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Theriogenology 81 (2014) 152–169

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40th Anniversary Special Issue

Forty years of embryo transfer in cattle: A review focusing on the journal
Theriogenology, the growth of the industry in North America, and
personal reminisces
John F. Hasler*
Bioniche Animal Health, Inc., Pullman, Washington, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 July 2013
Received in revised form 9 September 2013
Accepted 11 September 2013

After the first successful transfer of mammalian embryos in 1890, it was approximately 60
years before significant progress was reported in the basic technology of embryo transfer
(ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support
the founding of commercial ET programs in several countries. Today, well-established and
reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial
businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable
procedure, but improvements are needed for biopsied and in vitro-derived embryos. High
pregnancy rates are achieved when good quality embryos are transferred into suitable
recipients and low pregnancy rates are often owing to problems in recipient management
and not technology per se. In the future, unanticipated disease outbreaks and the everchanging economics of cattle and milk prices will continue to influence the ET industry.
The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily
resolved and the involvement of abnormal epigenetics associate with this technology
merits continued research. Last, genomic testing of bovine embryos is likely to be available
in the foreseeable future. This may markedly decrease the number of embryos that are
actually transferred and stimulate the evolution of more sophisticated ET businesses.
Ó 2014 Elsevier Inc. All rights reserved.

Embryo transfer
History of embryo transfer

1. Introduction
It is widely recognized that the first documented live
birth resulting from the transfer of mammalian embryos
was achieved by Walter Heape in 1890 [1]. In a subsequent paper, Heape described his technique for handling
rabbit embryos, which involved spearing them on the tip
of a needle and transferring them to a recipient without
an intermediary step of placing them in holding medium
[2]. Heape’s varied discoveries and successes in the field

* Corresponding author. Tel.: 970-222-5302; fax: 970-484-9860.
E-mail address:
0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.

of reproduction were described in detail by John Biggers,
who was awarded the Pioneer Award (PA) by the International Embryo Transfer Society (IETS) in 1990 [3].
Thirty years then passed before another success in producing mammalian offspring from embryo transfer (ET)
was reported and it again involved rabbits [4]. The first
birth of a calf resulting from ET is credited to E.L. Willet,
et al., at the University of Wisconsin [5] and occurred 63
years ago. The research by Willet and the other early
workers that led up to this first success in cattle is
elegantly described by Keith Betteridge (2003 IETS-PA) in
a paper published in Theriogenology in 2000 [6].
Protocols for the superovulation of cattle [7–9],
appropriate media (for review see [10,11]), and the

J. outline the growth of the commercial ET industry. but they span approximately 40 years. All of these units built and operated expensive surgical facilities for both the donors and recipients that underwent aseptic surgery under general anesthesia. I offer a sincere apology to any authors whose work I may have covered superficially or outright failed to include. Seeking to provide comprehensive coverage of ET in cattle. a group of scientists and practitioners representing both North America and Europe met at Cambridge. are covered in a number of reviews. Hasler / Theriogenology 81 (2014) 152–169 surgical recovery and transfer of embryos [9. could not be imported directly into the United States. These procedures. As technology rapidly improved and commercial ET units rapidly came on line. in January 2014. the contribution of practitioners will be acknowledged. Because of the breadth of the subject and space limitations. When possible. in 1982. Ontario. and 877 on bovine IVF.20]. Coincident with this 40th anniversary edition of Theriogenology. Three-quarter and even half-blood females sold for very high prices and these cattle fueled the early ET industry in North America. the development and growth of the commercial industry. no Angus and only 21 Holsteins were recorded by their respective breed associations. and Seidel (2008 IETS-PA) [16]. Using IVF as an example. and describe personal experiences in the growth and evolution of the commercial ET industry. I sat in my home office surrounded by reprints of at least 1000 published papers dealing with some aspect of bovine ET. for contributing much of the technology that got commercial bovine ET started in early 1970s. an excellent review edited by Cyril Adams of both the history and the current status of ET procedures was published [15]. 153 2. and Charolais. many of the participants in the Cambridge conference. which led to the establishment of the modern ET industry in the early 1970s. In addition to development of technology. and Codding Embryological Science. Modern Ova Trends in Norval. initially representing several breeds including Simmental. The resulting monograph [19] detailing the papers and discussions from this meeting and the Rowson publication from the Cambridge meeting [18] were enormously helpful in advancing ET science into the commercial realm. including some especially thorough contributions by Betteridge [13. A few years later. anecdotal form. or the research applications that are utilized. While preparing this manuscript. whether on the basis of the historical development of the technology. my experiences in the ET industry are not particularly unique or unusual.. and I believe a personal view may sometimes be more interesting and informative than limiting coverage to published scientific papers. Poland. Of the 33 cattle breeds . Although more than 1500 Simmentals were reported born during this period. Colorado State University (CSU) in Ft. they could qualify for movement into the United States. the 40th anniversary of the IETS will be celebrated at the annual meeting in Reno. it is not possible to include all relevant references.12] were developed primarily starting in the late 1940s and into the 1960s. many fine studies published in journals other than Theriogenology. Collins. a fair amount of technology and ET improvements from the private sector have not been published and are available only in verbal. Carnation Genetics in Hughson. Nevada. A book edited by Rowson detailing the presentations at this meeting was published by the European Commission of the European Communities [18]. Inc. Consequently. and much of the science described remains accurate and relevant today. but once they were in Canada. Adams [15]. not including a number of books and published proceedings.14]. Limousin. In July 1976. a second factor strongly influenced the early growth of the ET industry in North America. A recent survey of PubMed showed a total of 2051 references on bovine ET. which led to the establishment in the early 1970s of some of the first ET units in North America. plus a number of additional persons met to discuss ET during the 8th International Congress on Animal Reproduction in Krakow. including Alberta Livestock Transfer in Calgary Alberta. These cattle. However. The invitation to write this review for Theriogenology represents a daunting challenge. These early commercial programs greatly extended and improved the work started by research programs such as the station in Cambridge. there were two noteworthy meetings of academicians and practitioners involved with both ET research and commercial services. Foote (2002 IETS-PA) and Onuma [10]. I was afforded a good deal of freedom and I have chosen to emphasize the following: to briefly cover the early history and development of bovine ET. The Animal Research Council unit initiated surgical recovery and transfer of bovine embryos. Starting in 1973 through 1975. The legal importation of so-called exotic cattle from Europe was approved by the Canadian government [14. although I will emphasize key studies published in Theriogenology. ET on a commercial basis has grown into a mature industry that is active today on a worldwide basis. In cattle. In December 1975. However. Also. in Foraker.F. The modern livestock ET industry is a result of the pioneering efforts of two groups: The scientists who initially developed the procedures and techniques of ET. Great Britain. Oklahoma. there are 170 pages of references in Ian Gordon’s (1998 IETS-PA) 1994 comprehensive review of cattle IVF [17]. the commercial ET industry is today just a few years past 40 years of age. making it practical and available first to the cattle industry and then to other livestock species. California. 609 on bovine superovulation. and many that have appeared in Theriogenology are not included in this review. During this period of time there were also significant contributions being made from the growing ET industry in other areas of the world. is simply not possible in a short review paper. primarily in North America. the rapid increase in the number of Simmental calves born in the United States as a result of ET is shown in Table 1. and the commercial ET practitioners who modified this technology. The early days of commercial ET A large degree of recognition must be extended to Tim Rowson (1985 IETS-PA) and his colleagues at the Agricultural Research Council Unit of Animal Reproduction in Cambridge.

Collins. and the outflow was collected in graduated cylinders. Phil Dziuk (2001 IETS-PA). In fact. While in private practice he had met and worked with T. stating “there is a big future in embryo transfer. I was in the final year of working on my PhD at the University of Illinois. and Holstein Associations. Soon after joining the Colorado team. as a result. In addition to the need for expensive surgical facilities. The owner watched in dismay as George Seidel tried in vain to recover the embryos off the floor. Peter Elsden arrived at the CSU ET unit. George did not know that I had never taken a college course in the field of animal science. I met George Seidel. and compliments of the Simmental. my new ET career might have ended right then and there. who was on my PhD thesis committee at the University of Illinois. included in a 1989 report by Robert Baker [21]. they were readily shared with the visitors. there was considerable concern regarding donors (approximately 10%) rendered either infertile or subfertile by the surgery [23]. Many practitioners today use an almost identical protocol. In October of that year. . infant son. by 1988 the number of yearly ET-produced Simmental calves had dropped and both Angus and Holsteins increased greatly. my wife. nevertheless. As Peter recently related to me. but unfortunately my client had read grossly exaggerated reports in American cattle magazines and. where we transferred them into recipients starting in 1972. However. Peter shared with us his intention to develop a nonsurgical approach to embryo recovery. but means the occurrence and development of events by chance in a happy or beneficial way. While in Ft. The definition of “serendipity” goes beyond just the concept of “luck. Angus. In 1975. My research involved a study of environmental influences on reproduction in a Canadian arctic rodent. “My interest in ET was piqued with the Rowson-Cambridge publication in 1969 [22]. Collins and on his desk was a box of Foley cathetersdso I asked if I could have one and he said take the box.” Peter then extended the length of the Foley catheters with tubing and glue and further experimented with nonsurgically “flushing” donors. a rapidly growing number of visitors showed up. with the exception that specifically designed. where they described successful embryo recovery and embryo transfer and I thought this would be a method to discover some of the infertility problems which occur in general practice. bringing with him valuable personal experience with ET in Australia. and I knew that after 3 years and three countries visited this was the technique that would work without damaging the uterus if used correctly. Hasler / Theriogenology 81 (2014) 152–169 Table 1 Number of embryonic transfer (ET) calves registered by three US breed associations during selected years between 1973 and 1988. it may have been largely forgotten that the mid-ventral surgical approach made the collection of embryos from lactating dairy cattle impractical. he assumed that I was not performing up to standard. and in some cases remained for days or weeks. Sugie (1986 IETS-PA) and was convinced that surgical recoveries should be relegated to the past.” which can range from good to bad. if not into the next morning. often involving long days. . specifically Dicrostonyx groenlandicus. he subsequently offered me a post-doctoral fellowship in his laboratory. I dropped to the floor a glass Petri dish containing all 25 good and quite valuable embryos just collected from a donor. It is clear that by the late 1980s ET was being applied to many cattle breeds and that the initial surge applied mainly to Simmentals and other “exotic” breeds in North America had trailed off. As word spread of these successes. A relevant. If not for a forgiving environment. Following Rowson’s instructions. Later. .154 J. “I was visiting a MD friend in Ft. encouraged me to take the post-doc and make the jump from zoology to animal science. Obviously. Last. commercially available catheters and tubing are now available. ET had a huge role in establishing the Simmental breed in North America.F. As the techniques in the ET program evolved and improved. In 1973. known as the collared lemming. “We quickly discovered that all too frequently donors do not always produce embryos.” Peter stated. I clearly remember the challenge of finding embryos among the blood and mucus in some of the initial flushes. Simmentals were the most common breed represented early in the CSU program and donor owners frequently visited and observed the procedures. the equipment and Peter’s skill . As a result of meeting George and telling him about my work with lemming embryos. or that I really did not know the difference between Holstein and Hereford cattle.” The early months at CSU were a comprehensive learning experience for me and a physically demanding schedule for all the personnel in the ET unit. by 1988 there were seven beef breeds plus Holsteins that reported more calves than the Simmental breed. On mornings when a large number of embryos were recovered and suitable recipients were abundant. serendipitous event recalled by Peter follows. I was to participate in the newly established bovine ET program at the university. Birth year Simmental (n) Angus (n) Holstein (n) 1973-75 1976-78 1979-81 1982-84 1985 1988 1558 4163 7786 13916 4068 1879 0 53 1552 8303 4680 5940 21 1234 13103 43253 20991 22070 From [21]. we assumed that the surgical transfers would continue well into the night. for a short vacation and visit with my brother and his family. we recovered an embryo using a Foley catheter in my first successful nonsurgical flush. and I drove to Ft. were always made welcome.M. Colorado. only the Santa Gertrudis breed reported more than 90 ET calves from 1973 through 1975.” Several things stated by Peter have not changed in the last 40 years! Not long after his arrival at CSU. The flush medium was introduced into the uterus under pressure from gravity flow. Collins and owing to an entirely serendipitous series of events. who was just beginning his third year as an assistant professor at CSU and was busy setting up a bovine ET program with both commercial and research applications. we collected Charolais embryos in New Zealand and shipped them in an incubator to Australia.

and/or post-conference proceedings of the IETS. Serious money problems bedeviled the society in the early years and both George and Sarah Seidel provided quite significant personal time contributions in keeping the society going. where he helped establish American Embryos. Auld Croft Farms. our program concentrated on in-house services and reached a peak of approximately 300 donors boarded and approximately 1000 recipient heifers under our management. Great Britain. cattle boarding facilities. beef donors and company-owned recipients were boarded on a number of former nearby dairy farms. This agreement continued for 25 years. As an indication of the “state” of the science at that time. Bob had recently resigned a professorship at MacDonald College in SainteAnne-de-Bellevue. In time. With Bob’s departure.. et al’s paper. 1... Shortly after arriving with my family in Pennsylvania during a record-setting January blizzard in 1978. 1995. our business suffered from growing too large. my veterinarian colleague.. Michigan.J. Europe. there was growing interest in the formation of an ET scientific society. Alan McCauley (former faculty member at the Cornell University School of Veterinary Medicine) and I proceeded rather quickly to purchase the newly formed Via Pax business and rename it Em Tran. by building a spacious laboratory. which went on to Fig. In January 1978. In early 1977. Few of these large programs remain in business. another Ontario ET entity. ending with the January 2003 issue. Consequently. Inc. we hired and trained a number of veterinarians. Pennsylvania. with attendance at the early meetings going from 82 in 1975 to 156 in 1979. As a consequence of this cooperation. Upon returning to the United States. membership topped 1000 by the early 1990s. Ltd. a not uncommon characteristic of many early ET businesses. in Mississauga. and we worked together at Auld Croft in Ontario for nearly a year. my family and I were anxious to return to the United States and serendipity again influenced my career. Commensurate with the opening of several commercial ET businesses starting in the early 1970s. I received instructions to fly to Milan. we concentrated more on in-house services (Fig. and participate in a hectic. which currently stands at 748 members in 61 countries (compliments of the IETS). The formation of numerous more commercially based associations in a number of countries and regions began in the 1980s. the IETS was officially formed on May 26. leading to a widely cited publication on the technique [24]. There were a number of similar full-service embryo transfer business during the 1980s and 1990s in North America. Australia. in Middleville. As a consequence. a rapidly growing number of ET practitioners also provided on-farm services. I was employed by Via Pax. Inc. Quebec. With little competition and the demand for ET services literally exploding. . 3. The IETS grew somewhat fitfully in the early years. the 1978 to 2003 ‘IETS’ editions of Theriogenology contain a wealth of information related to ET. 25 individuals attended a meeting in Denver. there was a rapid increase in the number of commercial ET operations in North America. with the goal of establishing an ET business in Pennsylvania. including California. Hasler / Theriogenology 81 (2014) 152–169 evolved rapidly. A total of 82 individuals. Bob then left for the United States. roughly split in numbers between researchers and commercial practitioners. 2-week marathon flushing Italian Holsteins. and the first meeting was held over 2 days in Denver in January 1975. too fast. Lancaster County. While trying to provide on-farm ET services in more than a dozen states. I clearly remember seeing a number of attendees busily hand drawing renditions of an eight-cell bovine embryo photomicrograph shown on the screen during a talk by George Seidel. however. Italy. 1). and Japan. Ontario.. The IETS survived the early days and has held an international meeting every year since 1975. such as limited partnerships. In time. Inc. Although we continued to provide on-farm services. became charter members of the IETS at this meeting [28]. It should be pointed out that somewhat similar nonsurgical protocols were developed at the Universities of Wisconsin [25] and Utrecht [26] were also described in the same issue of Theriogenology as Elsden. Colorado. Many of them were supported by investment programs. I left Colorado and joined Robert Baker at a new ET company. In May 1974. 155 figure in an important ET patent battle. a number of these veterinarians went on to establish other ET businesses in Pennsylvania and several other states. Em Tran. Inc. a number of issues have carried the pre. to discuss the science and business of ET and to explore the idea of forming a society [27]. 1974. Another important link between the 40th anniversaries of Theriogenology and the IETS was the 1978 initiation of annual publication of the IETS proceedings in the January issue of the journal. In addition. a business we co-owned and operated until 2001. This has led to some retraction of membership in the IETS. and a milking parlor. with up to five teams on the road providing on-farm services many days. A number of similar large programs were established in North America during the 1980s and 1990s.F. The ET industry after the adoption of nonsurgical embryo recoveries After the three papers on nonsurgical embryo recovery cited above appeared in Theriogenology [24–26]. In addition.

with a number of ensuing studies [41]. is another account by Peter Elsden. as a consequence. Inc. the patent holder brought suits for patent infringement in US District Court in Chicago and injunctions forced three ET businesses to cease operation while legal proceedings took place. The advantages of using multiple injections of FSH versus a single injection of eCG were confirmed in a study by Boland. FSH was not available so I made my own crude extract from equine pituitaries collected from a local horse slaughterhouse. known in the past as pregnant mare’s serum gonadotropin. Augspurger. The mean number of transferrable embryos from reproductively normal cows has remained relatively unchanged during the past 30 years for both beef [33] and dairy cows [34]. FSH injections to initiate superovulation in cattle were started between Days 8 and 13 of the estrous cycle. which included an effective critique of the patent by George Seidel. The authors made the observations that the superovulation protocol resulted in great variation in response. it added another tool to methods of synchronizing donors for initiating superovulation. Overland Park. this study involved Keith Betteridge. an assessment on mean numbers of embryos recovered in four different commercial programs showed no change over a 20-years period [36]. Even though it is common practice to inject prostaglandin after embryo collection.13. supporting the data provided by the AETA. while managing . Consequently.40]. with or without hCG.0 for beef (>24. In the early 1990s. The earliest commercial work in North America utilized either commercially available FSH purified from porcine pituitaries (Armour-Baldwin Laboratories. and there was evidence of reduced responsiveness to repeat treatment. a Canadian pioneer in the development of bovine and equine ET and. For many years.14]. is only briefly covered herein.. including a significant percentage of virgin heifers as young as 8 months of age (personal communication).2 transferrable embryos between 1972 and 1975. plus the window of appropriate heat dates limited the ability to superovulate a group of cattle with scattered estrus dates. When progestin ear implants (Synchro-Mate-B. These studies have clearly shown that cattle can be effectively superovulated with the use of a progesterone-releasing device and estrogen. The first paper in Theriogenology involving bovine ET appeared in Volume 1. Looney. each superovulated for the first time. in Dublin [31]. embryo recovery rates were adversely affected by excessive ovulation rates. In 1981. Peter stated that the number of good embryos tended to be greater when FSH was used compared with eCG [30]. CEVA Laboratories.000 donors) in 2011. There have been several subsequent patents that might have had serious economic impact on the ET industry had they been upheld in court challenges. 50-mg Armour standard FSH units) or eCG. Also. and embryo numbers did not vary when donors were started on any of these days [34.156 J. the availability of silicone vaginal progesteronereleasing devices [38] represented a similar tool for controlling estrous cycles. Superovulation Like most of the subheadings in this review on ET. For many years. the common dogma among ET practitioners was that donors should be allowed to undergo two estrous cycles between superovulation attempts. However. plus two others of Augspurger’s. The lack of improvement in dairy cows is even more surprising when one considers that in the 1983 study [34].2 “good” embryos from more than 2000 beef donors representing 14 breeds and we reported [34] a mean of 6. 3. Nevertheless. a Michigan patent attorney with no personal experience in ET [29]. and I prepared pregnant mare’s serum gonadotropin from mares whose blood I collected at 80 to 120 days pregnant. generous ET practitioner purchased the patent. as discussed. problems with this protocol included the necessity of knowing the date of a donor’s last estrus. the US Patent office issued a patent entitled Reproductive processes for cellular bodies to L. These means can be compared with those provided by the annual census of the AETA. Substantial funds were raised by members of the American Embryo Transfer Association (AETA) and a vigorous defense was waged. Hasler / Theriogenology 81 (2014) 152–169 In 1974. Although production of Synchro-Mate-B eventually ceased. et al.000 donors) and 6. This approach was quite reliable. the latest year for which results are available (compliments of the AETA). [33] reported a mean of 6. KS) became available. with the stipulation that Augspurger dedicate the patents to public use. including some that were likely not technically possible. an anonymous.4 transferrable embryos from almost 700 individual Holstein donors.3 for dairy (>15. When victory for the Augspurger lawsuit seemed questionable. the author of a number of review papers related to ET [6. superovulation is covered comprehensibly in another paper in this issue of Theriogenology and. “During our early efforts in Australia. Issue 2 in February 1974 [32]. NB.F. after 5 years of commercial ET and research at CSU. with the widely recognized decrease in conception rates in dairy cattle after artificial insemination (AI) [35]. especially when used in conjunction with various estrogens. Omaha.37]. The resultant serum could only be used on a donor once because of the chance of anaphylactic shock. this protocol leads to an interflush interval of 60 or more days. The patent contained approximately 100 specific processes covering every conceivable technique in the field of ET. However. superovulated donors often do not return to estrus for 3 or more weeks.” Later. 45% of the donors were 7 years of age or older. we collected an average of 5.1. Not surprisingly. Relevant to this early use of eCG. whereas today there is a strong emphasis on flushing younger animals. it seems plausible that a drop in superovulation embryo numbers might have been anticipated. in which the means were 7. The study involved the superovulation of cows with eCG. plus many technical processes that had not yet been perfected. A good deal of the early work in this area was conducted by Gabriel Bo in Reuben Mapletoft’s (2010 IETS-PA) laboratory at the University of Saskatoon [39. The entire sequence of procedures involved in bovine ET usually starts with superovulation of the donor female. et al.

Some practitioners introduce the flushing medium into the uterus with the aid of gravity flow. some practitioners thread the catheter through the cervix and inflate the cuff in the internal cervical os.3. and flush each horn individually. the implant was removed and superovulation started mid-cycle after the next estrus. making them far superior to traditional Foley catheters.F. another implant was inserted and superovulation initiated five days later. In 47 cases of the total 264 attempted superovulations (17. Hasler / Theriogenology 81 (2014) 152–169 and flushing a large number of donor cows owned by Holland Genetics (CRV. however. in fact. parity. donors either did not come into estrus or produced zero freezable embryos but are included in the data totals.6 24 4.3 24 8. Inc. Using a stainless steel stylette to provide rigidity. Also. there was no decrease in the mean number (5. any single insemination protocol for superovulated cattle that is widely accepted as superior.5) of freezable embryos produced over a sequence of 11 superovulations repeated at a mean interval of 39.9 24 5.46] and in IVP systems have been encouraging [47]. a large number of other donors in the same herd failed over a period of years to produce as many embryos as Black Angus under the same management conditions. If estrus was not observed within 3 days of implant removal. many ET practitioners routinely repeat superovulation of donors on a 30. whereas others use a large syringe and sequentially add smaller volumes of medium. have longer cuffs than Foley catheters. FSH preparation and superovulation protocol. with strategically placed holes. 2). Others thread the catheter half way up one horn. Evidence for a positive relationship between sperm quality and both fertilization rate and embryo quality is supported by data from a commercial ET program [44]. 3.7 24 5. In addition. these donors received a progestin implant and were given prostaglandin immediately after flushing and then watched for standing estrus. A group of [24] Red Angus primiparous heifers boarded at Em Tran. by using progestin ear implants. The protocol. donor stress. even further.7 days. Embryo recovery For more than 35 years. Although there remains a general reluctance among ET practitioners to recommend the use of sexed semen by clients. utilizing hyaluronan as diluent. Superovulated cattle are often inseminated twice. It is not clear why these Red Angus and. The Netherlands). homologous serum was frequently used for both recovering and Table 2 Mean number of freezable embryos produced at 40-days intervals in sequential superovulations of 24 Red Angus primiparous heifers.5 23 5.. Sequence 1 2 3 4 5 6 7 8 9 10 11 12 No. There is not. The availability of sex-selected semen has opened new problems and opportunities for ET. Inc. The low numbers of accessory sperm observed in embryos recovered from superovulated cattle indicate that sperm numbers at the 157 site of fertilization are low [43]. embryos 24 5. Media: Flushing and holding As described in several reviews [6. The earliest models of embryo filters appeared in 1986 and up until then the flush medium was collected in a 1-L plastic. awkward cylinders during the flushing procedure. most if not all embryo recoveries in cattle have been achieved by nonsurgical flushing methods.5 24 6. Today. meaning that cattle need to be handled less. and are autoclavable. autoclavable graduated cylinder. 2). with one straw at 12 and 24 hours after the onset of 40-days interval. Of no less importance. and reproductive history.8 24 4. there now is a wide selection of embryo filters available that allow the flush medium to flow through and be discarded. nutrition. Silicone catheters in a variety of lengths and sizes are now manufactured and sold by several firms that specialize in ET equipment and supplies. Ten days later.2 24 4. was superovulated on this accelerated basis. are semen quality and the timing of AI and skill of the inseminator.8%). age. and the risk of injury to both cattle and human personnel. A wide variety of flushing techniques are practiced with apparently comparable success rates. we learned that their ET staff had found that embryo production was not decreased when donors were superovulated at shorter intervals.J. Embryo production for the slow-release formulation protocol was similar to the traditional protocol. climate.7 24 3. As a consequence. Arnhem. The greatest disadvantages of this system were the delay in proceeding with searching the flush for embryos and the challenge of handling large. The fluid was then allowed to settle for approximately 45 minutes and then all but the last 100 mL was syphoned off and discarded. or Erlenmeyer flask (Fig. These catheters are longer. This change in superovulation protocols increased by 50% the number of embryos that were recovered and frozen per unit time for export to Holland Genetics. thus reducing labor. we were able to superovulate the Dutch-owned donors at 40-days intervals without waiting for them exhibit two interflush estrous cycles. however. As with the Holland Genetics-owned Holsteins. was compared in a two-injection protocol versus the industrywide standard eight-injection protocol using saline as diluent [42]. A new protocol that has increased the efficiency of superovulation without increasing embryo production involves the use of a slow-release formulation diluent for the FSH. . such as breed.3 Unpublished data from Em Tran.2. 3. donors No. results in superovulated cattle [45.11]. As shown in Table 2. while trapping ova and embryos within the filter (Fig. Successful superovulation is influenced by many donor factors. or in some cases.5 24 5.8 24 4. and other management factors can influence superovulation outcome.

Experimental results demonstrating the syringe toxicity was later reported at the 1986 IETS conference [49. graduated cylinder and an Erlenmeyer flask. holding bovine embryos during the early days of ET research. As a result of these syringe problems. (B) Polystyrene embryo collection filter with stainless steel filter screen (70-m pores). In the study conducted at Em Tran. However. a number of specialized plastic and silicone products are available to the ET industry. there continue to be cases where the composition of plastics and other synthetics change without notice. 86-0268. Similar results were achieved in a mouse embryo study conducted in the laboratory of Robert Foote at Cornell University (unpublished data). The only testing of plastic syringes mandated by the FDA involved a rabbit skin test. mouse embryo development was extremely poor when embryos were cultured in media exposed to four brands of syringes for 16 hours. St. The early development of ET media was thoroughly described in reviews by Foote and Onuma [10] and Seidel [11]. (A) Medium (DPBS with phenol red) collected from a donor uterine flush in a 1-L. Louis. survival of mouse embryos exposed to the medium from full syringes was 67%. The medium was then stored through the working day in a 35-mL disposable syringe (Monoject. Most ET practitioners made their own media before the availability of commercially manufactured media specifically intended for ET. frozen in small aliquots. for many years. Civil Action No. Neither the U. Starting in late 1983 and early 1984. Food and Drug Administration (FDA) nor Sherwood Medical issued any warnings to veterinary consumers..25-mm syringe filter into Petri dishes as needed. Pennsylvania. the US Department of Health and Human Services had warned approximately 200 human genetic laboratories using similar syringes for collecting and storing amniotic samples owing to evidence of syringe toxicity.-owned Holstein heifers dropped from 71% in 1983 to 53% in 1984. and a 7-days jury trial was conducted in August 1986. In a comprehensive 1990 study of materials toxicity using mouse embryo development as an endpoint. Marquez. the Monoject syringes performed quite well with 1 hour of medium exposure [51]. Hasler / Theriogenology 81 (2014) 152–169 Fig. Sherwood Medical. By comparison. which is markedly less sensitive to toxicity compared with preimplantation embryos.F. modified Dulbecco’s phosphate-buffered saline (DPBS) with 1% heat-treated new-born calf serum for flushing and 10% new-born calf serum for holding embryos was made in the laboratory at Em Tran. a number of ET businesses in the United States reported a decrease in recipient pregnancy rates (personal communication). a suit claiming product liability was filed in Federal Middle District Court. facilitating the release of a toxic substance into the syringe contents... Inc. Toxicity became evident only after Sherwood Medical changed from ethylene oxide to gamma radiation to sterilize the syringes. but dropped to 0% when only 10 mL of disinfection by-products was held in the syringes (Table 3). Texas. and expressed through a 0. Holding medium was produced in multiliter quantities. Inc. The importance of this relative to the ET industry includes the following: The syringes had previously been nontoxic as used by the ET industry. Today. Utilizing reverse osmosis purified water. Evidence was introduced that gamma radiation affected the rubber gaskets. The jury found in favor of the plaintiff and ultimately damages were paid.S.. Because of the substantial financial losses incurred by Em Tran. and thawed daily for use. 2.) syringes was responsible for the sharp conception rate decline observed in their business. and as a result of the lawsuit the rubber composition of the Monoject syringes was changed. involving the effect of different volumes of DPBS stored in Monoject syringes for 3 hours. Inc. shared their concern (direct communication) that maintaining holding medium in Monoject (Sherwood Medical Co. Researchers at Granada Genetics. most ET practitioners currently only use . However.50]. MO). We later reported [48] that overall pregnancy rate in Em Tran. Inc. including Monoject. Inc.158 J. Most notable were numerous total failures in which no pregnancies were produced after transfers both on-farm and in-house. survival of control embryos was 84% in medium exposed to glass syringes and 86% in medium not exposed to any type of syringe. in Harrisburg.

This protocol was much faster than a midline transfer. The embryo rating descriptions in this paper have been widely adopted by both ET researchers and practitioners. there are a number of companies that manufacture media specifically designed for bovine flushing. Inc. However.F. Presently. and 3 unfertilized ova (International Embryo Transfer Society rating system). embryo photomicrographs and detailed descriptions of four different quality ratings of bovine embryos were published along with conception data from a commercial ET program in California [56].c 30 53c 23b. a. In 1983.c 20 31d 36d. 3.e 10 0a 100a Glass syringe No syringe exposure 35 84e 4f d 86e 3f Abbreviation: XB-HB.000 [59] transfers of fresh embryos. holding. surgical transfer via flank incision was widely utilized into the early 1980s. it prevented transfer into lactating dairy cattle. and 8 (hatched blastocyst) resulted in lower pregnancy rates than stages 6 and 7 (early and mid Fig. In the second study [59] involving a variety of business. Stages 4 (late morula). whereas embryos recovered from donors on Day 9 resulted in a lower pregnancy rate. Hasler / Theriogenology 81 (2014) 152–169 159 Table 3 Effect of medium exposed to plastic disposable syringes for 3 hours on development of mouse morulae (95 embryos per group).b Values on same row without common superscripts differ significantly (P < 0. Embryos were photographed using differential interference optics at 100 in a microdrop of medium in a special plastic Petri dish with a 1-mm-thick glass bottom. and cryopreservation. However. we published two retrospective analyses of Em Tran. data involving approximately 7000 [48] and later. 2. Also. A group of Day 7 bovine ova and embryos from one donor..) blastocysts) when the recipients were all Holstein heifers owned and managed by Em Tran. Serum is no longer a component of most ET media and has largely been replaced by either BSA or a synthetic surfactant such as polyvinyl alcohol. the sample size was too small to achieve significance. Despite the fact that nonsurgical flushing virtually universally replaced commercial surgical embryo recoveries starting in 1975 and 1976. are recognized by the IETS [57]. A thorough review describing the evaluation of embryo quality was covered by Merton [58]. While we were working together in Canada. 3. and are used for international labeling of embryos. without a rubber gasket on the plunger. There were highly significant differences in conception rate among quality 1. qualities 1. probably because the sample sizes were relatively small and the conception rate with the best embryos was only 45%.J. Embryo transfer and recipients Bovine embryos were almost exclusively transferred surgically via midline incision in the pioneering ET businesses in the early 1970s. quality 1 and 2. The take-home message regarding bovine embryo factors is that commercially acceptable pregnancy rates can be achieved after transfer of embryos representing a fairly wide range of donor breeds and ages. This photomicrograph illustrates the variation in stages (4 and 5) and qualities (1 and 2) often seen in embryos from a given donor. Unfortunately. recovered on Days 5 through 8 after estrus. stage. (Compliments of Brad Stroud. as mentioned.and client-owned recipients composed of both cows and heifers of both beef and dairy breeds. Embryos Because of the limitation of numbers. 7 (expanded blastocyst). There are a total of 10 good quality embryos. today there are some efficacious. the effects of these embryo variables were addressed in retrospective analyses of pregnancy data [53–55]. A relatively typical group of Day 7 embryos and ova from one donor is shown in Figure 3. traditionally it has been difficult to accumulate data in academic research programs on evaluations of the relationship between embryo age. In 1987. 2. as well as embryo stages and quality. [48]. syringes for media handling that are entirely composed of plastic. but neither embryo stage nor age was related to pregnancy rate. an analysis of an additional approximately 12. As commercial programs grew in number and size.5. specialized ET media available for bovine ET that are entirely synthetic and do not require refrigeration [52]. and 3 again resulted in different pregnancy rates. The scale used for grading embryos in this study was different than that recognized by the IETS in that we broke down IETS quality 1 (excellent and good) into quality 1 (excellent and quality) and 2 (good quality). and 3 embryos and although quality 4 was numerically lower. Embryos Volume (mL) of PBS-incubated in 35 mL Monoject syringes % embryos XB-HB Degenerate 35 67b 17b. Bob Baker introduced me to a transfer approach via flank incision utilizing a local anesthetic block. 3. could . there were not clear differences in conception rates relative to embryo stage and quality in this study [56].4. there were no differences in pregnancy rates among embryos. all stages combined. stages 4 and 5. Inc.005) [50]. and quality on pregnancy rates. Also. expanded blastocysts-hatched blastocysts. this approach eliminated the possibility of transfers being conducted on-farm or anywhere outside a well-equipped surgical facility.

Another characteristic of this transfer method is that most veterinarians learned it quickly and achieved high conception rates without having any previous experience. However. pregnancy rates were greater among recipients induced into estrus with prostaglandin compared with those exhibiting natural estrus. Inc. (B) The transfer of an embryo with a plastic pipette by a technician into the lumen of the uterine horn while the horn is exteriorized by a gloved surgeon. We made the transition at both Em Tran. including season. and the use of a recipient once versus twice (after an unsuccessful first transfer). Sreenan [63] reviewed the subject of nonsurgical transfers. we performed a retrospective analysis of more than 7600 flank transfers performed over a 6. be performed on-farm. but it was not until after IMV introduced a specific ET instrument (1984–85 in North America. Open-end AI guns were successfully utilized by some practitioners [55]. Lonergan et al. Early attempts at nonsurgical transfer were recited in some of the reviews cited. (Table 4) clearly showed with fresh embryos that the transition from surgical to nonsurgical transfers did not affect pregnancy rates in either recipient heifers or cows [59]. Inc. the presence of a palpable CL in the recipients in these two studies obviously was sufficient to provide an adequate concentration of progesterone. over a period of about 18 months. in the first issue of Theriogenology that carried the annual proceedings of the IETS (1978). [62] manipulated the level of progesterone in recipients receiving multiple embryos. and Em Tran-West. In a different approach to the question of progesterone and recipients. slowly phasing in the nonsurgicals starting in 1986. estrus synchrony between recipients and donors influenced pregnancy rate. there also was no influence of CL size on pregnancy rate. the side of the transfer. Although there were some encouraging results cited in the review. year. than did the now discontinued surgical approach. nor did luteal volume or progesterone concentration among recipients differ among recipients that did or did not become pregnant [61]. The paralumbar area of the recipient flank ipsilateral to the CL was clipped. Although this study did not address actual pregnancy rates. and beef heifers in one study [59]. Although transfers to the horn ipsilateral to the CL had a numerically greater conception rate (54%) than transfers to the contralateral side (39%). Although ET conception rates were greater in dairy heifers compared with cows in many studies. Also. and anesthetically locally blocked flank of a Holstein heifer. The results [48] included a pregnancy rate of 71% at 60 days of gestation and involved an analysis of 14 factors related to donors. et al. rates were similar among dairy heifers. Hasler / Theriogenology 81 (2014) 152–169 Fig. In a study using Angus beef recipients. The flank transfers proved to work quite well and resulted in high pregnancy rates. The ET practitioner exteriorized a loop of the uterine horn through a surgical incision and punctured the wall of the horn with a bone pin. surgical transfers predominantly resulted in somewhat higher pregnancy rates than nonsurgical transfers. several other factors have great latitude in North America. 4). significantly lower when frozen embryos were transferred nonsurgically into cows. 4. [60] looked at this question. Among recipient factors that did not influence conception rate were the size of the CL. We were reluctant to make the transition to nonsurgical transfers because we had achieved a consistently greater pregnancy rate using flank surgery. Surprisingly. The results at Em Tran-West.. One problem during the 1970s was the lack of a specific instrument designed for transfer of embryos.5-years period into Holstein recipients owned and managed by Em Tran. embryos. and synchrony between the embryo age and recipient estrus. specifically extensive experience in rectal palpation (AI experience is also advantageous). and surgically disinfected (Fig. compliments of IMV Technologies) that nonsurgicals completely replaced flank surgical transfers. One of the recipient factors of concern to early ET practitioners was the side of transfer relative to the side of the CL. A technician then transferred the embryo into the uterine lumen using a 20-ga catheter attached to a 1-mL syringe (Fig. in fact. the sample sizes were too small to attain significance. but failed to answer it conclusively. With statistical help from Bob Foote (2002 IETS-PA). Tervit. pregnancy rates for cows were numerically lower and. . Inc. As expected. beef cows. Inc.. (A) The clipped. surgically scrubbed.F. locally anesthetized. In an early issue of Theriogenology (1977). Successful nonsurgical transfer actually requires a great deal more experience. it showed that embryos transferred into recipients developed faster in a high progesterone environment. 4).160 J. and required a minimum of equipment. Also. and recipients. Although there obviously is a minimal level of progesterone necessary to establish and maintain pregnancy.

c vs. the sample sizes were rather small. Inc. Cattle owners often put a high priority on transferring all usable embryos. adequate cattle handling facilities. The additional 16 embryos were flown overnight to another ET facility where they were frozen in a somewhat experimental fashion. the technology evolved from glass ampules. Joe Wright [67] showed no differences in survival rate of frozen/thawed embryos ranging from stages 4 to 7. The successful programs we and most practitioners used in the early 1980s involved 0. nutrition. b. Windy Mont Matt Kathy.151 274.5 hours. Commercially. Kathy went on to produce more than 100 embryo-transferred calves in the next 5 years while continuing to carry three of her own calves.005) [59].060 (72) 12. embryos recovered and embryos frozen before transfer in the US and Canada in 2011. embryos frozen (% of recovered) 41. . and I returned home full of optimism.J. and a two-stage 3-hours cooling program involving cooling rates of both 0.965 59. flushing.4  C per minute that lasted only about 1. P < 0.F. In the years between my visit to Cambridge and a consistently successful protocol. Superovulated and flushed starting at age 5. serial additions of diluted DMSO.5-mL straws. and transfer. This is necessary because embryo stages can vary somewhat within a flush and donors that came into estrus over a 2 or even 3-days range may need to be flushed as a group on the same day. embryos equilibrated directly in 10% glycerol and a onestage cooling program at about 0. we discarded more than 500 embryos of 4772 collected because of a lack of recipients. Factors Fresh embryos No. compliments of the AETA and CETA). including disease status. Early in the ET industry. dairy. P < 0. donors collected No. but it was about 5 more years before I was successful in establishing a reliable cryopreservation program. 65%. The 16 embryos were only half of the total number (32) of transferrable embryos produced from the flush. before reliable cryopreservation protocols. there was an emphasis on achieving as many embryos as possible transferred into suitable recipients. transfers Surgical Heifers 1485 Cows 491 Nonsurgical Heifers 590 Cows 84 Frozen embryos % Pregnant No. Inc. Management can encompass many factors.A comprehensive review of the early reports involving cryopreservation of bovine embryos was covered in detail by Ralph Maurer [66] in the first proceedings (1978) of the IETS printed in Theriogenology. 81%. embryos recovered No.969 87.887 197. Hasler / Theriogenology 81 (2014) 152–169 Table 4 Comparison of 60-days conception rates after surgical or nonsurgical transfer of fresh or frozen-thawed embryos to cows and heifers at Em Tran-West. is recipient management. In 1980 and 1981 at Em Tran. and reproductive health.3  C per minute. Likewise. 3. assuming good quality embryos and practitioner competence.001. however. Holstein cow with 10 calves born after transfer of 16 embryos in 1978.041 (67) Compliments of the American Embryo Transfer Association and Canadian Embryo Transfer Association. In the most recent datasets available (2011) from the AETA and Canadian Embryo Transfer Association (CETA).1  C and 0. with none surviving when they were subsequently thawed. transfers % Pregnant 80a 70b 586 90 71c 71c 79a 61b 72 26 60 39d a. I was able to visit Steen Willadsen (2005 IETS-PA) in his laboratory in Cambridge. respectively) and is similar for Canada (beef. The importance today of reliable cryopreservation to the ET industry is shown in Table 5. d. approximately 70% of all bovine embryos collected were frozen and not 161 Table 5 Numbers of reported donors collected. The percentage frozen has risen steadily over the years and in the United States is higher for beef versus dairy embryos (79% and 59%. Successful incorporation of cryopreservation into commercial ET lagged behind the basic procedures of superovulation. Cryopreservation The announcement in 1972 of the survival of mouse embryos after cryopreservation at 196  C and thawing was groundbreaking [64]. embryos selected for cryopreservation include most good quality embryos ranging from stages 4 to 7. but only 16 recipients were available. before commercial cryopreservation of embryos.b Values in columns without common superscripts differ significantly (a vs.6. In September 1977. Factors USA Canada No. Perhaps the most important factor influencing the success of transferring embryos.. Success with bovine embryos followed shortly thereafter and in 1973 the birth of the first calf resulting from transfer of a cryopreserved embryo was announced by Wilmut (2011 IETS-PA) and Rowson [65]. we detected no Fig. Approximately 30 years ago. He was generous with his time and advice on freezing cattle embryos. transferred fresh. Figure 5 is an extremely productive donor cow with 10 calves produced from 16 embryos transferred. Stroud and Hasler [44] provided a number of examples of how various management problems negatively impact pregnancy rates in ET recipients. 5.

These reductions in pregnancy rate do not entirely eliminate the commercial value of freezing stage 6 and 7 embryos. there is minimal room for improvement of freezing technology. but intimidating-looking Alsatian. slow freezing is faster than vitrification when calculated on a per embryo basis. Hasler / Theriogenology 81 (2014) 152–169 differences in survival of stages 4 to 7. whereas stage 6 was also slightly but significantly lower (52. Stage 7 embryos had a lower pregnancy rate (43. and after seeding.25-mL straws. Further research on developing a protocol for these expanded blastocysts is needed. have achieved satisfactory pregnancy rates [71. Vitrification of one or a few embryos is quite convenient and considerably faster than slow freezing the same number of embryos. After each frustrating attempted transfer of an embryo. The announcement in 1992 of a direct transfer (DT) cryopreservation system using ethylene glycol (EG) as a cryoprotectant had a rapid and positive effect on the world-wide ET industry [69]. One afternoon in September 1977. we had to attend a ‘celebratory’ dinner in the village café with a dozen government veterinarians eating and toasting the success of our program until late in the evening. In the last survey on the subject in 2009. which involves the use of highly concentrated aqueous solutions of cryoprotective agents that prevent ice crystal formation during cooling. In my pocket were five glass vials containing 30 embryos from separate Holstein donors that had been flushed the previous morning on farms near Toronto. The downside of this system is that upon thawing the embryos had to be removed from straws and rehydrated through a series of sucrose dilutions and then reloaded into straws for transfer to recipients. assuming that frozen embryo pregnancy rates are unlikely to exceed those of fresh embryos. although in vitro storage in liquid medium was used more commonly. is also characterized by exceedingly fast cooling. removed after 4 days. For years. The protocols became greatly shortened and simplified by loading 0. and penicillin in 10-mL ampules. a group of government veterinarians in business suits toasted the event with liquor that was offered off a tray held all night by a young woman from the farm. Although they are frozen in a manner very similar to glycerol in straws.7% and 55. However. Import/export of embryos It was first demonstrated by the Cambridge group in 1972 that bovine embryos could be transferred to the oviducts of rabbits. a single scalpel that was sharpened on a whet stone. but the number of stage 7 embryos was small [59]. we had to make due with a traditional straight razor.8%) than stages 4 and 5 (53. After nearly 3 hours in customs and immigration. however. DT straws are loaded directly into a transfer gun after thawing. Incredibly. This is true whether the embryos are frozen in glycerol or EG (unpublished). respectively). Once the number of embryos exceeds a small number.7. Cryopreservation by vitrification.6  C per minute to 32  C followed by plunging into liquid nitrogen. However. In 2011. The World Bank financed the program for Hungary.0  C. plus the speed of the actual chilling step in the protocol. the pregnancy rate from frozen–thawed bovine in vivo-derived embryos is only about 10 percentage points lower than fresh embryos of the same quality rating [59]. more than 99% of beef and 94% of dairy embryos in the United States were frozen in EG for DT (AETA). This technique was used on a small scale for export of embryos into the early 1970s [74].F. Studies involving vitrification of in vivo-derived embryos in 0. Some weeks later we were not too surprised to hear that only one recipient had died of peritonitis and there was one confirmed pregnancy! The transfer conditions had been such that we would not have been surprised with more deaths and no pregnancies. lidocaine in 5-mL glass ampules along with a glass syringe and a single dull needle. Instead of the detailed list of equipment and supplies that we had been promised.162 J. Most vitrification systems utilize ‘carrier tools’ other than straws to achieve rapid chilling rates [70]. 3. the evidence now clearly supports avoiding freezing stage 7 embryos whenever possible. to facilitate DT. increasing numbers of ET practitioners had declared a reluctance to freeze stage 7 embryos. despite my forceful protestations and before starting transfer of the embryos. once straws are loaded and placed in a programmable freezer. Ontario. For more than 10 years. placing them in a programmable freezer holding at 6. thus saving time and the need for a microscope and experienced embryologist. the ET practitioner or technician is free to handle other tasks. I acquired a large dataset (w72.3%. This account is probably not surprising to those readers who participated in some of the early fresh embryo export adventures to import locations with no experience in ET.25-mL straws with embryos that had been exposed to 10% glycerol for approximately 10 minutes.000 transfers) from four ET North American businesses of pregnancy rates versus frozen embryo stages [68]. the pregnancy rates were no larger than those achieved with standard slow frozen DT/EG straws.4  C to 0. we squeezed into a Russian Lada and headed for a state collective farm in the country. Another advantage often attributed to vitrification is the lack of the need for a programmable freezer. A long night of frustrating work at the state farm followed that dinner. The development of reliable bovine embryo cryopreservation protocols . I came down the stairs of a Malev Airlines Tupolev jet with a veterinary colleague to be greeted on the Budapest tarmac by two Russian soldiers armed with submachine guns and accompanied by a leashed. it is not uncommon in commercial ET to freeze 10 to as many as 100 or more embryos in a day’s work that is often conducted on-farm. transferred into recipients. However. Vitrification is sometimes touted as a superior method of cryopreserving in vivo-derived bovine embryos compared with traditional ‘slow’ freezing. glycerol was the favored cryoprotectant for bovine embryo cryopreservation. with a column of diluent included in the straw.1%). Consequently. In addition.72]. However. and result in the birth of live calves [73]. lowering the temperature at 0. but it was up to us to organize and make it successful.

This avoided the long days that are so typical when additional ET procedures. Few commercial ET practitioners adopted the technology. A plethora of papers dealing with sexing embryos utilizing polymerase chain reaction and electrophoresis was published throughout the 1990s. known as “demi.. and used a permanent micromanipulation set up in our home laboratory. long-term relationship during which we boarded more than 200 Holland Genetics-purchased and -owned cows that produced some 10. usually. In this protocol. Three skinny cows soon arrived with hooves in such serious need of trimming that they basically skied down the truck ramp. This technique required no electrophoresis and thus was faster than conventional protocols and.8. Undoubtedly.79]. Inc. the ET industry responded accordingly [81. When it became clear that conception rates with zona-free demi embryos were comparable to those with zona-clad demis. Inc. sex determination of embryo biopsies by polymerase chain reaction is another technology utilized by the ET industry. we were not the only ET business that could have provided the needed services to Holland Genetics. There are undoubtedly more published papers dealing with sexing of bovine embryos (97 listed by PubMed) than there are practitioners actually offering the service . The IETS took a lead role in listing specific diseases into risk categories based on published studies. This protocol necessitated the use of two micromanipulators and the availability of a second zona. both former students at CSU. he asked us if we would be willing to provide management.” it involved producing identical half. but we just happened to be the first business that Jake Chardon visited on his tour of the United States ET industry. [80].J. avoided the risk of DNA contamination of the work/laboratory site. We avoided trying to use sexing on-farm. This technology was adapted to relatively simple systems for sexing bovine embryos in the late 1980s [83]. paid an unexpected visit to Em Tran.” embryos by splitting the embryonic mass with a micro-blade. The resulting demi embryos are then transferred zona-free. the embryo adheres to the bottom of a Petri dish in surfactant-free medium and the embryo is split from above with a micro-blade attached to a single micromanipulator or handheld. embryo freezing. other than flushing. This would have represented approximately 300 embryos. Inc. provided significant on-site help in developing a successful embryo splitting program at Em Tran. importantly. a great degree of success in the export/import of embryos. however. Initial enthusiasm for embryo splitting technology among dairy cattle breeders rather quickly diminished when it became difficult to justify the costs associated with increasing the number of bull calves. Embryo micromanipulation In the early 1980s. a new technology became available to the ET industry. however.82]. we were able to either freeze or transfer the desired sex embryos in the afternoon. which was characterized by high conception rates for demi 163 embryos transferred either surgically or nonsurgically. 3. Another serendipitous event involving my ET career occurred in 1985 when Jake Chardon. Espoo. Many countries no longer require specific testing of donors for diseases that are listed by the IETS as Category 1 (“those for which sufficient evidence has accrued to show that the risk of transmission is negligible”) [55]. the decreased demand for splitting is also likely owing to the inconvenience of on-farm splitting as well as the increased emphasis on freezing a majority of embryos from most donors. The history of this subject was thoroughly covered in a fine review [75]. Rather quickly. with half of them males and a nearly 50% conception rate for the female demis.F. What followed was a mutually beneficial. Tim Williams and Techan Takeda. Finland) quite successfully [85]. Remarkably. Production of zona-free embryos is both quicker and requires less equipment. Years of collaboration and continuing friendship followed and we utilized Peter’s system (Finnzymes Oy.000 frozen embryos exported to The Netherlands. some of the pioneering efforts leading to commercial splitting of bovine embryos were conducted at the Animal Research Station in Cambridge [76. The first commercial applications of embryo splitting involved the use of two micromanipulators and the transfer of both demi embryos clad in a zona [78. Within a month of arrival. usually obtained by removing the vittelus from unfertilized ova. Hasler / Theriogenology 81 (2014) 152–169 allowed a rapid growth and. The frozen embryos in the first few shipments from Em Tran. Unhappy with the management and embryo production of some Holland Genetics-owned Holstein cows housed in another state. We basically avoided it owing to some of the horror stories circulating about the time involved in sexing a group of embryos plus the issue of DNA contamination that led to false male identifications. infectious disease transmission via embryos was addressed by various agencies within a number of countries.77]. A specific protocol for the handling and washing of embryos destined for export is also prescribed by the IETS and widely accepted internationally. a total of only 36 dairy embryos of all breeds were reported split in the AETA survey. Like so many of the techniques and protocols involved in ET. embryos from all three cows were stored in liquid nitrogen. within 2 weeks of inquiring about the technology to Peter Bredbacka. there were few established health regulations relating specifically to embryos. Early in the embryo export/import programs. and export to The Netherlands. transfer and/or freezing are conducted on-farm. by limiting biopsy procedures to the morning. A peak in commercial embryo splitting occurred about 1986 when the Holstein Association reported the registration of 158 female calves. the CEO of Holland Genetics. he was in my laboratory teaching us the technique. In addition to splitting. I read a paper and was impressed with the description of a new approach to sex determination by polymerase chain reaction developed in Finland [84]. to The Netherlands were thawed and transferred into quarantined recipients that then calved while still in quarantine. the senior author of the paper. In addition to the problem of increasing the number of dairy bull calves. In 2011. In 1995. Termed “splitting.

IVF. The announcement that Granada Genetics had entered bankruptcy was an unexpected shock to both the ET industry and to the suddenly unemployed. comprehensive studies on the schedule and frequency of OPU with and without FSH stimulation have been conducted [94. but a small number of very active Canadian practitioners may account for this 10-fold difference.3% of the embryos collected in 2011 were biopsied for sex (AETA). A percentage point loss of 5 or 8 points. In 1959. The first calves produced entirely from IVM. with mature cows the greatest. who at that time was completing his PhD at Cornell. Data such as these generated by the ET industry clearly show why it is so difficult to detect small but important effects in studies which are limited by animal numbers. and IVC were born in 1987 [92]. however. (unpublished data).1 [98] and 5. The rest is history. a sizable IVP industry developed in Japan and Brazil and. When the program was close to abandonment.3 in a similar program as reported by Charles Looney at Granada Genetics [99]. It is not clear why the procedure has been used more extensively in Canada compared with the United States. when the conception rate is around 50% translates in an actual decrease of 10% to 16% fewer pregnancies.000 IVP embryos were reported as having been transferred in South America. followed by heifers over 9 . there are significant differences related to donor age.7% of the embryos were sexed in 2012 (CETA). Inc. because the mouse did not prove to be a good procedural model. the rabbit was the first mammalian species in which live offspring were known to have been produced by IVF (M. fertilization rates were low. During the period when the emphasis in North America was still on using IVP from problem donors. 3. in Italy and The Netherlands. This observation also is supported by data recently provided by Trans Ova Genetics. Pregnancies also were achieved after transfer of IVM–IVF bovine embryos cultured in the oviduct of a sheep for 5 days [90]. Subsequent progress with in vitro technology in mice. Although cleavage percentage did not differ. the mean number of oocytes collected via OPU was only 4.164 J. and IVC.000 in Asia. By 2002. The first live calf resulting from IVF was born in 1981 as the result of the transfer of a four-cell embryo into the oviduct of a recipient cow [89].C. The IETS does not collect data on embryo biopsy/ sexing. 3. Chang. several breakthroughs in the 1980s involving all steps in the IVP process set the stage for adoption of the technology by the commercial ET industry. In Vitro Fertilization In vitro fertilization has been adopted as a generic phrase that often includes the procedures of IVM. Hasler / Theriogenology 81 (2014) 152–169 in the North America and probably internationally as well. However. This technique has become widely known as ovum pick-up (OPU). but in the United States only 0. This program almost exclusively involved OPU of oocytes from donors with fertility problems. to some degree. whereas in most IVP programs Day 0 relates to the day of IVF.95]. Several hundred donors that had performed unsatisfactorily in conventional superovulation/flush programs entered the program and almost all of them produced at least one pregnancy. In Canada. However. efficient technique involving transvaginal ultrasound-guided aspiration was developed in 1988 [93] and has become the predominant technique for oocyte collection from living cattle.9. The first repeatable. a 1 recipient (Day 6) is not just 1 day younger than a Day 7 recipient. Two calves were born after transfer of embryos resulting from IVF with sperm capacitated using calcium ionophore and the resulting zygotes cultured in rabbit oviduct [91]. Japan reported that more than 4600 IVP embryos were produced. this unfortunate business failure turned out to be hugely beneficial to my business because I was able to quickly bring Charles Looney into our laboratory and he effected an immediate and great improvement in all aspects of our IVF program. 1983 IETS-PA) [86]. often did not extend to livestock species. showing a loss of 8 percentage points when IVP embryos were transferred into Day 6 versus Day 7 or 8 recipients. All three procedures usually are conducted in sequence to produce embryos exclusively in vitro (IVP). progress was slow. 22. and 11. The IETS did not start tabulating statistics on IVP embryos until the annual report in 1996 (compliments of the IETS). talented staff members of the company. and few embryos developed in our experimental IVC system. However. No statistics on IVP embryos were available from North or South America. but is in fact actually 2 days in asynchrony. throughout the 1980s bovine in vitro techniques remained primarily research technology with little utilization by the ET industry. For example. Because Day 0 in relation to heat is the day of estrus. IVF. More recently. IVF in cattle was first accomplished with semen capacitated in the oviduct or uterus of cows in estrus or the uterus of a rabbit [88]. sexing has been utilized on a wider basis. serendipity struck again. However. the percentage of good embryos relative to the percentage cleaved varied significantly relative to age in Holsteins. In 1977. We made a decision to explore the use of IVP in our commercial program in 1991 and I received a good deal of help from Jerry Yang. In addition to OPU.000 in Europe. A retrospective analysis of the pregnancy rate of more than 4500 IVP ETs showed that a significantly higher pregnancy rate of 5 percentage points was achieved by transfer of Day 7 IVP embryos into Day 7 or 8 recipients compared with Day 6 recipients [100]. the numbers had changed dramatically and more than 48. IVP embryos are in fact actually 1 day older than in vivo-derived embryos usually considered to be the same age.F. and the results of the first few years of our IVF program were published in 1995 and presented at the annual IETS conference [98]. which was the greatest number among any of the countries covered in the report. In addition to breed differences in success of OPU/IVP. Perhaps two of the most noteworthy findings were the discovery that IVF of bovine oocytes proceeds most efficiently at 39  C [96] and the development of in vitro capacitation techniques utilizing heparin [97]. The next reported success was with laboratory mice in 1968 [87]. In that year.

Inc. The Brazilian IVP industry has continued to expand and is now operating in several other South American countries and South Africa. Inc.F. All of the North American IVF programs include stimulation of most donors with FSH and OPU performed once every 14 days. Texas. there is a lack of peer-reviewed published data describing the current status of LOS problems in the commercial ET-IVP industry. with three major companies offering services (OvaTech LLC. and one company has recently initiated a large program in Russia (In Vitro Brazil. Inc.000 IVP embryos transferred in 2011 (IETS) compared with 35. Gainesville.602 from 8689 OPUs in 2011 (AETA). 6.107] and then in cattle [98. Worldwide. representing the first mammal successfully cloned from a somatic cell [114]. including cattle. (Compliments of the International Embryo Transfer Society. the large oocyte production via OPU of B indicus cattle. Larger fetuses and heavier birth weights are only one. Trans Ova Genetics is currently the largest producer of IVP embryos in the United States. do not receive FSH. Brazil accounted for approximately 86% of all the IVP transfers in 2011. total production of IVP embryos for the country went from just 287 in 2004 to 4453 in 2012 (CETA). The head of the laboratory. The leveling off of conventional ET and the rapid rise in production of IVP embryos is shown in Figure 6. Navasota. Cyagra was purchased by an Argentinian businessman and moved into the former facilities of Em Tran. and Maryland. is probably the current leader in production of IVP embryos from OPU in Canada. Navasota. created worldwide coverage in the lay press. in Pennsylvania. with a maximum of 128 per nonstimulated OPU session [103]. Proposals have been made for dealing with some of the problems associated with IVP embryos [105].. Hasler / Theriogenology 81 (2014) 152–169 Fig. and modest labor costs. Various co-culture IVC systems containing serum have been shown to support a high rate of blastocyst development and thus there was some reluctance by the ET industry to discontinue the use of serum and/or co-culture. personal communication). a B indicus dairy breed. IVP production from OPU totaled 2078 in 2004 and reached 40. Projected production for 2013 includes approximately 15. this has resulted in the collection of 18 to 20 oocytes per OPU.. sorted. L’Alliance Boviteq. and an extremely large increase in the incidence of hydroallantois are among the conditions recorded. FL. Increased abortions throughout gestation. which ceased operation in the United States in 2010. abnormal placentas. and oocytes from many donors are fertilized with “resorted” semen (previously unsexed. Much of the early growth of the Brazilian ET market was fueled by Nelore beef donors. Trans Ova operates under a license (XY. Starting in the late 1990s. with and without co-culture. North America. of the problems associated with LOS. Unfortunately. At Trans Ova. which in one study produced a mean of 23 usable oocytes. Sioux Center. Semidefined systems in which BSA replaced serum have been shown to perform nearly equally to co-culture [112]. Several hundred cattle clones were . with a 25% blastocyst development rate. and then used for IVF immediately). The replacement of traditional ET in Brazil by OPU/IVP started in 1998 and 1999 [102] and grew quite rapidly. frozen semen which is thawed. and not necessarily the most serious.116]. Inc. In addition. IA).10. In 2002. As first shown in sheep [106. problems associated with pregnancies and neonatal issues are often included in what has become known as the “large offspring syndrome” (LOS). with laboratories in Iowa. primarily in Brazil. The LOS syndrome largely has been attributed to IVC systems containing serum.. Optimism regarding commercial applications of cloning by somatic cell nuclear transfer (SCNT) soon followed [115. One issue that has not been entirely resolved among the problems that are associated with production and commercial utilization of IVP embryos is the question of the normalcy of IVP embryos and ensuing pregnancies after transfer. In the United States. Hong Wei. Europe. Some of the problems greatly decreased when serum was replaced by BSA [113]. reported that 165 cleavage and blastocyst development rates are larger with resorted semen compared with sexed semen available from commercial bull studs. was established in 1999 as a division of Advanced Cell Technology in Massachusetts. Sexing Technologies.000 OPUs resulting in more than 70000 IVP embryos (unpublished data.. including OPU on cows in early pregnancy. 3. TX. TX) to sex semen.) months of age. Cyagra. several businesses were established in the United States to offer commercial cloning services for a number of species. The emphasis in this program is on reproductive healthy cows and heifers. Gir cows. produced more oocytes than Holstein dairy cows [104]. and OPU is performed weekly. including the lower number of embryos produced by superovulation of Bos indicus compared with B taurus breeds. Cloning The announcement of the birth in 1996 of “Dolly” the lamb. which were the lowest [101].563 in vivo-derived embryos transferred. This is in contrast with Brazil. heifers age 6 to 8 months and calves age 2 to 3 months. The change in the Brazilian ET industry has been attributed to a number of factors. with the remarkable total of more than 318. personal communication). and Trans Ova Genetics. Comparison of the annual number of in vivo-derived and in vitro embryo produced embryos transferred internationally since 2000 through 2011. and many thousands of IVP-derived pregnancies and calves have been produced in the past few years.108– 111]. and Japan. where most B indicus donors.J. delayed and poor labor in recipients. the relatively high cost of FSH. characterized by large ovarian follicle counts.

and in vitro technology. there remains a voluntary agreement on the part of the main producers of bovine clones in the United States not to market meat or milk from clones because consumers remain wary of such food. without question. in addition to a number of other relevant subjects. Today. and slaughterhouse collection of oocytes was permitted only for research purposes in five European countries. and genomics services. offspring from cloned cows were not marketed and semen from bulls was not frozen and sold for AI. meat and. The discovery prompted the United States and other nations to ban imports of Canadian cattle and beef. For example. Although there has been some recovery.130]. was founded in Austin. Significant improvements in these technologies will be necessary for any substantial increase in utilization to occur. pigs. the beef ET business has never fully recovered in Canada. Inc. the CETA reported a mean of approximately 3260 beef donors flushed annually. unpredictable disease outbreaks can have profound. especially the transfer of frozen embryos. in turn. The costs associated with performing ET services have risen somewhat dramatically over the years. The ET business has become quite competitive and. had significant impact on commercial ET. and porcine gene banking. does not mimic the changing environment encountered by embryos descending the oviduct and entering the uterus. depending on the country or state. in 2002 to provide commercial bovine. Growth in the number of IVP embryos produced yearly in the commercial ET industry has increased the demand for improving all aspects of IVF technology. and the cloning data base contributed to the risk assessment by the Center for Veterinary Medicine division of the FDA. and goats. whether in large volumes or microdrops. In 1978.000 to $20. have been utilized commercially only on a limited basis. Using genomics. the growth of commercial IVP and the potential problems with epigenetic effects on in vitro embryos. 179) entitled “Use of Animal Clones and Clone Progeny for Human Food and Animal Feed. The only other large. After the import ban. in May 2003 Canada confirmed its first native case of bovine spongiform encephalopathy disease. unpredictable events affect commercial ET. 4. Much as the periodic drop in milk prices has negatively impacted the dairy ET business in the United States. Before this. Genomic evaluation of cattle recently has been reviewed [129. the cost of the available FSH preparations exceeds $100 per dose and the ET practitioner fees are half or less than what they were 35 years ago. North American cloning company.50 and the ET practitioner charge per donor superovulated and flushed was $500. I pointed out three European countries outright that prohibited collection of oocytes via OPU. and this seems to have greatly limited the utilization of the technology by cattle breeders. From 2000 to 2002. The well-publicized and publically supported closing of all horse slaughterhouses in the United States has made in vitro research in horses frustratingly difficult.126] in the context of both IVP and SCNT bovine embryos and other mammalian species. but also to energize and strengthen the resistance of animal rights groups to ET and other reproductive technology. and it is unlikely that the majority of commercial ET practitioners either have had or will have any involvement with transferring cloned embryos in the foreseeable future. in some cases. Among the problems raised in my 2003 paper dealing with the future of ET [122]. In addition.. George Seidel listed 10 recent technologies of “pertinence” to embryo technology. Texas. the cost per dose of the original Armour FSH-P was $6. In additional to changes in technology. the cost of commercially producing cloned cattle is variously estimated at $15. In January. abnormalities in epigenetic control of gene expression recently were reviewed [125. embryos cultured in conventional Petri dishes.S. Unexpected disease outbreaks have the potential to have a direct and negative impact on ET.F. non-veterinarians are providing an increasing share of the procedures. The future Several papers have addressed the future of ET [121– 124]. Several technologies. In his 1991 paper entitled “Embryo transfer: the next 100 years” [121]. conventional ET procedures are unlikely to be affected by the development of microfluidics technology. Hasler / Theriogenology 81 (2014) 152–169 produced at Cyagra. cloning.166 J. many other papers have dealt with single. It is widely agreed that IVC. In his 2006 paper dealing with ET “achievements and perspectives” [124].. the number of Canadian beef donors flushed dropped to approximately 2600 in 2003 and only approximately 2000 in 2004.128]. milk. ViaGen acquired ProLinia of Athens. including estrus and ovulation synchronization. there have been optimistic proposals for improving the technology [120]. equine. to Cyagra. It is likely that an increasing proportion of ET procedures will be performed by noncredentialed technicians in the future. in 2012 to Trans Ova Genetics in Iowa. and the growing importance of AI sire genomic selection is undisputed. regulatory barrier to the marketing of meat and milk from cloned cattle. Georgia. specific technologies that may eventually become part of the ET industry. However. Keith Betteridge discussed.” The report concluded that meat and milk from healthy cloned animals or their offspring are as safe as those from ordinary animals. Currently. Although numerous anatomic. The author’s only experience with cloning was the sale of Em Tran. and epigenetic problems have been documented for SCNT-derived pregnancies and calves [117–119]. more bull calves are being screened and fewer calves are . However.000 apiece. Microfluidic culture systems have been proposed as providing solutions to some of these problems [127. which happened to be in an Alberta cow. may not be exposed to an ideal ratio of media volume to embryo mass. and then was sold. whether based on single or sequential media. sperm sexing. Inc. Several of his choices have. Pertinent to this subject. such as cloning and transgenics. effectively removing the last U. 2008 the FDA released a 968-page Final Risk Assessment (no. In addition. negative effects on the ET industry. ViaGen. physiologic. cryopreservation technology. In 2003.

Inc. genomic information is strongest for Angus cattle. Del Campo MR. I thank my former business partners at Em Tran. genomic information is also being used for selection of Jersey and Brown Swiss bull calves (Select Sires. Science 1981. Onuma H.62:1–13.. [10] Foote RH. [13] Betteridge KJ. Brand A. [33] Looney CR. Moor RM. However. 16.61:171. 20–4.F. Monograph no. Superovulating cows with follicle stimulating hormone and pregnant mare’s serum gonadotropin. Superovulation. In fact. Embryo transfer calves produced in the United States from 1973 to 1988. French LR. Ottawa: Canada Department of Agriculture. In addition. along with all the other veterinarians and embryologists who worked with us over the years. Roche JF. [32] Betteridge KJ. Monograph No. effect of method. US Patent no. Minnesota Tech Bull 1958. Vet Rec 1949.62:178–83. genomic evaluation of embryos will decrease generation intervals. Embryo transfer in farm animals. Superovulatory responses of Holstein cows. Short-term maintenance and culture of embryos. An historical look at embryo transfer. Embryo transfer in farm animals. Theriogenology 1976. Theriogenology 1980. Plain City.113:247.000 different SNPs. Embryo collection by surgical methods. editor.16. Inc. Among beef breeds. commercially available chips that respond to as many as 800. 1974. Without their efforts. Undoubtedly. Superovulation in beef females. J Reprod Fertil 1991. vitrification is often proposed for cryopreservation of bovine IVP. Science 1951. [26] Drost M. Proc Roy Soc London B 1890. Inc. Wallingford. [3] Biggers JD. 1976. Reproduction processes for cellular bodies. Black WG. [12] Rowson LEA. Theriogenology 1991. Luxembourg: The Commission of the European Communities. my career would have been entirely different and most probably I would not have been invited to write this review. 53:3–10. Theriogenology 1978. and biopsied in vivo-derived embryos.7:260–70. which is now available using several different. an efficacious and practical DT vitrification system for biopsied bovine embryos will be valuable. Reflections on the golden anniversary of the first embryo transfer to produce a calf. Theriogenology 1974. 16. Boyd Henderson. 1982. J Dairy Sci 1970. 2012. Sex. Stone WH. recipients. Buckner PG. p. Petersen WE. editor. Vitrification has become the system of choice for cryopreservation of human embryos [133].13:3–6. and unwanted calves.9:17–26. I thank both George Seidel and Peter Elsden for helping shape my early career in ET. FL: CRC Press. followed by Herefords and Simmentals. Several ET/ reproductive technology businesses are currently working toward introducing genomic analysis of embryos to cattle breeders in the foreseeable future (unpublished data). medium and synchronization of estrus. Successful transplantation of a fertilized bovine ovum. Laboratory production of cattle embryos. Further note on the transplantation and growth of mammalian ova within a uterine foster-mother. [25] Rowe RW. p. Hasler / Theriogenology 81 (2014) 152–169 actually being purchased by bull studs and undergoing progeny testing [131].6:523–32. Walter Heape.79:203–44. In: Proceedings of the 5th Annual Convention of the AETA.48:457–9. SCNT. In addition. and Em Tran-West. Z Geburtshilfe Gynäk 1922. this technology will appeal to many cattle breeders. J Reprod Fertil 1969. Ottawa: Canadian Department of Agriculture. Hofstätter R. [30] Elsden RP. Non-surgical recovery of bovine eggs. Superovulation and embryo transfer in cattle. Hasler JF. Seidel Jr GE. [28] Schultz RH.211:351–8. In: Proceedings of the 8th Annual Convention of the AETA. [7] Dziuk PG. Ginther OJ. Monograph no. Donker JD. [8] Rowson LEA. Aarts MH. Egg transfer in cattle. Foote RH. a review. [11] Seidel Jr GE. Theriogenology 1983. A single cannula technique for nonsurgical collection of ova from cattle. United Kingdom: CAB International.84:59–130. History of the International Embryo Transfer Society – Part 1. Problems associated with transfer of ova between cattle. . Schermerhorn EC. [21] Baker RD. Fertility following egg transfer in the cow. Nichols JR. ovum collection. A device for non-surgical recovery of bovine embryos. [18] Rowson LEA.25-mL straws as the container have not resulted in satisfactory pregnancy rates (unpublished data). McCauley AD.93:173–86. J Endocrinol 1951. currently available vitrification protocols using 0. Experimentelle Studien u Einnistung und Weiterentwicklung des Eies im Uterus. References [1] Heape W. [31] Boland MP. [9] Umbaugh RE. A apparatus for the extraction of fertilized eggs from the living cow. [15] Adams CE. Anim Reprod Sci 2003. Ft. } ber die [4] Biedl A. Alternative gonadotropins for superovulation in cattle. [27] Carmichael RA. Superovulation and ovum transfer in cattle. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother.13:7–11. With the use of single nucleotide polymorphism (SNP) chips. Theriogenology 1976.19: 83–99. [20] Pickett BW. [6] Betteridge KJ. Dowling DF. [29] Augspurger LL (assignee). Lawson RAS. Colorado State University. In addition. science and survival in Academe. [5] Willett EL. 1977. AETA 1986:16–32. embryo genomic information will appeal to cattle breeders because they will save money on unwanted ET service costs.1:69–82. and Jimmy Webb. 1977. Mitchell D. AETA 1989:105–116. [22] Rowson LEA. 10–3. Am J Vet Res 1949. Winch RP. Goulding D.6:471–83. [34] Hasler JF. The advantages of applying genomic selection to embryos were recently covered by Seidel [132].470.6:503–7. Nelson LD. a satisfactory genomic profile is almost a necessity today to secure a sales contract on a dairy bull calf from a bull stud. Inc. [19] Betteridge KJ. Drs Alan McCauley. Theriogenology 2000. A history of farm animal embryo transfer and some associated techniques. Identification of SNPs in embryos requires whole genome amplification of the embryo biopsy. Embryo transfer in farm animals. culture and transfer. [23] Elsden RP. In: Betteridge JK. Acknowledgments I thank George Seidel and Jennifer Barfield for reading the manuscript and offering helpful suggestions. Theriogenology 1980. but it will also force a rather drastic reorganization of ET businesses who want to offer this service. J Reprod Fertil 1981. Collins: Animal Reproduction and Biotechnology Laboratory.18:517–23. Boca Raton. . OH). FRS: a pioneer in reproductive biology. The power of genomic selection is greatest for the Holstein breed owing to the enormous amount of production data and sire information available for cows and bulls of this breed.. 222:1–75. Peters H.10:295–305. Seidel Jr GE. Centenary of his embryo transfer experiments. Casida LE.35:5–17. Eilts CL. 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