Intrauterine embryo transfer with canine embryos cryopreserved by the slow freezing and the Cryotop method

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P < 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos

Download Full-text

Physical health of singleton children born after frozen embryo transfer using slow freezing: a 3-year follow-up study

Human Reproduction ◽

10.1093/humrep/dev203 ◽

2015 ◽

Vol 30 (10) ◽

pp. 2411-2418 ◽

Cited By ~ 15

Author(s):

S. Pelkonen ◽

M. Gissler ◽

S. Koivurova ◽

S. Lehtinen ◽

H. Martikainen ◽

...

Keyword(s):

Physical Health ◽

Embryo Transfer ◽

Frozen Embryo Transfer ◽

Slow Freezing ◽

Follow Up Study

Download Full-text

Transcervical intrauterine embryo transfer (ET) with a new everting catheter and “peel-back” technique

Journal of Assisted Reproduction and Genetics ◽

10.1007/bf02066182 ◽

1996 ◽

Vol 13 (5) ◽

pp. 452-455

Author(s):

C. C. Zoll ◽

O. Bauer ◽

S.Al Hasani ◽

M. Kaisi ◽

Ch. Diedrich ◽

...

Keyword(s):

Embryo Transfer ◽

Intrauterine Embryo Transfer

Download Full-text

70 ONE-STEP CRYOPROTECTANT DILUTION FOLLOWING VITRIFICATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab70 ◽

2013 ◽

Vol 25 (1) ◽

pp. 182

Author(s):

R. Morató ◽

T. Mogas

Keyword(s):

Embryo Transfer ◽

Statistical Significance ◽

Survival Rates ◽

Blastocyst Stage ◽

Slow Freezing ◽

Embryo Cryopreservation ◽

Dilution Method ◽

Bovine Embryo ◽

One Step

Although slow freezing continues to be the most widely used technique of cryopreservation for bovine in vivo- and in vitro-produced embryos, vitrification has been tested in different species with good results, especially when dealing with in vitro-produced embryos. Vitrification represents a minor expense in time and equipment associated with cryopreservation compared with conventional slow freezing. However, vitrification, which is the most common method for human embryo cryopreservation, has not been widely adopted by embryo-transfer practitioners for commercial use in cattle. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting, and it is difficult to perform in the field. The objective of this study was to develop a one-step dilution method suitable for one-step bovine embryo transfer using the cryotop vitrification method. Embryos produced in vitro by standard procedures were vitrified at the blastocyst stage at Day 7 post-insemination in a mixture of 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose using cryotop devices. Embryos were randomly assigned to 1 of 3 warming methods: (1) W3: warming was carried out following the cryotop method (1 M sucrose for 1 min, 0.5 M sucrose for 3 min, and 0 M sucrose for 6 min); (2) W1/0.5: embryos were warmed directly in 0.5 M sucrose for 3 min; and (3) W1/0: embryos were warmed directly in 0 M sucrose for 5 min. Survival rates were assessed in terms of blastocyst re-expansion, hatching, and hatched status at 3 and 24 h after warming. Data were analyzed using the statistical analysis systems package (SAS, v9.1). Data from at least 3 replicates were collected. Comparisons of vitrified–warmed blastocyst survival rates between groups were performed using the chi-squared test. The level of statistical significance was set at P < 0.05. When embryo survival was evaluated at 3 h postwarming, embryos warmed using the 3-step dilution protocol and those warmed directly in 0.5 M sucrose showed higher percentages of survival (W3: 89.8%, n = 98; W1/0.5: 87.5%, n = 64; P < 0.05) than those blastocysts that were warmed directly in 0 M sucrose (W1/0: 66.4%, n = 146). However, similar rates irrespective of the warming procedure were observed at 24 h postwarming (W3: 85.7%, W1/0.5: 88.2%, W1/0: 70.5%). Warmed in vitro-produced embryos exposed to W3 (47.6%) and W1/0.5 (35.6%) achieved higher percentages of embryos developing to the hatched blastocyst stage after 24 h of culture than those embryos warmed in W1/0 (20.4%; P < 0.05). Our results indicate that direct warming and dilution of cyotop-vitrified embryos in 0.5 M sucrose for 3 min may enable one-step bovine embryo transfer without requirement of a microscope or other laboratory equipment, simplifying the embryo-transfer procedure of vitrified embryos on farm at the same level of complexity as carrying out AI. Support came from Spanish MEC (RZ2010-00015-0-00; AGL2010-19069) and Generalitat de Catalunya (2009 SGR 621).

Download Full-text

84 Effect of Embryo Stage and Cryopreservation Method on Pregnancy Rates Obtained Following the Transfer of In Vivo-Derived Ovine Embryos on Small-Scale Farms in Thailand

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab84 ◽

2018 ◽

Vol 30 (1) ◽

pp. 181

Author(s):

S. Khunmanee ◽

J. Suwimonteerabutr ◽

M. Techakumphu ◽

T. Swangchan-Uthai

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Blastocyst Stage ◽

Pregnancy Rates ◽

Slow Freezing ◽

Small Scale ◽

Tropical Conditions ◽

Frozen Embryos ◽

Embryo Stage ◽

Cryopreservation Method

Assisted reproductive technologies including superovulation, laparoscopic AI (LAI), and embryo transfer (ET) are important tools for genetic improvement in the sheep industry. The present study aimed to determine the effects of embryo stage and cryopreservation method on field trial outcomes of embryo transfer on small-scale farms in Thailand. Black Dorper ewes (n = 16) were used as donors and mixed breed ewes (n = 21) were used as recipients. Donors were superovulated as previously described (Tríbulo et al. 2012 Theriogenology 77, 1679-1685, 10.1016/j.theriogenology.2011.12.013) and inseminated by LAI within 22 to 24 h after standing heat (Day 0). Donors females were flushed on Day 2 to recover 2- to 8-cell embryos (n = 8) or on Day 6 to recover blastocyst-stage embryos (n = 8). Recovered embryos were randomly cryopreserved by either slow freezing or vitrification methods. Into 21 recipients was inserted an intravaginal device impregnated with CIDR® that was left in place for 10 days. Those received 300 IU of pregnant mare serum gonadotropin (PMSG) on Day 9 and randomly assigned to receive embryos on either Day 2 or 6 after oestrus. Two- to 8-cell embryos were thawed and transferred into the ipsilateral oviduct (n = 1-5 embryos/recipient) on Day 2. Five recipients received 15 slow-frozen embryos and 5 received 13 vitrified embryos. Blastocyst stage embryos were thawed and transferred into the ipsilateral uterine horn (n = 1-4 embryos/recipient) on Day 6. Five recipients received 11 slow-frozen embryos and 4 received 7 embryos. Pregnancy diagnosis was determined by ultrasonography 45 days after embryo transfer. Pregnancy rate was calculated as the proportion of ewes with at least one pregnancy out of the total number of ewes that received embryos. Chi-squared analysis was used to determine the effects of embryo freezing technique and embryo stage on pregnancy rate (SAS 9.2, SAS Institute Inc., Cary, NC, USA). All donor ewes responded to the superovulation program (donor had 3 to 6 corpora lutea). The mean number of viable embryos recovered was 4.3 ± 2.4 and 3.1 ± 3.7 for Day 2 and Day 6, respectively. Nineteen of the 21 recipient ewes responded to the synchronization program and received embryos in the study. There was no effect (P > 0.05) of embryo stage (5/10 = 50% v. 3/9 = 33.3% for 2- to 8-cell v. blastocyst, respectively) or cryopreservation method (4/10 = 40% v. 4/9 = 44.4% for slow-freezing and vitrification, respectively) on pregnancy rate following embryo transfer. Results of the present study suggest that similar pregnancy rates following embryo transfer in sheep under tropical conditions in Thailand can be obtained using either 2- to 8-cell embryos or blastocyst-stage embryos and with embryos that have been cryopreserved by slow-freezing or vitrification. Further research with larger numbers of animals is necessary to confirm the preliminary results of the present study.

Download Full-text