88 SURVIVAL AFTER FREEZING OF IN VITRO AND IN VIVO BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
C. Y. Choe ◽  
J. J. Son ◽  
H. J. Kim ◽  
...  
Keyword(s):  
Cell Number ◽  
Water Bath ◽  
Bovine Embryos ◽  
Estrous Cycles ◽  
Embryo Freezing ◽  
Frozen Embryos ◽  
In Vitro Survival ◽  
Medium Culture

The present study was conducted to investigate the survivability of post-thawed bovine embryos for direct transfer. Bovine ovaries were collected at a local slaughterhouse. The cumulus-oocyte complexes (COC) were aspirated from 2 to 8 mm antral follicles using a syringe with an 18-gauge needle. Selected COC were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 15 COC were matured for 22 h in 50-μL droplets of TCM-199 supplemented with 5% FBS, 10 μg mL-1 of LH, 10 μg mL-1 of FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Mature COC were fertilized with frozen-thawed semen treated with BO medium (Brackett and Oliphants Biol. Reprod. 12, 260-274). All oocytes and embryos were placed in CR1aa medium culture system for in vivo embryo production. The Korean native cows that were between days 8 and 12 of their estrous cycles were superovulated with 28 mg of porcine follicle stimulating hormone (FSH, Antorine-R10; Kawasaki Mitaka Pharmaceutical, Tokyo, Japan) in twice daily i.m. injections, with a gradual decrease over 4 days. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.5 M, and 1.8 M ethylene glycol(EG) was used as a cryoprotectant. Embryo was loaded into 0.25 mL straw and directly into a cooling chamber (CL-863, USA) and kept at -7°C for 10 min, including time for seeding, and further cooled to -35°C at -0.3°C. After 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in water bath at 35°C and 37°C. Embryos were evaluated at 24, 48, and 72 h post thawing. Embryos that survived were recorded as either blastocysts that had expanded or hatched at 24 h or had hatched at 72 h. All of the results were analyzed by ANOVA using the STATVIEW program. After frozen the blastocysts cultured without serum, better survivability for frozen embryos was seen in the 1.8 M EG with 0.5% BSA (bovine serum albumin) group than 1.5 M EG with 0.5% BSA (75.7 v. 72.7). The survivability of frozen-thawed embryos was significantly higher in the 37°C water bath than 35°C (85.7% v. 70.8%). However, there was no difference in the total cell number of thawed embryos (142 ± 13 v. 137 ± 12), and chromosome abnormality higher than in vivo frozen-thawed embryos. In conclusion, the results suggest that the thawing temperature at 37°C may be optimal for better in vitro survival of frozen-thawed embryos produced in vitro and in vivo. Furthermore, the data suggest that embryo freezing system may provide reasonable conditions for embryo transfer.

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 >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 >10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P>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>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<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

149 IN VITRO BOVINE EMBRYOS FROZEN BY DIRECT TRANSFER METHOD WITH ETHYLENE GLYCOL

2015 ◽  
Vol 27 (1) ◽  
pp. 166
Author(s):  
S. H. Kizil ◽  
M. Satilmis ◽  
N. Akyol ◽  
T. Karasahin
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Hatching Rate ◽  
Direct Transfer ◽  
Embryo Freezing ◽  
Frozen Embryos ◽  
Transfer Method ◽  
Phosphate Buffered Saline ◽  
In Vitro Survival

The objective of this study was to search for capability of freezing by ethylene glycol direct transfer method of in vitro-produced cattle embryos. Fifty-six in vitro-produced good-quality cow embryos were frozen by direct transfer method with ethylene glycol in this study. Cattle ovaries were collected from a slaughterhouse and oocytes were aspirated from follicles with 2 to 8 mm diameters. Then oocytes were let for maturation of 20 to 22 h in 100-μL microdroplets of TCM-199 with 0.1 mM β-mercaptoethanol and 20% FCS. After 5 to 6 h of fertilization in Bracket Oliphant (BO), they were cultured for 7 days in 100 µL of CR1aa medium with 5% FCS under 5% CO2, 98% relative humidity, and 38.5°C in a CO2 incubator. Embryos were equilibrated for 15 min in room temperature in 1.8 M ethylene glycol + 0.1 M sucrose in Dulbecco's phosphate buffered saline (D-PBS) supplemented with 20% FCS. Embryos were then loaded individually into a 0.25-mL straw and placed directly into a cooling chamber of a programmable freezer with methyl alcohol precooled to –7°C. After 2 min, the straw was seeded and maintained at –7°C for 8 min more. Then it was cooled to –30°C at 0.3°C min–1 before plunging into liquid nitrogen. The frozen embryos were thawed by allowing the straw to stand in air for 5 to 6 s and then immersing them in a 30°C water bath for 10 s. After thawing, embryos were transferred into TCM-199 + 0.1 mM β-mercaptoethanol + 20% FCS medium to check in vitro survival rates at 48 h post-thawing. The re-expansion and hatching rate of blastocysts was 64.28% (36 blastocysts). This result indicated that ethylene glycol can be used effectively for cow embryo freezing as a suitable cryoprotectant for direct transfer method.

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<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

171 LASER-ASSISTED ZONA PELLUCIDA HATCHING IN FROZEN - THAWED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 244 ◽  
Author(s):  
M. K. Chiasson ◽  
J. A. Carter ◽  
K. R. Bondioli ◽  
R. A. Godke ◽  
G. T. Gentry
Keyword(s):  
Calf Serum ◽  
Pregnancy Rates ◽  
Water Bath ◽  
Assisted Hatching ◽  
Direct Transfer ◽  
Pulse Treatment ◽  
Bovine Embryos ◽  
Hatching Rates

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo derived frozen-thawed bovine embryo hatching rates. In Exp. 1, direct-transfer beef cattle embryos were air-thawed for 15 s, placed in a 30°C water bath for 15 s, then held in TALP-HEPES, evaluated for stage and grade (1 = good to 3 = poor) and randomly applied to treatments. Embryos (n = 156) received either 2 or 3 symmetrical rents 40% through the outer zona surface using the XYClone diode laser (Hamilton Thorne, Beverly, MA, USA) at 90% power with a 600 μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were then cultured in vitro in CR1aa supplemented with 10% calf serum at 39°C in 5% CO2 and 5% O2 for 4 d. Embryo hatching rates were 47% for Treatment A and 53% for Treatment B. In Exp. 2, in vivo produced, nonsurgically collected direct-transfer Hereford embryos (n = 64) were utilized. In Exp. 3, in vivo produced nonsurgically collected glycerol frozen Brangus embryos (n= 46) were utilized. Embryos utilized in Exp. 2 and 3 were air-thawed for 15 s, placed in a 30°C water bath for 15 s, and then held in 1 M sucrose for 7 min. Embryos were then held in phosphate-buffered saline with 10% calf serum (Exp. 2) or ViGRO Holding Plus (Bioniche, Pullman, WA, USA) (Exp. 3), evaluated for stage and grade before being randomly assigned to either Treatment A or B. Embryos received either 3 symmetrical rents 40% through the outer zona surface using the XYClone laser at 90% power with a 600-μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were transferred nonsurgically (1 embryo/female) by the same technician into synchronized mixed breed recipient beef cows on Day 7 of the estrous cycle. Pregnancy status was determined at 35 days and 60 days via ultrasonography. In Exp. 2, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Also, treatment did not affect pregnancy rates at 35 or 60 days (41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively). Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Exp. 3, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Pregnancy rates at 35 and 60 days were not affected by treatment (65% and 65% for Treatment A and 76% and 59% for Treatment B, respectively). Calving rates for those recipients in Exp. 3 were not available at the time of abstract preparation. Based on the data presented herein, it does not appear that laser-assisted hatching with the XYClone laser increases the number of in vivo derived frozen-thawed embryos that hatch following in vitro culture or increase pregnancy rates after transfer to recipient cattle.

40 IN VITRO SURVIVAL RATES OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED BY SLOW CONTROLLED FREEZING OR VITRIFICATION

2012 ◽  
Vol 24 (1) ◽  
pp. 132 ◽  
Author(s):  
P. Rodriguez Villamil ◽  
D. Lozano ◽  
G. A. Bó
Keyword(s):  
Solid Surface ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Bovine Embryos ◽  
Controlled Freezing ◽  
In Vitro Survival ◽  
Hatching Rates

Although slow programmable freezing is currently the standard method for bovine embryo cryopreservation, vitrification has become an alternative for in vitro-produced embryos. A study was designed to compare the in vitro survival rates of in vivo- and in vitro-produced bovine embryos with 1 of 2 commercially available methods of cryopreservation: slow freezing and solid surface vitrification. In vivo-produced Grade 1 blastocysts (n = 210) collected from superovulated donor cows 7 days post-insemination and in vitro-produced Grade 1 blastocysts (n = 122) from slaughterhouse oocytes, produced with the procedure described by Chaubal et al. (2007 Theriogenology 67, 719–728) were randomly allocated in 2 groups. Group 1 (slow freezing) embryos were exposed to 1.5 M ethylene glycol (ViGro EG; Bioniche Animal Health USA Inc., Pullman, WA, USA) for 5 min and loaded in 0.25-mL plastic straws. The straws were placed in a Freeze Control CL 5500 freezer (CryoLogic, Victoria, Australia) at –6.5°C, seeded and after 10 min of equilibration, cooled at –0.6°C min–1 until –CE°C, before plunging into liquid nitrogen. Group B (vitrification) embryos were exposed to a AE% EG+0.BEM trehalose solution for A min and then into C0% EG+AM trehalose solution for C0 sec at room temperature to be vitrified using the CVM system (CryoLogic). The CVM used a cryohook and the solutions with the embryos are exposed to a metal solid surface cooled at –AIF°C. The vitrification solution was chosen after a toxicity test in which several EG and trehalose combinations were tested (Rodriguez Villamil et al. Ith IRAC Symposium, Argentina B0AA). After at least 1 wk of storage, embryos in the slow freezing groups were thawed in water bath at C0°C for AB s, placed in holding medium for E min and then cultured in SOF. Vitrified embryos were placed directly in a 0.BE M sucrose solution for E min (at CG°C) and then cultured in SOF medium. Re-expansion and hatching rates were evaluated at BD and GB h, respectively. Data was analyzed by nonparametric tests with type of embryo and cryopreservation procedure as main effects, using the software Infostat (UNC, Argentina, B0A0). In vivo-produced embryos had higher (P < 0.0A) re-expansion (AGI/BB0, HA% vs FI/ABB, EF%) and hatching rates (AEI/BB0, GB% vs EC/ABB, DC%) than in vitro-produced embryos, regardless of cryopreservation method. However, re-expansion (DE/FC, GA%) and hatching (CI/FC, FB%) rates were higher (P < 0.0A) with in vitro-produced vitrified embryos than in vitro-produced embryos in the slow freezing group (re-expansion: BD/EI, D0% and hatching: AD/EI, BD%). Although similar re-expansion rates (IC/AA0, HE% vs HF/A00, HF%) were obtained with in vivo- produced embryos cryopreserved by the 2 systems, hatching rates tended to be lower (P = 0.0I) with in vivo-produced embryos that were vitrified compared with slow freezing (GH/AA0, GA% vs HA/AA0, HA%). In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro-produced embryos compared with the conventional slow, controlled freezing procedure.

scholarly journals MATURAÇÃO E FECUNDAÇÃO IN VITRO DE OÓCITOS BOVINOS EM TUBOS PREVIAMENTE GASEIFICADOS E MANTIDOS EM BANHO-MARIA

2002 ◽  
Vol 7 (2) ◽  
Author(s):  
M. KURTZ FILHO ◽  
L. M. SILVA ◽  
B. MOREIRA ◽  
D. S. BRUM ◽  
F. G. LEIVAS ◽  
...  
Keyword(s):  
Water Bath ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Embryo Production ◽  
Vitro Production

A produção in vitro (PIV) de embriões bovinos alcançada com vacas de matadouros ou de aspiração folicular in vivo (OPU) é uma prática cada vez mais difundida e a sua simplificação poderia baixar os custos de produção. O objetivo desta pesquisa foi comparar a produção in vitro de embriões em estufa com temperatura, umidade relativa e atmosfera controlada (controle), com tubos de poliestireno gaseificados e mantidos em banho-maria (tratamento). Oócitos obtidos de ovários de vacas abatidas foram maturados in vitro em TCM- 199 modificado com 25mM de N-2-hidroxietilpiperazina-N -2-ácido etanosulfônico (HEPES); 0,025mg/ml de piruvato de sódio, 0,01UI de rFSHh/ml, 0,5µg/ml de LHs e 10% de soro de vaca em estro. Na fecundação in vitro utilizou-se Talp-Fert com 0,06mg/ml de albumina sérica bovina, 0,022mg/ml de piruvato de sódio e 10µg/ml de heparina. O cultivo foi conduzido em placas de 4 poços em SOF com 5% de soro de vaca em estro, 20µl/ml de aminoácidos essenciais e 10µl/ml de aminoácidos não essenciais, sob óleo mineral, em estufa com atmosfera de 5% de CO2, umidade saturada e 39°C, por 9 dias. Na maturação não houve diferença (P>0,05) entre o tratamento e o controle. Porém, a maturação e a fecundação ou somente a fecundação in vitro em tubos mantidos em banho-maria não demonstrou ser uma alternativa recomendada para a produção de embriões bovinos. In vitro maturation and fertilization of bovine oocytes in tubes previously gasified kept in water bath Abstract In vitro bovine embryo production either obtained from oocytes of slaughtered cows or in vivo follicular aspiration (OPU) is a well-known technique and it’s simplification might reduce the cost of embryo production. The aim of this study was to compare the cleavage rate and embryo development of the in vitro production of bovine embryos using standard culture system (temperature, gas phase and controlled humidity) versus gasified polystyrene tubes kept in water bath. Oocytes obtained from ovaries of slaughtered cows were in vitro maturated in TCM- 199’modified with 25 mM of N-2-hidroxyethylpiperazine-N’-2-ethanosulfonic acid (HEPES); containing 0.01UI rFSHh/ml and 0.5µg/ml LHs, 0.025mg/ml sodium pyruvate and 10% estrous cow serum. The in vitro fertilization was carried out in Talp-Fert containing 0.06mg/ml BSA, 0.022mg/ml sodium pyruvate and 10µg/ml heparin. The culture was performed in SOF medium with 20µl/ml essential aminoacids, 10µl/ml, non-essential aminoacids and 5% estrous cow serum, with oil overlay, in 4 well dishes and incubated with 5% CO2, maximum humidity at 39°C, for 9 days. The results of this study showed no difference (P>0.05) between the treatment and control groups during the maturation process. However, the maturation and fertilization or only the fertilization in tubes do not represent a viable alternative for the in vitro production of bovine embryos.

Relaxation of Orthodontic Elastomeric Chains and Modules In Vitro and In Vivo

1978 ◽  
Vol 57 (5-6) ◽  
pp. 685-690 ◽  
Author(s):  
J.L. Ash ◽  
R.J. Nikolai
Keyword(s):  
Water Bath ◽  
Dry Air ◽  
Initial Activation ◽  
Elastomeric Chains

Relaxation patterns for two orthodontic polyurethane-based elastics have been quantified in dry air and water bath environments and in vivo. Water bath simulation of in vivo behavior is apparently valid for up to a week following initial activation, but it becomes somewhat erroneous thereafter.

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