89 OPEN PULLED STRAW VITRIFICATION FOR IN VITRO-PRODUCED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 153 ◽  
Author(s):  
A. De Rosa ◽  
R. Di Palo ◽  
L. Attanasio ◽  
E. Monaco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Developmental Stages ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Embryo Survival ◽  
Vitro Fertilization ◽  
Arcsine Transformation ◽  
Further Development

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos at different developmental stages by the open pulled straw (OPS) method. In method A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min before being placed into 1.4 M glycerol + 3.6 M ethylene glycol (EG) for 5 min. Then, embryos were transferred into 3.4 M glycerol + 4.6 M EG for 25 s and loaded into the OPSs. For warming, OPSs were briefly immersed in a 0.5 M sucrose solution; the embryos were exposed to 0.25 M sucrose for 5 min before transfer to SOF medium for culture. In Method B, we examined the vitrification and warming solutions previously used for OPS vitrification of cattle embryos (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Buffalo embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO and 0.5 M sucrose. After 25 s, they were loaded into the OPSs. For warming, embryos were recovered in a 0.25 M sucrose solution and transferred into a 0.15 M sucrose solution for 5 min before being placed in SOF medium. A total of 293 IVP buffalo embryos (eight replicates) were vitrified at Day 7 of culture (Day 0 = in vitro fertilization). Embryos were vitrified at the following developmental stages: early blastocyst (eBL, n = 26 and 34 with methods A and B, respectively), blastocyst (Bl, n = 31 and 35 for Methods A and B, respectively), expanded blastocyst (XBl, n = 29 and 38 for Methods A and B, respectively), and hatched blastocyst (HBl, n = 46 and 54 for Methods A and B, respectively). Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by ANOVA following arcsine transformation of data. The overall embryo survival rate recorded at 24 h was not significantly different between Methods A and B (70% vs. 62%, respectively). Specifically, no differences were observed in embryos vitrified at the eBL (70% vs. 73%, A and B, respectively), Bl (69% vs. 70%, A and B, respectively), and HBl (46% vs. 36%, A and B, respectively) stages. In contrast, a significantly higher survival rate was recorded for XBl-stage embryos vitrified-warmed by Method A as compared to Method B (90% vs. 53%, respectively; P < 0.01). In Method A, survival rate of XBl was significantly higher than that of HBl (P < 0.05), but it was not different from that of eBl and Bl. Within Method B, the survival efficiency was similar for eBL, BL, and XBl, whereas survival rate of HBl was significantly lower (P < 0.05). In conclusion, although overall embryo survival in vitro was similar between methods, the combination of cryoprotectants used in Method A seemed more suitable for vitrification of IVP buffalo embryos at the XBl stage.

114 CRYOTOP VITRIFICATION FOR IN VITRO-PRODUCED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 174 ◽  
Author(s):  
A. De Rosa ◽  
L. Attanasio ◽  
L. Boccia ◽  
G. Pellerano ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
Liquid Nitrogen ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Thin Strip ◽  
Embryo Survival ◽  
Vitro Fertilization ◽  
Group A ◽  
Group B

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (&lt;0.1 �L) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide � 20 mm long � 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P &lt; 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts.

Selection of Reference miRNAs for Relative Quantification in Buffalo (Bubalus bubalis) Blastocysts Produced by Hand-Made Cloning and In Vitro Fertilization

2019 ◽  
Vol 21 (4) ◽  
pp. 200-209 ◽  
Author(s):  
Swati Viviyan Lagah ◽  
Tanushri Jerath Sood ◽  
Prabhat Palta ◽  
Manishi Mukesh ◽  
Manmohan Singh Chauhan ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Bubalus Bubalis ◽  
Relative Quantification ◽  
Vitro Fertilization ◽  
Selection Of ◽  
Reference Mirnas

RNA sequencing and transcriptome analysis of buffalo ( Bubalus bubalis ) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization

2019 ◽  
Vol 86 (9) ◽  
pp. 1149-1167 ◽  
Author(s):  
Tanushri Jerath Sood ◽  
Swati Viviyan Lagah ◽  
Manishi Mukesh ◽  
Suresh Kumar Singla ◽  
Manmohan Singh Chauhan ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Rna Sequencing ◽  
Transcriptome Analysis ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bubalus Bubalis ◽  
Vitro Fertilization

scholarly journals Global microRNA Expression Profiling of Buffalo (Bubalus Bubalis) Embryos at Different Developmental Stages Produced by Somatic Cell Nuclear Transfer and in Vitro Fertilization Using Rna Sequencing

2021 ◽  
Author(s):  
Pallavi Goel ◽  
Shivani Malpotra ◽  
Songyukta Shyam ◽  
Deepak Kumar ◽  
Manoj Kumar Singh ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Developmental Stages ◽  
Wnt Signaling Pathway ◽  
Bubalus Bubalis ◽  
Mirna Profile ◽  
Vitro Fertilization ◽  
Wide Scale ◽  
Mirnas Expression

Abstract Despite the success of cloning technology in production of offspring across several species, its application on a wide scale is severely limited by the very low offspring rate obtained with cloned embryos. MicroRNAs (miRNAs) expression profile in cloned embryos throughout the embryonic development are reported to deviate from regular pattern. In present study, aimed at determining the dynamics of global expression of miRNA profile in cloned and IVF pre-implantation embryos at different developmental stages i.e 2-cell, 8-cell and blastocyst using next-generation sequencing. The results of this study suggested that there is a profound difference in the global miRNA profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. The cloned embryos differ from their IVF counterparts in the enriched GO terms of Biological Process, Molecular Function, Cellular Component and Protein Class categories in terms of the targets of differentially expressed miRNAs. The major pathways related to embryonic development such as Wnt signaling pathway, apoptosis signaling pathway, FGF signaling pathway, p53 pathway etc. were found to be affected in cloned relative to IVF embryos.

68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

PSXI-10 How to use ultrasonic cutter on frozen semen for multiple embryo production

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 248-249
Author(s):  
Sung Woo Kim ◽  
Jae-Yeong Lee ◽  
Chan-Lan Kim ◽  
In-Sul Hwang ◽  
Yeoung-Gyu Ko ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Endangered Species ◽  
Genetic Resources ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
Economical Method ◽  
Frozen Semen ◽  
Ultrasonic Cutting

Abstract The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Sign in / Sign up

Export Citation Format

Share Document