Transfer Of Frozen-thawed Bovine Embryos Research Articles

Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management.

Embryo transfer entails many procedures and techniques, of which embryo freezing is an important component in bovine embryo transfer. Embryo freezing techniques have been developed over the last 40 years, allowing practical availability, and have become essential for cattle reproduction management under field conditions. The direct transfer methods of frozen-thawed, in vivo-derived, and in vitro-produced (IVF) bovine embryos using 1.5 M ethylene glycol (EG) with or without sucrose (SUC) are used widely under on-farm conditions, not only in Japan but also globally. The direct transfer method using 1.5 M glycerol (GLY) and 0.25 M SUC (GLY-SUC) is used mainly in Japan. The pregnancy rate with direct transfer of frozen-thawed bovine embryos in either EG or GLY-SUC has been found to not differ from conventional freezing with GLY and traditional dilution techniques. Pregnancy rates following direct transfer of frozen-thawed bovine embryos were affected by the developmental stage of the embryos and the parity of the recipients. The use of ultrasound-guided on-farm ovum pickup is ushering in a new revolution for the commercial application of IVF embryos. Globally, for the first time more IVF bovine embryos were transferred in 2017 than produced in vivo. More than 60% of IVF embryos were transferred fresh due to a low pregnancy rate of frozen-thawed IVF embryos. Many factors seemed to be involved in improving the survival rate of frozen-thawed IVF embryos. Therefore, further research is needed to improve the freezing tolerance of IVF embryos to develop efficient direct transfer methods analogous to those used for in vivo embryos.

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

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.

牛凍結胚の子宮角への移植部位と受胎率

牛凍結胚の子宮角への移植部位と受胎率

Birth of calves after direct transfer of thawed bovine embryos stored frozen in ethylene glycol

The present study investigated the use of ethylene glycol (EG) as a cryoprotectant before the direct transfer of frozen-thawed bovine embryos. Embryos at the morula to blastocyst stages collected on Days 6–8 (estrus designated Day 0) in phosphate buffered saline+20% newborn calf serum were placed into 1.8 M EG or in 1.8 MEG+0.25 M sucrose (EG+SUC), and equilibrated for 13–93 min at room temperature (20–25°C) or 37°C. Each embryo was then loaded into a 0.25 ml straw, and directly placed in the cooling chamber of a freezer precooled at −7°C. After 2 min at −7°C, the samples were seeded, then held for a further 8 min at −7°C, and cooled to −25 or −30°C at −0.3°C min −1 before being plunged into liquid nitrogen. Control embryos were also frozen with 1.4 M glycerol +0.25 M sucrose (GLY+SUC). Frozen embryos were thawed in a water bath at 37°C (EG and EG+SUC) or 20°C (GLY+SUC). After thawing the straws containing an embryo frozen in EG, EG+SUC or GLY+SUC were directly mounted into an embryo transfer gun and transferred to recipients without diluting of the cryoprotectants. The pregnancy rates were 69% ( 20 29 ), 52% ( 13 25 ) and 60% ( 15 25 ) for EG, EG+SUC and control medium (GLY+SUC), respectively. The pregnant recipient animals delivered healthy calves except for five which aborted. These results indicated that EG is a suitable cryoprotectant in which to store embryos before they are directly thawed and transferred to recipients.

Birth of live calves after transfer of frozen-thawed bovine embryos fertilised in vitro.

Follicular oocytes were aspirated from bovine ovaries collected at a local abattoir. The cumulus-intact oocytes were matured, fertilised and subsequently cultured in vitro. Of 2297 oocytes exposed to in vitro procedures during a 30-day experimental period, 92 per cent matured, 83 per cent were fertilised, 73 per cent cleaved, 48 per cent developed to the morulae and 14 per cent developed to the expanded blastocyst stage. During this experimental period, 300 similar embryos fertilised in vitro were frozen at different post in vitro block developmental stages. After approximately one year of storage in liquid nitrogen, 98 of these embryos were thawed and cultured either for up to four hours or for two days in tissue culture medium-199. Culturing the embryos for up to four hours was not as successful for in vitro development as culturing for two days. Of the 40 embryos cultured for two days, 67 per cent of early blastocyst stage embryos developed to expanded blastocysts in vitro and 46 per cent of morula stage embryos developed to expanded blastocysts, whereas only 8 per cent of 16-cell stage embryos developed to expanded blastocysts. A 50 per cent pregnancy rate resulted when frozen-thawed embryos were co-cultured for two days before transfer compared with 20 per cent for frozen-thawed embryos cultured for up to four hours before transfer. Five calves were born after a normal gestation period with birthweights ranging from 37.3 to 54.5 kg.

Direct Transfer of Frozen-Thawed Bovine Embryos Derived from In Vivo or <I>In Vitro</I> Fertilization Using Ethylene Glycol

Summary. Twenty six of in vivo embryos were collected from 7 Holstein cows and 1 Japanese black cows by single or superovulated flushing. Thirty one of in vitro embryos were produced using in vitro fertilization techniques using Japanese black oocytes aspilated from ovaries obtained local slaughter house. These embryos were suspended in 1.8 M ethylene glycol (EG) supplemented with 10% superovulated cows serum (scs) at 25 C and held 10 min, then embryos were loaded into 0.25 ml plastic straws, placed into an alcohol bath freezer at 0°C, cooled from 0°C to -7°C at 1°C/min, seeded, held for 10 min, cooled at 0.3 C/min or 0.5°C/min to -30°C. Straws were then plunged and stored in liquid nitrogen. After a 20 day storage period in liquid nitrogen, the cryopreserved straws were placed in air for 6 sec and plunged into 30°C water bath for thawing and transfered immediately into syncronized recipients. The pregnancy rates of 0.3°C/min [90% (9/10)] were significantly higher (P<0.01) than those of 0.5 C/min [12.5% (2/16)] for cooling rates in vivo embryos. And the pregnancy rates of in vitro frozen embryos were 63% (12/19).

Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes ( EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at ∼ 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.

Direct Transfer of Frozen-Thawed Bovine Embryos Freezed by Using Ethlene Glycol as a Cryoprotectant

Direct Transfer of Frozen-Thawed Bovine Embryos Freezed by Using Ethlene Glycol as a Cryoprotectant

Direct transfer of frozen-thawed bovine embryos

The ability to transfer frozen embryos directly to recipient females after thawing would be a convenience in embryo transfer and would facilitate marketing embryos produced in large quantities by in vitro procedures. Techniques for direct transfer of bovine embryos use sucrose as an osmotic buffer to remove glycerol while the embryo is in the straw prior to transfer or use sucrose added to the cryoprotectant solution allowing transfer without additional steps. These methods are reviewed as well as a new approach to freezing embryos for direct transfer. The new procedure for direct transfer of frozen embryos uses 1.5 M ethylene glycol as a cryoprotectant. Embryos frozen in ethylene glycol can be rehydrated directly in holding medium without step-wise dilution of the cryoprotectant yielding viability equivalent to that of embryos frozen in glycerol and rehydrated in a step-wise manner. In addition, the pregnancy rate for nonsurgically recovered embryos frozen in 1.5 M ethylene glycol in straws containing additional columns of holding medium, thawed and transferred directly to recipients (50%) was comparable to the pregnancy rate for embryos frozen in glycerol and rehydrated using a step-wise procedure prior to transfer. Ethylene glycol can be used effectively as a cryoprotectant for bovine embryos permitting thawed embryos to be rehydrated directly in holding medium or transferred directly to recipients. Identifying effective cryoprotectants to which bovine embryos are highly permeable is an alternative approach for developing direct transfer procedures.

Field trials of non-surgical transfer of frozen-thawed bovine embryos by improved one step straw method.

Field trials of non-surgical transfer of frozen-thawed bovine embryos by improved one step straw method.

Pregnancy rate after synchronous and asynchronous transfer of frozen-thawed bovine embryos

Day 7 bovine embryos, collected from superovulated donors, were frozen-thawed and those badly injured judging by morphological criteria were discarded. Then 127 of 153 embryos were transferred surgically to exactly synchronous recipients and to recipients +1 or −1 day out of phase. The highest pregnancy rate, 48%, was established after synchronous transfer. A marked drop in pregnancy (24.4%) resulted from the transfer of Day 7 embryos to Day 6 recipients. After transfer of Day 7 embryos to Day 8 recipients the pregnancy rate was still lower. It was concluded that freezing and thawing procedures to some extent decrease the capability of embryos to overcome the asynchrony and hence proper attention should be paid to the stage of the estrous cycle of the recipient relative to the age of the embryos.

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos

One impediment to the more widespread use of freezing as an adjunct to embryo transfer in cattle has been the method used to remove the protective compound from the thawed embryos. Recently, a method has been developed that permits bovine embryos to be diluted out of the protective solution within the plastic straw in which the embryos were originally rrozen and thawed. The method requires only about 10 minutes to perform and does not require a microscope or other laboratory equipment. Therefore, frozen bovine embryos can be thawed, diluted, and transferred nonsurgically into recipient cattle under conditions quite similar to those used for artificial insemination. A large number of field trials of this method have been performed during the past 2 1 2 years. A total of 327 pregnancies have been established by the nonsurgical transfer of 1259 embryos that had been frozen, thawed, and diluted by this method.

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. I. Basic studies

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. I. Basic studies

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. II. Applied studies

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. II. Applied studies