models in nude mice. </P><P> Methods: HCT-116 cells were cultured in vitro via CCK-8 assays and the absorbance was measured at 450 nm. Apoptosis and cell cycle were assessed by Flow Cytometry (FCM) in vitro. HCT-116 human colorectal cancer cells inoculated subcutaneously in nude mice of treatment with PBS (GG), ACBP, LOHP, ACBP+LOHP (A+L) in vivo. The quality of life was assessed by dietary amount of nude mice, the weight of nude mice, inhibition rates, tumor weight and tumor volume. Immunohistochemistry and RT-qPCR method was conducted to determine the levels of apoptosisregulating proteins/genes in transplanted tumors. </P><P> Results: ACBP induced substantial reductions in viable cell numbers and apoptosis of HCT116 cells in combined with LOHP in vitro. Compared with the control GG group, ACBP combined low dose oxaliplatin (U) group demonstrated significantly different tumor volume, the rate of apoptosis, the expression levels of Cyt-C, caspase-3,8,9 proteins and corresponding RNAs (P<0.05). The expression of pro-apoptotic proteins in the cytoplasm around the nucleus was significantly enhanced by ACBP. Short term intermittent use of ACBP alone indicted a certain inhibitory effect on tumor growth, and improve the quality of life of tumor bearing nude mice. </P><P> Conclusion: ACBP significantly increased the anti-cancer responses of low dose oxaliplatin (L-LOHP), thus, significantly improving the quality of life of tumor-bearing nude mice.</P>

scholarly journals Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2428
Author(s):  
Frank Liang ◽  
Azar Rezapour ◽  
Peter Falk ◽  
Eva Angenete ◽  
Ulf Yrlid
Keyword(s):  
Rectal Cancer ◽  
T Cells ◽  
Γδ T Cells ◽  
T Cell Subsets ◽  
Functional Evaluation ◽  
Mait Cells ◽  
Ifn Γ ◽  
Tumor Biopsies

TILs comprise functionally distinct conventional and unconventional T cell subsets and their role in responses to CRC treatments is poorly understood. We explored recovery of viable TILs from cryopreserved tumor biopsies of (chemo)-radiated patients with rectal cancer to establish a platform for retrospective TIL analyses of frozen tumors from pre-selected study cohorts. Frequencies of TIL subsets and their capacity to mount IFN-γ responses in cell suspensions of fresh vs. cryopreserved portions of the same tumor biopsies were determined for platform validation. The percentages and proportions of CD4+ TILs and CD8+ cytotoxic T lymphocytes (CTLs) among total TILs were not affected by cryopreservation. While recovery of unconventional γδ T cells and mucosal-associated invariant T cells (MAIT cells) was stable after cryopreservation, the regulatory T cells (Tregs) were reduced, but in sufficient yields for quantification. IFN-γ production by in vitro-stimulated CD4+ TILs, CTLs, γδ T cells, and MAIT cells were proportionally similar in fresh and cryopreserved tumor portions, albeit the latter displayed lower levels. Thus, the proposed platform intended for TIL analyses on cryopreserved tumor biobank biopsies holds promises for studies linking the quantity and quality of TIL subsets with specific clinical outcome after CRC treatment.

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