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 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

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

Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Zenon Oikonomou ◽  
Katerina Chatzimeletiou ◽  
Antonia Sioga ◽  
Louisa Oikonomou ◽  
Basil C. Tarlatzis ◽  
...  
Keyword(s):  
Survival Rates ◽  
Multivariable Analysis ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
High Survival ◽  
Fluorescent Markers ◽  
Morula Stage ◽  
Confocal Scanning ◽  
Fresh Embryos

SummaryVitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.

138 DEVELOPMENTAL COMPETENCE OF BIOPSIED AND SPLIT BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 161
Author(s):  
M. Reichenbach ◽  
S. Jung ◽  
R. Fries ◽  
E. Wolf ◽  
C. Gschoederer ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Early Stage ◽  
Survival Rates ◽  
Dna Amplification ◽  
Developmental Competence ◽  
Breeding Value ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival

The aim of the present study was to develop a reliable method to simultaneously split and biopsy valuable bovine embryos for a complete genomic evaluation (gender, polledness, and hereditary abnormalities) and to estimate the breeding value of progeny for traits of economic importance immediately after embryo recovery. A total of 208 good quality embryos collected from superovulated German Simmental animals were biopsied immediately after recovery using an inverse microscope (Zeiss, Germany) at 50× magnification with a single-use steel blade mounted on a holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Biopsy was performed either by splitting the embryo and cutting of one-third of a half [G1: morulae (M), n = 50; early blastocysts (EB), n = 24; blastocysts (B), n = 16], by just splitting in equal halves (G2: M, n = 16; B, n = 2), or by cutting of just a small biopsy of the embryo (G3: M, n = 53) or of the trophoblast (G3: EB, n = 19; B, n = 28). Biopsied cells were immediately used for DNA amplification. Biopsied embryos (E) and demi-embryos (DE) were in vitro cultured in SOF, under mineral oil, at 39°C and 5% CO2, 5% O2, 90% N2 for 24 h, after which survival was recorded. Survival rate of G1 (survival of at least 1 DE: M, 98.0%; EB, 100.0%; B, 93.8%), G2 (survival of DE: M, 75.0%; B, 100.0%), and G3 (embryo survival: M, 96.3%; EB, 100.0%; B, 96.4%) were similar, but in relation to the number of original embryo the highest ratio of DE was obtained in G1 (1.67) v. G2 (0.88) and G3 (0.97; G1:G2/G3; P < 0.01). Within G1, the highest ration to the original number of embryos was by using M (1.78), followed by EB (1.75) and B (1.19; M/EB:B; P < 0.05). To verify the viability of biopsied embryos some DE from G1 (1, the nonbiopsied DE, n = 7, or 2, the biopsied and the nonbiopsied DE per recipient, n = 21), G2 (1 DE per recipient, n = 13), and G3 (1 E per recipient, n = 8) were transferred after 24 h of culture. Overall pregnancy rate (Day 42) of G1, G2, and G3 was 64.3, 23.1, and 50.0%, respectively (G1 : G2; P < 0.05). In G1, pregnancy rates (Day 42) of biopsied embryos differed significantly if either 1 or 2 DE were transferred per recipient (28.6 v. 76.2%, respectively; P < 0.05). A twin pregnancy rate of 38.9% was observed by ultrasonography in recipients when 2 DE were transferred. The results suggest that high survival rates can be obtained with the G1 technique, and splitting during biopsy can increase productivity in programs aimed to evaluate the genomic constitution of early stage embryos. Funded by the Bayerische Forschungsstiftung (AZ-1031-12).

scholarly journals Interferon tau secretion in cattle embryos in vitro fertilized before and after cryopreservation

2005 ◽  
Vol 57 (6) ◽  
pp. 752-756
Author(s):  
M.C.C. Araújo ◽  
V.R. Vale Filho ◽  
A.M. Ferreira ◽  
W.F. Sá ◽  
J.B. Barreto Filho ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Media Culture ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Individual Culture ◽  
Frozen Embryos ◽  
Before And After ◽  
The Media ◽  
Fresh Embryos

The effect of cryopreservation in IFN-tau, from bovine embryos produced in vitro was evaluated. Two treated groups (G1= fresh bovine embryos, n=59 and G2= freezed embryos, n=84) were used to study the effect of cryopreservation on IFN-tau secretion. After reaching the blastocyst phase, the embryos were kept on individual culture for additional period of 7 days. On days 3 and 7 after the beginning of embryos cultivation, samples of the media culture were taken for IFN-tau secretion titration. Oocysts taken from follicles ranging from 3 to 5mm in diameter were obtained from ovaries of females at slaughterhouse. The embryos were frozen, after being dehydrated with ethylene glycol (1.8m), conditioned on 0.5ml palletes and frozen. Frozen embryos secreted lower IFN-tau than fresh embryos (P<0.05). At day 7 it was registered higher IFN-tau secretion from trophoblast than at day 3 (P<0.05). The increasing of IFN-tau secretion was observed when the blastocyst began to longed and it was directly related to the embryos development. The synthesis of IFN-tau is related to the capability of development of the blastocyst. Cryopreservation is a method that affects the maternal recognition of pregnancy and the post-freezing embryo development.

scholarly journals 183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 213 ◽  
Author(s):  
J. Small ◽  
M. Colazo ◽  
D. Ambrose ◽  
R. Mapletoft ◽  
J. Reeb ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Animal Health ◽  
Beef Cows ◽  
Water Bath ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Frozen Embryos

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

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 Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

2020 ◽  
Vol 36 (3) ◽  
pp. 251-270
Author(s):  
Van Do ◽  
Andrew Taylor-Robinson
Keyword(s):  
Slow Rate ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cattle Industry

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

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.

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