scholarly journals Pre-screening the intrinsic angiogenic capacity of biomaterials in an optimised ex ovo chorioallantoic membrane model

2020 ◽  
Vol 11 ◽  
pp. 204173142090162 ◽  
Author(s):  
Nupur Kohli ◽  
Prasad Sawadkar ◽  
Sonia Ho ◽  
Vaibhav Sharma ◽  
Martyn Snow ◽  
...  
Keyword(s):  
Survival Rates ◽  
Chorioallantoic Membrane ◽  
Natural Polymers ◽  
Embryo Survival ◽  
Tissue Repair And Regeneration ◽  
Natural Composites ◽  
Successful Clinical Outcome ◽  
Cling Film ◽  
Set Up

Biomaterial development for clinical applications is currently on the rise. This necessitates adequate in vitro testing, where the structure and composition of biomaterials must be specifically tailored to withstand in situ repair and regeneration responses for a successful clinical outcome. The chorioallantoic membrane of chicken embryos has been previously used to study angiogenesis, a prerequisite for most tissue repair and regeneration. In this study, we report an optimised ex ovo method using a glass-cling film set-up that yields increased embryo survival rates and has an improved protocol for harvesting biomaterials. Furthermore, we used this method to examine the intrinsic angiogenic capacity of a variety of biomaterials categorised as natural, synthetic, natural/synthetic and natural/natural composites with varying porosities. We detected significant differences in biomaterials’ angiogenesis with natural polymers and polymers with a high overall porosity showing a greater vascularisation compared to synthetic polymers. Therefore, our proposed ex ovo chorioallantoic membrane method can be effectively used to pre-screen biomaterials intended for clinical application.

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
O. Sarv ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
In Vitro Fertilisation ◽  
Ministry Of Education ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Embryo Survival ◽  
Serum Free

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

scholarly journals 97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 199 ◽  
Author(s):  
B. Peachey ◽  
K. Hartwich ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Slow Freezing ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Frozen Embryos ◽  
Morula Stage ◽  
Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

scholarly journals High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sergio Ledda ◽  
Jen M. Kelly ◽  
Stefano Nieddu ◽  
Daniela Bebbere ◽  
Federica Ariu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
High Survival ◽  
Apoptotic Cells ◽  
Embryo Survival ◽  
New Device ◽  
In Vitro Survival

Abstract Background To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named “E.Vit”, composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. Results Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. Conclusions A high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

114 VITRIFICATION OF BOVINE EMBRYOS IN MEDIUM WITH POLYVINYL ALCOHOL REPLACING BSA

2006 ◽  
Vol 18 (2) ◽  
pp. 165
Author(s):  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Ethylene Glycol ◽  
Polyvinyl Alcohol ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Sodium Hyaluronate ◽  
Blastocyst Stage ◽  
Embryo Survival ◽  
Better Than

Embryos vitrified in medium supplemented with 4.25 μg/mL sodium hyaluronate (SH) and 0.1% polyvinyl alcohol (PVA) survived vitrification better than embryos vitrified in medium supplemented with 0.25% FAF-BSA (Walker and Seidel 2005 Reprod. Fert. Dev. 17, 153). The purpose of the present study was to determine if the small amount of SH was beneficial to in vitro survival and to examine the effects of different concentrations of PVA in vitrification solutions. Day 7 blastocysts (n = 360) were produced in vitro with semen from three bulls, two replicates each. Cryoprotectant solutions were prepared in a 2 × 3 factorial combination with two SH concentrations (0 or 4.25 μg/mL) and three PVA concentrations (0.05, 0.1%, or 0.2%). For vitrification, embryos were placed into chemically defined HEPES-buffered medium (HCDM-2) at room temperature (22–24°C) and then transferred to V1 (5 m ethylene glycol in HCDM-2) for 3 min. Next, embryos were placed in a 6 μL drop of V2 (7 m ethylene glycol, 0.5 m galactose, and 18% w/v Ficoll 70 in HCDM-2) for 45 s. During these 45 s, dilution medium (0.5 m galactose in HCDM-2) was aspirated into 0.25-mL straws, followed by the 6 μL drop of V2 plus embryos and a final short column of dilution medium. When 45 s had elapsed, the heat-sealed end of straw was dipped into liquid nitrogen to cover the embryo, and then the remainder of the straw was immersed slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Next, straws were shaken like a clinical thermometer four times to mix columns, and held in 37°C water for 10 min before embryos were expelled, rinsed and cultured in CDM-2 + 5% FCS. At 48 h, embryo survival (as determined by expansion of blastocysts), embryo quality (1 = excellent, 2 = fair, 3 = poor), inner cell mass (ICM) quality (1 = large and compact, 2 = clearly visible, 3 = not discernable) and blastocyst stage (5 = early, 6 = full, 7 = expanded, 8 = hatching, 9 = hatched) were evaluated and replicate averages were analyzed by ANOVA. Neither bull nor SH concentration nor PVA concentration significantly affected any response (P > 0.10). Averaged over PVA concentrations, vitrification of embryos in 0 μg/mL or 4.25 μg/mL SH resulted in similar survival rates (67% vs. 62%, respectively). When averaged over SH concentrations, 0.2% PVA had a numerically higher survival rate of blastocysts as compared to 0.1% or 0.05% (71% vs. 63% and 60%, respectively). The main effects of 0 μg/mL SH and 0.2% PVA also resulted in numerically higher, but nonsignificant improvements in quality score, ICM score and blastocyst stage as compared to the other doses of SH and PVA. Vitrification of Day 7 in vitro-produced bovine blastocysts in medium containing 0.2% PVA in the absence of SH resulted in a subclass mean of 80% embryo survival. Results of this experiment show no benefit of 4.25 μg/mL SH and that 0.2% PVA may be slightly better than 0.05% or 0.1% in terms of embryo survival. Therefore, our results indicate that 0.2% PVA can be used alone as an effective alternative to animal products in this vitrification procedure for in vitro-derived bovine blastocysts.

22 Vitrification of bovine embryo using antifreeze polyamino acid

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
T. Fujikawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Basal Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Polyamino Acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

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

24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device

2019 ◽  
Vol 31 (1) ◽  
pp. 138
Author(s):  
N. González ◽  
J. Scherzer ◽  
M. Reichenbach ◽  
C. Otzdorff ◽  
H. Zerbe
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Embryo Transfer ◽  
Zona Pellucida ◽  
Survival Rates ◽  
Petri Dish ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Survival

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

scholarly journals Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

2021 ◽  
Vol 8 ◽  
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Alejandro González-Plaza ◽  
Inmaculada Parrilla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Pathways ◽  
Survival Rates ◽  
Gene Expression Pattern ◽  
Control Group ◽  
High Survival ◽  
Embryo Survival ◽  
Developmental Potential

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted &lt;0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

scholarly journals 119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL

2004 ◽  
Vol 16 (2) ◽  
pp. 182
Author(s):  
B. Shangguan ◽  
N. Yang ◽  
R. Vanderwal ◽  
M.D. Darrow
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Field Trials ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Ag Addition ◽  
Freezing Medium ◽  
Frozen Embryos

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P&lt;0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos. Table 1 Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM

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