Recap: AETA/AASRP 2019 Small Ruminant Embryo Transfer Seminar

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Published on: July 26, 2019

The AETA/AASRP 2019 Small Ruminant Embryo Transfer Seminar was held June 19 to 22, 2019, at The Ohio State University Large Animal Services satellite veterinary teaching hospital in Marysville, Ohio. The meeting was organized by Dr. Eric Gordon.

The course started on Wednesday, June 19, with a review of small ruminant reproductive physiology by Dr. Sherri Clark. Dr. Bill Croushore and Dr. Dave Dixon discussed embryos processing, grading, and cryopreservation. Drs. Mattes and Shipley discussed embryo collection, anesthesia, and sync methods as well as more reproductive physiology and handling of semen. Later in the day, a goat was surgically flushed as a demonstration.

On Thursday, June 20, and Friday, June 21, course participants broke out into teams of three and flushed three goats or sheep each day. All flushing occurred under gas anesthesia. After each flush, the teams searched embryos, and viable embryos were cryopreserved. Later in the day, Dr. Shipley discussed semen collection and cryopreservation. He also elaborated on reproductive physiology. On Saturday, June 22, teams of three practitioners each laparoscopically inseminated three ewes.

The meeting went very well, and it is felt that participants were quite satisfied with the value of this course. Dr. Eric Gordon at OSU CVM at Marysville deserves a huge thank you for his efforts in bringing this course together and in convincing clients to provide animals to flush. Dr. Justin Kieffer with OSU Animal Science was a huge help in bringing in his technician staff and in assisting with planning for animal usage in this CE meeting and for providing, via the animal science department, some of the animals used.

Preliminary trials of a specific gravity technique in the determination of early embryo growth potential†

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Published on: July 26, 2019

Read full article here

S.D. Prien,1,2,* C.E. Wessels,2 and L.L. Penrose1

. 2015 Sep; 30(9): 2076–2083.
Published online 2015 Jul 22. doi: 10.1093/humrep/dev178
PMCID: PMC4542720
PMID: 26202920

Abstract

STUDY QUESTION

Can a modified specific gravity technique be used to distinguish viable from nonviable embryos?

SUMMARY ANSWER

Preliminary data suggests a modified specific gravity technique can be used to determine embryo viability and potential for future development.

WHAT IS KNOWN ALREADY

Single embryo transfer (SET) is fast becoming the standard of practice. However, there is currently no reliable method to ensure development of the embryo transferred.

STUDY DESIGN, SIZE, DURATION

A preliminary, animal-based in vitro study of specific gravity as a predictor of embryo development using a mouse model.

PARTICIPANTS/MATERIALS, SETTING, METHODS

After a brief study to demonstrate embryo recovery, experiments were conducted to assess the ability of the specific gravity system (SGS) to distinguish between viable and nonviable embryos. In the first study, 1-cell mouse embryos were exposed to the SGS with or without previous exposure to an extreme heat source (60°C); measurements were repeated daily for 5 days. In the second experiment, larger pools of 1-cell embryos were either placed directly in culture or passed through the SGS and then placed in culture and monitored for 4 days.

MAIN RESULTS AND THE ROLE OF CHANCE

In the first experiment, viable embryos demonstrated a predictable pattern of descent time over the first 48 h of development (similar to previous experience with the SGS), while embryos that were heat killed demonstrated significantly altered drop patterns (P < 0.001); first descending faster. In the second experiment, average descent times were different for embryos that stalled early versus those that developed to blastocyst (P < 0.001). Interestingly, more embryos dropped through the SGS developed to blastocyst than the culture control (P < 0.01).

LIMITATIONS, REASONS FOR CAUTION

As this is a preliminary report of the SGS technology determining viability, a larger embryo population will be needed. Further, the current in vitro study will need to be followed by fecundity studies prior to application to a human population.

WIDER IMPLICATIONS OF THE FINDINGS

If proven, the SGS would provide a noninvasive means of assessing embryos prior to transfer after assisted reproductive technologies procedures, thereby improving fecundity and allowing more reliable SET.

STUDY FUNDING/COMPETING INTEREST(S)

The authors gratefully acknowledge the funding support of the U.S. Jersey Association, the Laura W. Bush Institute for Women’s Health and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University. None of the authors have any conflict of interest regarding this work.

TRIAL REGISTRATION NUMBER

none.

Keywords: embryo development, embryo selection, embryo viability, specific gravity, buoyance, noninvasive, zygote, blastocyst

Comparison of iSperm to current accepted methods for raw semen analysis

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Published on: April 17, 2019

Download the poster

New Module on Frozen Semen Evaluation Available

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Published on: December 27, 2018

The AETA Education Committee has partnered with Brad Stroud, DVM, and is pleased to announce that a new educational module is available to AETA members.

To access the new Frozen Semen Spot Test, please log in to the AETA site and follow this link: https://www.aeta.org/edu-teaching-module-frozen-semen.asp. You can view the module in your browser, on your phone, or download it (it is a large file).

Thank you to Brad Stroud, DVM.

 

8 Questions You May Have About Cryopreserving Bovine In Vivo–Derived Embryos

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Published on: December 27, 2017

John F. Hasler
Jfhasler05@msn.com
John.hasler@vetoqinol.com
Cell: 970-222-5302

Dr. Pat Comyn, the new chair of the AETA Education Committee, asked me to write a short piece clarifying some issues concerning the cryopreservation of bovine embryos for inclusion in the December issue of A Closer Look. The AETA has come a long way since our humble beginnings in 1983, and our 2017 membership now totals 556, including a large increase in the number of new members. The following facts and suggestions will be of most interest to our new and less experienced members. Not only are there many variables involved in successfully freezing and thawing bovine embryos, there also are many variations on most of the steps that do not notably detract from success rates. Having worked with many ET practitioners in 17 different states and a number of foreign countries, I have a pretty good idea of what works well and what does not. The following points are either based on published data that I deem to be replicable or based on my own experience and observations. Please feel free to contact me should you want advice or clarification.

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VitaFerm Article: Preparing Cows for Embryo Transfer

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Published on: December 27, 2017

Prepare Cows for Embryo Transfer

Embryo transfer (E.T.) is an important tool to propagate outstanding genetic influence within the herd, with the potential to produce multiple offspring of the same mating in the same year. Because of the time, labor and expense involved in creating these genetics, we reached out to Trans Ova Genetics, a leader in reproductive technologies, to provide useful information to prepare your cows for a successful E.T. program.

According to Trans Ova, successful E.T. programs require intensive management and attention to detail. The results you achieve are highly variable and the level of success is based on your ability to manage all aspects of the operation.

Get Your Recips Ready

Preparing recipient cows for their role of carrying and growing the embryo is not a lot different than preparing cows to be bred naturally. You want to keep them in a low-stress environment, be consistent in daily management practices, give all vaccinations prior to estrus and make sure their nutrition program is supplemented with high levels of trace minerals like copper, zinc and manganese that impact reproductive success.

“Nutrition is without a doubt one of the most important areas of donor and recipient management,” said Jon Schmidt, DVM and Chief Operations Officer at Trans Ova. “First of all, I believe the nutritional management of your cattle needs to be a year-long process. Attention should be placed on meeting their demands for the entire season including gestation and lactation.”

The most critical and demanding time however, includes the month before calving through the first three to four months after calving. This is the most stressful and nutritionally demanding time to allow that cow to produce a healthy calf via colostrum production, begin lactation to raise that calf and become pregnant.

Reproduction is not an essential process in survivability of that cow, and consequently suffers first if nutritional needs are not met. Maintenance and milk production will partition available energy supplies with reproduction suffering at their expense. Therefore, it is critical to meet their requirements. Ensure cows are fed a high-quality mineral especially one that optimizes zinc, selenium and copper as they are critical for successful embryo transfer outcomes. Avoid rations that are high in distiller’s byproducts or sulfur-containing forages. Avoid diets high in Urea.

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Ultrasonography and Embryo Transfer Workshops at the University of Saskatchewan

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Published on: March 21, 2017

Ultrasonography and Embryo Transfer Workshops are being conducted in tandem May 7 – 12, 2017 at the Western College of Veterinary Medicine, University of Saskatchewan with the intention of providing essential knowledge and hands-on experience for veterinary practitioners, research scientists and graduate students. The Ultrasound Workshop is a two and a half-day course covering principles of ultrasonography and equipment operation and imaging, imaging of the reproductive tract in large animals, and OPU/IVF. The Embryo Transfer Workshop is a three and a half-day course covering all aspects of embryo transfer technology in cattle.

Principal instructors for the Ultrasonography Workshop are Drs. Gregg Adams and Jaswant Singh and principal instructors for the Embryo Transfer Workshop are Drs. Reuben Mapletoft, Marcos Colazo, Gregg Adams and Jaswant Singh. Both workshops will involve lectures and hands-on lab sessions. Participants may register for one or both of the workshops, or for individual days.

For more information, contact Reuben Mapletoft (reuben.mapletoft@usask.ca), tel: (306) 222-6152, or Gregg Adams (gregg.adams@usask.ca), tel: (306) 966-7411.

To register contact: Jackie Bahnmann (Jackie.bahnmann@usask.ca), tel: (306) 966-7108, fax: (306) 966-8747.

http://www.usask.ca/wcvm/news_events/events/conferences/wcvm-ultrasound-and-embryo-transfer-workshops.php

Ask John: Question on freezer malfunction

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Published on: September 13, 2016

By John F.Hasler

A few years ago I was asked to change my newsletter column from “Ask John” to “Evidence Based ET”.  However, for this Sept. 2016 edition we are back to some questions submitted to me for which there are not really any hard data available.

For our younger members, some background on my experience in freezing embryos follows. In 1977, my first year out in commercial ET practice after finishing my post-doc at CSU, I flew to Cambridge, England and met with Dr. Steen Willadsen. One year earlier in 1976, Steen had published a paper that described freezing sheep embryos followed by the production of pregnancies after the embryos were thawed and transferred. This followed the first report of successful freezing of mammal embryos (mice) in 1972 by Whittingham, Leibo and Mazur. During my visit to Cambridge, Steen showed me how to make ampules from glass tubing and how to make freezing medium containing DMSO as the cryoprotectant.  I came home enthusiastic and optimistic, but spent a couple of very frustrating years with ampules blowing up not infrequently upon at thawing and very few embryos appearing to survive. We finally gave up on slowing lowering the ampules into the neck of a liquid nitrogen tank and Alan McCauley and I paid $12,000 (equivalent to about $30,000 in today’s dollars) for a Planer freezer.  However, we still had to make our own freezing media and the recommended freezing program was over 3 hours long.  Lastly, the biggest mistake we made for several more years was to transfer the very best embryos into available recipients and freeze what was left, often late at night after we returned to home base.  That old Planer freezer was much too big and unwieldy to haul around from farm to farm.

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Recipient Management and Synchronization: A Veterinarian’s Perspective

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Published on: December 16, 2015

by Pat Comyn, Madison, VA

 

Introduction

Successful embryo transfer comprises two somewhat independent components. The first component is to produce or recover grade and select embryos for immediate placement or cryopreservation. The second component is to transfer the selected fresh or frozen embryos into fertile recipients and obtain viable pregnancies and eventually calves.

This section will focus on the second component, primarily recipient selection and management and use of appropriate protocols to match a recipient’s endometrium with the chronological age of the embryo being transferred.

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Evidence-based ET: What is the best protocol for cryopreserving IVF-derived bovine embryos?

Evidence-based ET: What is the best protocol for cryopreserving IVF-derived bovine embryos?

John F. Hasler

Note: The title of this column was suggested by our AETA board of directors

There has been concern regarding the best way to cryopreserve bovine IVF-derived embryos ever since commercial in vitro embryo production (IVP) started in the early 1990s. Donors in the early days were primarily infertile, problem donors, and annual IVP embryo production in the United States was limited to a few thousand embryos, at most. Production of IVP embryos has increased substantially in recent years, and in 2013, it was reported that 48,112 embryos were produced from OPU collections compared with 301,671 in vivo embryos collected from superovulated cattle. Thus, 13.8% of the total embryos produced were from IVF procedures and 55% of them were reported to have been frozen. It is anticipated that reported IVP production will be substantially higher in 2014. Understandably, the companies providing IVF services are reluctant to share details of their cryopreservation services. However, because fresh IVP embryos are often shipped overnight to ET practitioners/donor owners for transfer on-farm, embryo numbers sometimes exceed the number of available recipients. Consequently, not infrequently, practitioners are faced with cryopreserving leftover IVP embryos. Even today, however, there does not seem to be any widely agreed upon, best protocol for cryopreserving IVP embryos. In the last few years it has been publically reported that IVP embryos have been commercially cryopreserved by vitrification and slow freezing with both ethylene glycol (EG) and glycerol used as the cryoprotectants.

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Evidence-based ET: What is the best synchrony between IVP embryos and recipients?

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Published on: September 10, 2014

Evidence-based ET

John F. Hasler

Note: I have been using subjects suggested by our board of directors for this column for the past two years.  I would be happy to attempt covering any subjects that the membership might suggest. Let me hear from you!

What is the best synchrony between IVP embryos and recipients?

There is a great deal of evidence that synchrony between the age of in vivo-derived embryos and the day of the estrous cycle in recipients at the time of transfer is affected very little, if at all, when synchrony is within the three day period of 0, plus one or minus one day (zero, meaning day of estrus and age of embryo are the same).  Consequently, the pregnancy rate is not affected when day 7 embryos are transferred into day 6, 7, or 8 recipients.  This holds true for both fresh and frozen in vivo-derived embryos (Hasler, 2001).

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Evidence-based ET: Does the inclusion of sucrose in EG freezing medium improve embryo survival and pregnancy rates?

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Published on: June 17, 2014

Evidence-based ET

John F. Hasler

Does the inclusion of sucrose in EG freezing medium improve embryo survival and pregnancy rates?

As pointed out previously in this column, efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as has been the case for some years now, more than 70% of embryos collected in the US are frozen, while fewer than 30% are transferred fresh. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.

Several companies provide 1.5 M EG freezing media with or without the inclusion of 0.1 M sucrose. The question posed here is whether including sucrose in the freezing medium makes any difference. In the normal range of ambient temperatures under which most embryos are placed in freezing media and loaded into straws, sucrose does not enter the cells.  Although sucrose readily diffuses through the zona, it remains outside the cells and, at the relatively low concentration of 0.1 M, exerts a mild osmotic imbalance, thereby pulling some water out of the cells. Whether this improves embryo survival is in question and may, in fact, be somewhat related to embryo stage of development. (more…)

Evidence-Based ET: Where does the transferred embryo have to be placed in order to maximize pregnancy rate?

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Published on: March 13, 2014

Note: This is the fourth in the series of topics suggested by the AETA Board of Directors. Should any readers want to suggest future topics, please email them to me.

John F. Hasler

Jfhasler05@msn.com

Where does the transferred embryo have to be placed in order to maximize pregnancy rate?

Well, we know where a bovine embryo belongs when it first travels through the UT junction!  Initially, it will be in the tip of the uterine horn, and if the embryo resulted from fertilization of the animal’s own ovulated oocyte, then the embryo will be in the horn on the same side (ipsilateral) as the CL. However, that is not necessarily where the embryo will end up and implant.  Embryo migration within the uterus is well-known in litter producing domestic animals, such as swine, but it also has been shown to occur in single ovulating cattle at a frequency of 2 to 3% (for review see McMillan and Peterson, 1999), although Scanlon (1972) reported no trans-uterine migration in more than 600 single ovulating cows. He did, however, note that a series of previous published reports indicated a migration percentage of 0.3 to 1.6%. Migration has not been reported in cattle following the non-surgical transfer of a single embryo transferred to the horn either to the ipsilateral or contralateral to the ovary with the CL, although the sample sizes were small. (more…)

Evidence-Based ET: Does corpus luteum (CL) quality in bovine ET recipients matter?

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Published on: December 19, 2013

John F. Hasler

Yes, of course it does! Obviously, however, the accuracy of this statement depends on the definition of CL ‘quality’. In many cases, assessing the ‘quality’ of the corpus luteum is not a particularly accurate endeavor whether it is based on gross morphology, rectal palpation or ultrasound examination. When considering the outcome of ET in bovine recipients, the most important single criterion involving the CL is whether or not there is sufficient progesterone to support a pregnancy. Based on this, and for the purposes of this discussion, I am defining CL ‘quality’ as being related to progesterone secretion.  However, it is not practical to determine progesterone levels in recipients at the time of ET.  In addition, numerous studies have demonstrated that there is a very wide range of progesterone levels at the time of ET that successfully support pregnancy. Consequently, ET usually is based on physical characteristics of the CL at the time of transfer.

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Evidence-based ET: Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

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Published on: April 30, 2013

Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

John F. Hasler

Efficient and efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as shown by the most recent AETA statistics (2011), 72% of embryos were frozen following collection versus only 28% that were transferred fresh into recipients. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.

During the past 20 years there has been a continuing debate among ET practitioners regarding the question of whether EG is more toxic than glycerol to bovine embryos. This concern has led some practitioners to limit exposure time of embryos to EG for a maximum of 5 min prior to chilling and seeding. I am not aware of any published reports showing that EG is any more toxic to bovine embryos than is glycerol. Voelkel and Hu (1992) reported that 100% of IVF blastocysts exposed for 20 min to 1.5M EG or 1.5M propylene glycol were viable 48 h after being rehydrated in holding medium and cultured in vitro. The authors’ interpretation was that “neither of the cryoprotectants was overtly toxic to bovine embryos”. Also, in the original US patent filed by Voelkel in 1992, an exposure time of 10 to 20 min to EG prior to freezing, at a temperature of 18° to 25°C, was recommended.

Two studies demonstrated that exposure of in vitro-produced (IVP) embryos to EG for up to 40 min (Hasler et al., 1997) or 60 min (Takagi et al., 1993a) prior to freezing did not decrease survival rate of embryos following thawing and return to in vitro culture.  Obviously, there is also embryo exposure to EG after thawing and prior to transfer. In an additional study, Takagi et al. (1993b) reported no differences in the survival of IVP embryos exposed to 1.8M EG for 15 min, frozen in a direct transfer protocol and then thawed and held in the straws for <1, 10 and 30 min at 20-25°C before being cultured in vitro. Matoba, et al. (2004) also examined the effect of EG exposure after thawing of IVP embryos and included temperature as another factor.  There were no differences in embryo survival after 0, 10, 20 or 30 min exposure at 26.0°C or of 0, 10 or 20 min exposure at 38.5°C. However, at 38.5°C embryo survival decreased when exposure time exceeded 30 min.  Lastly, a recent study in Argentina that involved large numbers of in vivo-derived embryos exposed to EG for different periods of time prior to freezing and then in vitro culture after thawing failed to detect an influence of exposure time (Tribulo, et al., 2012). Specifically, blastocyst re-expansion and hatching rates were similar for embryos exposed to EG for 5, 10, 20 and 30 minutes

Enough on in vitro culture, how about pregnancy results of actual transfers into recipients?

Martinez, et al. (2002) reported that there was no significant difference in the pregnancy rates or calving rates of recipients after transfer of in vivo-derived embryos frozen for DT after 5 min versus 20 min exposure to EG.  A study in Japan resulted in very similar pregnancy rates after transfer of embryos exposed to EG for periods ranging from 5 to 45 min compared to embryos frozen in glycerol that were thawed and then diluted in a step-wise protocol (Dochi et al. 1998). Contemporary comparisons of exposure time to EG within one laboratory or ET program provide the most powerful evidence. However, comparisons between ET programs also provide some indication that EG is not toxic when exposure time is more than 5 min. In the data set that I collected in 2012 from 5 large commercial ET units in Canada and the US, there was no evidence that exposure time to EG ranging from 4 minutes to 40 minutes had an influence on conception rates (Hasler, J.F., 2012).  One last data set was reported from Canada early in the commercial usage of EG for embryo freezing. McIntosh and Hazeleger (1994) reported a pregnancy rate of 59% for a large number of embryos that were exposed to EG for 10 to 20 min prior to freezing in EG.

 

The data that I have been able to uncover strongly support the principle that toxicity is not an issue when embryos are exposed to EG for periods up to 30 or more minutes. However, there is some evidence that somewhat shorter maximal exposures of 20 min or so should be used at elevated temperatures.

 

I do not recommend that anyone currently using minimal exposure times of 5 min or so lengthen the timing of their protocol. If a protocol is working well and does not involve serious inconvenience, there is no reason to change, even if science supports the efficacy of different protocols.

 

References

Dochi, O., Yamamoto, Y., Saga, H., et al. 1998. Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program. Theriogenology 49:1051-1058.

Hasler, J.F. 2012. Effect of embryo stage on pregnancy rate following direct transfer of bovine embryos frozen in ethylene glycol. IETS, January 2012, Phoenix, Arizona. (Reprod. Fertil. Dev. 24:131, 2012).

 

Hasler, J.F., Hurtgen, P.J., Jin, Z.Q. and Stokes, J.E. 1997. Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol. Theriogenology 48:563-579.

McIntosh, A. and N.L. Hazeleger. 1994. The use of ethylene glycol for freezing bovine embryos. Theriogenology 41:253.

Matoba, S., Imai, K., Mimaki, Y., Marita, M., Tagawa, M., Dochi, O. and Saito, N. 2004. Toxicity of ethylene glycol on frozen and thawed IVP embryos in direct transfer method. Reprod. Fert. Dev. 16:175-176.

Martínez, A.G., Brogliatti, G.M., Valcarcel, M.A. de las Heras. 2002. Pregnancy rates after transfer of frozen bovine embryos: a field trial. Theriogenology 58:963-972.

Takagi, M., Boediono, A., Saha, S. and Suzuki, T. 1993a. Survival of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants. Cryobiology 30:306-312.

Takagi, M., Otoi, T., and Suzuki, T. 1993b. Survival of frozen-thawed bovine IVM/IVF embryos in relation to post-thaw exposure time in two cryoprotectants.

Tribulo, H., Rodriguez, P. , Oviedo, J., Ongarato, F., Cuervo, R., Mapletoft, R., and Bó, G.A. 2013. Survival of in vivo-produced bovine embryos exposed to 1.5M ethylene glycol for different periods of time prior to conventional cryopreservation. Proc. of the 17th ICAR, Reprod. In Dom. Anim. 47:456-456.

Voelkel, S.A. and Hu, Y.X. 1992. Direct transfer of frozen-thawed bovine embryos. Theriogenology 37:23-37.

Evidence-based ET: For what time span can bovine embryos be maintained between recovery and freezing without compromising viability?

Categories: Evidence-Based ET
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Published on: December 21, 2012

John F. Hasler

Introduction – After our annual conference in Winnipeg, our AETA Board of Directors asked me if I would be willing to start a column in A Closer Look that deals with practical ET problems and provides published documentation for the subjects and issues in question. I agreed to do this and the following is my first ‘column’. All of us hold personal opinions on numerous technical aspects of our ET profession. In some cases these opinions are based on well-established principles. However, in some cases we all base opinions primarily on ‘clinical impressions’. I believe that such impressions can sometimes be entirely accurate, but I also know, from personal experience, that unless one accumulates an adequate data base, clinical impressions can sometimes turn out to be wrong. Consequently, this column will present information that has some published documentation and is based on more than just my personal opinion or, in fact, undocumented opinions period. Our board members have submitted a number of suggested topics and have given me the freedom to pick among them. I will attempt to adequately document any subject that is covered in this column. Also, please feel free to suggest future subjects that you feel are of interest to most of our membership.

For what time span can bovine embryos be maintained between recovery and freezing without compromising viability?

For a 10 year period from the mid-1980s to the mid-1990s, Em Tran, Inc. shipped many thousands of frozen embryos from Holstein donors in the US to Holland Genetics, The Netherlands. During one phase of this program, we kept very precise track of the time, down to the minute, between flushing and freezing, out to a maximum of 3 hr or 180 min. Of a total of 2,102 embryos frozen in glycerol during this period, the pregnancy rate for embryos frozen between 0-30 min after flushing was 54.1% and did not vary significantly among 30 minute blocks of time out to the 150-180 min group, which had a pregnancy rate of 55.7%.  These embryos were held at ambient lab temperature in Dulbecco’s modified PBS, which we purchased in the powder form and reconstituted in MilliQ water in our lab, adding antibiotics and 10% FCS. The recipients were primarily Holstein cows.

Another data set generated from 3,570 embryos frozen in the Netherlands at Holland Genetics and transferred within the country showed a slightly different outcome (Otter, 1994). The pregnancy rates were 71%, 66% and 57% for embryos held for 0-2, 2-4 or 4-6 hr respectively. These differences were statistically significant.

I recently uncovered a publication on this subject that I had long forgotten about. At the 6th AETA convention in Orlando FL, in 1987, our former AETA president Stan Coley presented some very interesting frozen embryo data from his ET practice in GA. Stan presented data indicating a very high pregnancy rate (75%) for 300 embryos held in in modified PBS + 0.4% BSA and frozen in glycerol within 4 hr post collection. The pregnancy rate was slightly lower for 76 embryos held at ambient temperatures for 4-8 hr before freezing and lower yet (39%) for a small group of 31 embryos held for over 8 hr. Maintaining a total of 91 embryos at 4°C for over 8 hr did not substantially improve the pregnancy rate (46%) compared to the 8 hr group held at ambient temperature.

Pregnancy rates of frozen-thawed embryos are the best index of embryo viability. However, in vitro culture after thawing also can provide meaningful information. In a study conducted at Virginia Polytech (Jousan, 2003), embryos were recovered from donors, maintained in holding medium (OCM; ECHM-500, Immuno-Chemical Products, Ltd.), at either 5 or 22°C for 2, 6 or 12 hr and then frozen in EG. After thawing the embryos were cultured in a commercial IVC medium and examined after 72 hr of culture. There was no difference in the development rates of embryos held for 2 versus 6 hr prior to freezing (86 vs. 79% respectively), but embryos held for 12 hr had a significantly lower survival rate (54%). Holding embryos at 5 versus 22°C prior to freezing did not improve the survival rates for any of the holding periods.

Unfortunately, none of these studies compared different holding media along with different holding times. Certainly, holding medium quality would enter into this issue as perhaps would temperature if it was highly elevated.

If I was still directly involved with freezing embryos commercially, I would feel most comfortable sticking to a 3-4 hr maximum holding time (in a commercial holding medium) whenever possible. However, a few more hours in holding medium might lower subsequent pregnancy rates but probably would not lead to a ‘disaster’.

References

Coley, Stanley L. 1987. Improving pregnancy rates from frozen embryos. Proceedings of the 6th Annual Convention of the AETA, Orlando, Florida. pp.40-50

Hasler, John F.  2001. Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology 56:1401-1415.

Jousan, F.D., Utt, M.D., Whitman, S.S., Hinshaw, R.H. and Beal, W.E. 2003. Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro. Theriogenology 61:1193-1201.

Otter, T. 1994. Pregnancy rate of fresh and frozen-thawed cattle embryos. Proceedings of the 10th Scientific Meeting of the European Embryo Transfer Association (AETE), Lyon, France. pp.228.

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