1

EMBRYO TRANSFER TECHNOLOGY (ETT) IN CATTLE: A REVIEW 1. Embryo tra !"#r 1.1. I tro$%&t'o Bovine embryo transfer has been applied widely around the world. This technology increases the number of offspring obtained from donors with high genetic value and is used to disseminate desirable genetics (Baruselli et al. 2006). Embryo transfer is a multi step process that involves the production and collection of pre implantation embryos from genetically superior females (called donors) and the subse!uent transfer of the harvested embryos into reproductively healthy females (called recipients)" for the purpose of establishing pregnancies and producing live offspring (#oungs" 200$). Embryo transfer procedures have been used to increase the reproductive performance of particular females of agriculturally important species" such as cattle" horse" goat and sheep. %n domestic animals transfers were reported by &arwic' and Berry (()*)) with sheep and goats" by +vasnic'ii ((),() with pigs" and by &illett et al. ((),() with cattle. The first successful embryo transfers in livestoc' species were performed on sheep and goats in Te-as (.omano"20(/). 0lthough the basic procedures (superovulation" embryo recovery" storage1free2ing and transfer) employed in the domestic animal (especially cattle) ET are now well established" there is considerable research for improvement in various areas of ET technology. Throughout the world over the past year" more than (00"000 donor cows were super stimulated and more than ,00"000 bovine embryos were transferred (Thibier" 200,). This technology is influencing the direction of cattle breeding industries3 the numbers are small but the impact is high. 4ommercial cattle breeders must recogni2e that they can benefit from well designed embryo transfer programs providing selection criteria are appropriate for their environment and individual breeding ob5ectives. Embryos also can be produced in the laboratory via techni!ues such as in vitro fertili2ation (%67) or somatic cell cloning. But the actual transfer of an embryo is only one step in a series of processes that may include some or all of the following8 superovulation and insemination of donors" collection of embryos" isolation" evaluation and short term storage of embryos" micromanipulation and genetic testing of embryos" free2ing of embryos and embryo transfer (9asler" 200*). 1.1.1 H'!tory o" #mbryo tra !"#r Embryo transfer in cattle has recently gained considerable popularity with seed stoc' cattle. :ost of the applicable embryo transfer technology was developed in the ()$0;s and ()<0;s. 9owever" the history of the concept goes bac' much farther. 4redit for the first successful transfer of mammalian embryos is given to &alter 9eape" an Englishmen who had an ama2ingly wide range of interests that included animal breeding (9asler" 200/). %n (<)0" 9eape transferred two four cell 0ngora rabbit embryos into an inseminated Belgian doe" which subse!uently gave birth to four Belgian and two 0ngora young (Betteridge" 200/). 0s documented by Betteridge (()<()" there is no further record of any successful ETs until the ()20s" when several investigators again reported on transfers in rabbits. =uring this period" better understanding of the relationship between the pituitary and ovaries led to significant advancements in reproductive technology. 0lso" development of techni!ues for superovulation" estrous synchroni2ation and artificial insemination (0%) set the stage during the ()/0s and ()*0s for successful ET in a number of species (9asler" 200/). Embryo transfer in food animals began in the ()/0;s using sheep and goats" but it was not until the (),0;s that successful embryo transfers were reported in cattle and pigs by >im .owson at 4ambridge" England. %ndeed" Betteridge (200/) has referred to .owson as a founding father of embryo transfer in farm animals. The first commercial embryo transfers were done in the early ()$0;s. %nitially" embryos were recovered from valuable donors and transferred to recipient animals using surgical procedures. %t was not until nonsurgical methods were developed in the late

()$0;s that embryo transfer grew in popularity. 4ommercial embryo transfer is now a large" international business (9asler" 200*). 1.1.( Embryo tra !"#r amo ) t*# !am# !+#&'#! :ost of embryo transfers have been carried out among the same species (intra specific embryo transfer)" and usually it was for the purpose of propagating an animal with desirable characteristics of the two parental species. 0 listing of the species includes rabbit" rat" sheep" mouse" goat" cattle" pig" hamster" ferret" min'" horse" baboon" cat" dog" and water buffalo. Embryo transfer is now commonly used to produce artificial insemination (0%) sires from highly proven cows and bulls. 0lthough technical costs would seem to preclude the use of ET techni!ues for anything but seed stoc' production at this time" the commercial cattle industry can benefit by the use of bulls produced through well designed :?ET (:ultiple ?vulation and Embryo Transfer) program (.uane and @mith" ()<)). 1.1., Embryo tra !"#r amo ) $'""#r# t !+#&'#! Embryo transfers among different species (inter specific embryo transfer) procedures allow the establishment of true inter specific pregnancy (0nderson" ()<<). %nter specific or cross species pregnancy is the condition of carrying the embryo of one species in the uterus of different species. %n natural breeding" inter specific hybridi2ation was able to occur in some species combinations. 0dam (()<2) reviewed inter specific embryo transfer that had been reported up to that time and noted that mammalian embryos usually are tolerant of a foreign species during development to the blastocyst stage" thereafter" embryonic mortality is observed. :ore recently" several inter specific combinations have been intensively studied in various species. 1.1.- .ot# t'a/ 0a/%# o" #mbryo tra !"#r ' &att/# The reproductive potential of each normal newborn calf is enormous. There are an estimated (,0"000 potential AeggsB or ova in the cow and billions of sperm produced by each bull. By natural breeding" only a fraction of the reproductive potential of an outstanding individual is reali2ed. The average herd bull will sire (, to ,0 calves per year" and the average cow will have one calf per year. &ith artificial insemination" it is possible to e-ploit the vast number of sperm produced by a genetically superior bull3 however" the reproductive potential of the female has been largely unutili2ed. Cnder normal management programs" a cow produces an average of eight to ten calves in her lifetime. Di'e artificial insemination has done for the bull" embryo transfer is a techni!ue that can greatly increase the number of offspring a genetically important cow can produce (#oungs" 200$). Embryo transfer is now commonly used to produce 0% sires from proven cows and bulls (Teep'er and +eller" ()<)). %n addition" new genomic techni!ues are being used increasingly to select embryo donors" especially for selection of dairy bull dams for super stimulation" where a genomic analysis is becoming essential (@eidel" 20(0). @uperovulation and 0% followed by embryo recovery and transfer allows the genetic contribution of outstanding cows to be increased. This embryo transfer technology has also contributed to the acceleration of genetic improvement by producing sires in multiple ovulation and embryo transfer (:?ET) program. 7urthermore" embryos are an e-cellent way of moving germplasm from one region to another without introducing new diseases (:orris et al." 200(). =uring this entire process" many new bio techni!ues have been developed. These techni!ues include optimal ovarian stimulation scheme" oocyte pic'ing up (?EC) or oocyte retrieval" in vitro maturation (%6:) of immature oocyte" in vitro fertili2ation (%67) and intracytoplasmic sperm in5ection (%4@%)" pre implantation genetic diagnosis (EF=)" blastocyst embryo culture technology" identification of optimal uterine environment etc (Bin" 20(2).

1.(. G# #ra/ +ro&#$%ra/ !t#+! The donor may be inseminated naturally or artificially and embryos will be collected non surgically sito eight days after breeding. 7ollowing collection" embryos must be identified" evaluated and maintained in a suitable medium prior to transfer. 0t this point" they may also be sub5ected to manipulations" such as splitting and se-ing" and may be cooled or fro2en for longer periods of storage (9asler" 200/). The steps involved in ETT are8 G @election of donor female G @uperovulation in donor female G %nsemination of donor female G Embryo collection in vivo or G Embryo collection from ovaries in vitro and maturation of oocytes G In vitro fertili2ation and embryo production G Erocessing and preservation G Transfer 1.(.1 .*y!'o/o)y a $ # $o&r' o/o)y o" t*# orma/ bo0' # #!tro%! &y&/# The endogenous control of the bovine estrous cycle involves the interrelated secretion of a number of hormones from the hypothalamus" anterior pituitary" ovaries and uterus. These include gonadotrophin releasing hormone (Fn.9) from the hypothalamus" follicle stimulating hormone (7@9) and luteini2ing hormone (D9) from the pituitary gland" estrogen" progesterone and inhibin from the ovary and prostaglandin 72H (EF72H) from the uterus. The primary timing mechanism of the bovine estrous cycle is the demise of the corpus luteum (4D)" which occurs about =ay ($ (< in the normal cycling" non pregnant cow. The simplest hypothesis for regression of the 4D is that the non pregnant uterus secretes a luteolytic agent into the uterine venous blood. This material is transferred through a local veno arterial pathway to the ovarian artery whereby it reaches the ovary and causes luteolysis. EF7 2H has been proposed as the natural luteolytic agent although definitive proof and details of the mechanism(s) of action are unclear. .egression of the 4D results in a rapid fall in serum progesterone concentrations to values less than ( ng1ml. D9 pulse fre!uency increases and follicular growth is further stimulated. The growth and maturation of the preovulatory follicle results in increasing secretion of estradiol" which causes estrogenic changes in the oviduct and uterus" behavioral estrus" and a preovulatory release of D9. The preovulatory D9 pea' results in resumption of oocyte meiosis" ovulation 2* /2 hr. later (the D9 pea' occurs around the onset of estrus) and luteini2ation of the ovulated follicle to form a secreting corpus hemorrhagicum. Frowth and development of the corpus hemorrhagicum into a fully functional 4D results in progestational changes in the oviduct and uterus that are conducive to embryonic development and establishment of pregnancy. @hould pregnancy not occur" the cycle will begin again with the demise of the 4D on about =ay ($ (< following ovulation. %t has now been shown by ultrasonography that follicles in cattle develop in a wave li'e fashion (Eierson and Finther" ()<*). Bovine estrous cycles are composed of 2 or / waves of follicular development. 0 follicular wave consists of a group of growing antral follicles / 6 mm in diameter from which a dominant follicle is selected while the remaining follicles become subordinate and undergo atresia (Finther et al." ()<)). %n both 2 and / wave estrous cycles" emergence of the first follicular wave occurs on the day of ovulation (=ay 0) while the second wave emerges ) or (0 days after ovulation in 2 wave cycles" and on < or ) days after ovulation in / wave cycles" with a third wave emerging on =ay (, or (6.

=uration of the estrous cycle is appro-imately 20 days in 2 wave cycles and 2/ days in / wave cycles. The dominant follicle present at the time of luteolysis will become the ovulatory follicle" and emergence of the ne-t wave is delayed until the ensuing ovulation. The proportion of animals with 2 vs. / wave cycles are probably more or less e!ually distributed" and follicular waves have been reported in heifers before puberty (0dam" ())<)" and postpartum cows before the first ovulation (0dam" ())<). 7ollicle waves persist in pregnant animals until appro-imately the last / wee's before parturition. .ecruitment of follicular waves and selection of a dominant follicle is based on differential responsiveness to 7@9 and D9 (0dam et al." ())2). @urges in plasma 7@9 are responsible for eliciting the emergence of a follicular wave. 7@9 is subse!uently suppressed by products of the growing follicles (e.g." estradiol and inhibin). %n each wave" the follicle that first ac!uires D9 receptors becomes the dominant follicle while subordinates undergo atresia. @uppression of D9 as a conse!uence of progesterone secretion by the 4D causes the dominant follicle to eventually cease its metabolic functions and it begins to regress. This leads to 7@9 release and emergence of a new follicular wave. Duteal regression allows D9 pulse fre!uency to increase" the dominant follicle increases its growth and dramatically higher concentrations of estradiol result in a positive feedbac' on the hypothalamo pituitary a-is and a surge of D9 followed by ovulation.

F')%r# 18 Events during the estrous cycle in cows with two wave cycle

F')%r# (8 Events during the estrous cycle in cows with three wave cycle 1.(.( S#/#&t'o o" $o or &o1 The first step is the selection of the donor cow. Embryo transfer is not a Acure all.B %t does not ma'e average cattle good or good cattle better. %t is suitable for a limited number of seed stoc' producers with beef or dairy cattle that can be breed or species AimproversB for one or more economically important traits. The potential donor cow should be reproductively sound to produce ma-imal results. This means that she should have a normal reproductive tract on rectal palpation and have a normal postpartum history. 4ows should be at least 60 days postpartum before the transfer procedure begins. =onor cows for ET should be selected on following basis8 a) .egular heat cycles commencing at a young age. b) 0 history of no more than two breeding per conception. c) Erevious calves were born at appro-imately /6, day intervals. d) Io parturition difficulties or reproductive irregularities. e) Io conformational or detectable genetic defects. 1.(., E!tr%! !y &*ro '2at'o ' $o or! =elicate and comple-ly balanced level of gonadotropin releasing hormone (Fn.9)" gonadotropins (7@9 and D9)" ovarian steroids (estrogen and progesterone) and prostaglandin 72J by endocrine and neuro endocrine systems are responsible for overall cyclic activity during estrus cycle of animals (4unningham and +lein" 200$8 Ioa'es et al. 200(8 +o5ima" 200/). The estrus in donors is synchroni2ed for two purposes. 7irst" by synchroni2ing we can collect embryos from several donors at the same time" and the second" it helps to bring both the donors and recipient animals in same stage of cycle which is a must for physiological adaption of embryo from donor in the uterus of recipients (@enger" 200/). :a5or organ manipulated is the 4D and either lifespan of 4D is shortened or prolonged for estrus synchroni2ation by using prostaglandin or progestagen respectively (Bearden and 7u!uay" 2000). 0dministration of prostaglandin results in low blood progesterone level by regression of 4D which in turn causes elevated

level of endogenous gonadotropins (7@9 and D9) due to higher Fn.9 secretion (@enger 200/" Bearden and 7u!uay" 2000) which in turn causes all of the animals with functional 4D of , to ($ days come into estrus within * days (Bearden and 7u!uay" 2000).To cover animals with immature 4D (age of less than , days)" two in5ections of EF72J in (( (* days apart is used (Bearden and 7u!uay" 2000). Erogestagen (or progestin) method was used to synchroni2e estrus in both donors and recipients in this study. %n progestagen method" progesterone is used for appro-imately $ days which inhibits release of Fn.9 and gonadotropin with halted follicular growth and ovulation (Bearden and 7u!uay" 2000" Iiasari Iasla5i" ())6) in animals with functional 4Ds which regress within progesterone supply period (@enger" 200/3 Bearden and 7u!uay" 2000). .emoval of progesterone causes spontaneous estrus in these animals while the other animals with functional 4D till the end of progesterone supply are brought to estrus in / to , days by in5ecting EF72J (@enger" 200/3 Bearden and 7u!uay" 20003 >ainudeen et al. 2000). %ntravaginal controlled internal drug release (4%=.) inserts were used as the source of progesterone. 1.(.- S%+#ro0%/at'o To ac!uire the benefit from embryo transfer program" it is essential to produce more number of oocytes in single cycle from an elite dam to increase genetic gain by increasing selection intensity in female. @uperovulation is the release of multiple eggs at a single estrus. The process of embryo transfer includes a donor hormone treatment referred to as superovulation" which results in appro-imately (0 ova ovulated at estrus" instead of the usual one egg. Typically" ,0K of the ovulation results in transferable embryos. The single most important component in ET is superovulation. The physiology and biochemistry involved in successful superovulation are not completely understood. %n general" <,K of all Lnormal" fertileL donors will respond to superovulation treatment with an average of five transferable embryos" and can be repeatedly superovulated at 60 day intervals with a slight decrease in embryo production over time. This multiple ovulation was achieved by in5ecting follicle stimulating hormone (7@9) which either recruit new follicles for ovulation by inhibiting the function of inhibin (4unningham and +lein" 200$) or prevent already growing follicle to become atretic (Iiasari Iasla5i" ())6). @uperovulation protocols also comprised of EF72H to regress pre e-isting 4Ds and a heat or estrus to occur appro-imately *< to 60 hours later. @uperovulation protocol in donors has been initiated by ma'ing sure that the reproductive cycle of donors and recipients coincide (Bearden and 7u!uay" 2000). =etail protocol of estrus synchroni2ation and superovulation program in donors is as3 Tab/#1. 3#ta'/ +roto&o/ "or !y &*ro '2at'o o" #!tr%! a $ !%+#ro0%/at'o ' $o or!
3ay 0 2 6 $ < ) Hormo #4$#0'&# 4%=. Fn.9 7@9 ( and 219eat detector 7@9 / and * 7@9 , and 61 .emove 4%=. 7@9 $ and <1 EF72H No.43o!#! ?ne *ml am and pm %1m / ml am and pm %1m 2 ml am and pm %1m 7@9 ( ml am and pm %1m EF72H , ml am and pm %1m

So%r&#: (Ba5agai" 20(/)

F')%r# ,: =etail protocol for estrus synchroni2ation and superovulation in donors 1.(.5 R#&'+'# t !y &*ro '2at'o Before consideration is given to superovulation and embryo recovery" the surrogate host (recipient) must be discussed. 9ighest conception is achieved when an embryo is transplanted to a uterine environment that most closely resembles the environment that the embryo originated from. a) =ecide if recipients are needed on a daily1wee'ly year round basis" or once1twice per year. b) 4onfirm how many open recipients are available at the ranch" dairy" or ET center. c) @elect a synchroni2ation program. d) ?rgani2e the necessary drugs" supplies" and labor needs. Tab/#(. 3#ta'/ +roto&o/ "or !y &*ro '2at'o o" #!tr%! a $ !%+#ro0%/at'o '
3ay 0 $ < Hormo #4$#0'&# 4%=. EF72H1 9eat detector .emove 4%=.

r#&'+'# t

No.43o!#! ?ne , ml %1m

So%r&#: (Ba5agai" 20(/)

F')%r# -: =etail protocol for estrus synchroni2ation in recipients 1.(.6 Art'"'&'a/ ' !#m' at'o Because of the release of many ova from the multiple follicles on the ovaries over a period of several hours" there is a greater than normal need to be certain that viable sperm cells reach the oviducts of the superovulated females. Therefore" many embryo transfer technicians will choose to inseminate the cow several times during and after estrus. Csing high !uality semen with a high percentage of normal" motile cells is a very critical step in any embryo transfer program. The correct site for semen placement is in the body of the uterus. @emen is placed either in the body of the uterus or at the entrance into each uterine horn. 0n artificial insemination program that yields a high embryo fertili2ation rate is the product of four factors8 9eat detection @emen !uality @emen handling @emen placement ?va are randomly released (ovulated) from superovulated ovaries over a period of si- to twelve hours. 7or this reason" the donor must be inseminated at least twice. The first breeding should occur (2 hours after the onset of standing heat. The second breeding occurs (2 hours after the first insemination. %f the donor is still in standing heat after the second insemination" she should then be in5ected with * ml of Fonadotropin releasing hormone (Fn.9" Cystorelirz). Breed the donor a third time (2 hours after in5ecting Fn.9. The insemination procedure is followed as3 6erify sire and dam blood type compatibility from breed association. ?rder semen and inventory the purchase after its arrival. Einpoint the donorMs onset of standing heat. %dentify the sire" and then thaw semen at /,N4 for *0 seconds. Doad semen into a pre warmed insemination rod.

&earing a shoulder length plastic sleeve" clean out the rectum if necessary" and then grasp the cervi-. &ipe the vulva clean with a paper towel. Eart the vulva by placing a clean" folded paper towel between the vulva lips. Eass the breeding rod through the cervi-" and stop when the rod has 5ust entered the uterus. @lowly (five seconds) plunge semen into the uterus. 0void pushing the rod forward while plunging. &ithdraw the insemination rod. .ecord the breeding by noting the complete name and registration number of both sire and dam sire code number" date" and name of inseminator.

1.(.7 Embryo r#&o0#ry 0fter donors have been artificially inseminated" fertili2ation will occur and pre implantation embryonic development will begin. The embryos reside in the oviduct for appro-imately four days before migrating to the tips of the uterine horns (&inters et al. (),/). ?nce embryos are found in the uterus" it is possible to recover them using nonsurgical embryo recovery techni!ues. Ion surgical embryo recovery methods emerged in the mid ()$0s (=rost et al. ()$63 Elsden et al. ()$6) and revolutioni2ed the bovine embryo transfer industry. There are two different non surgical embryo recovery methods. ?ne method is the gravity flow method. &ith this method" the sterile flushing medium is suspended above the donor cow and the force of gravity introduces medium into the uterine horn. 0dvantages of this method are over filling of the uterine horns seldom occurs and the system is AclosedB" minimi2ing li'elihood of contamination of the flushing medium and embryos. The second method" 'nown as the syringe method" utili2es 60 cc syringes to introduce and remove medium from the uterine horns. 0dvantages of this method include 'nowing the e-act volume of medium introduced and recovered from the uterus" as well as the ability to more forcefully e-tract flushing medium from the uterus. ?ne of the disadvantages is it is an Aopen systemB (the syringe is connected and disconnected from the catheter multiple times for each uterine horn)" providing an opportunity for potential contamination of the embryos. This may be of particular concern for embryos destined for e-port. 1.(.7.1 Co//#&t'o $at# a $ r#&'+'# t '$# t'"'&at'o Bovine embryo collection is generally attempted on day $ after estrus" which yields a development stage suitable for free2ing. Embryos are fre!uently located in the oviducts prior to day 6 and therefore can;t be collected by the nonsurgical recovery techni!ue. By day <.," the clear sphere (2ona pellucida) surrounding the embryonic cellular mass has ruptured" leaving an embryo too mature for free2ing. Eregnancy rates from fresh (not fro2en) embryos collected and transferred 6 to < days after heat are not different. 9owever" in order to correctly free2e embryos produced in e-cess of recipients on a particular day" collection should be scheduled for day $. &hether collection is done on farm or at the ET center" the first activity on collection day (or the evening before) will be recipient selection. 0ssuming a day $ collection" obtain a list of all recipients that are 6" $" or < days after heat. Ealpate both ovaries of each animal to identify the presence of a well formed 4D. 7or the most accurate and sensitive palpation" first remove the fingertips from a shoulder length plastic sleeves" put the sleeve on" and then pull on a snug fitting late- e-amination glove. This combination offers ine-pensive" disposable" full length protection and permits accurate identification of ovarian structures. ?nce a well formed 4D is identified" use a cattle mar'ing crayon to place a slash on the hip corresponding to the ovary (right or left) that the 4D is located on. 0 record should be 'ept on all useable recipients (those with a well formed 4D) for

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convenient recipient1embryo matching later in the collection day. The useable recipient list identifies how many embryos can be transferred fresh" and subse!uently when to free2e e-cess embryos. 1.(.7.( E8%'+m# t a $ $o or +r#+arat'o 0fter recipient identification" the ne-t step is to organi2e and prepare the collection" free2ing" and transfer e!uipment and supplies. 1.(.7., Embryo )ra$' ) 0s the individual embryos are located using the microscope" they are evaluated for their !uality and classified numerically as to the potential li'elihood of success if transferred to a recipient female. The ma5or criteria for evaluation include8 .egularity of shape of the embryo 4ompactness of the blastomeres (the dividing cells within the boundaries of the embryo). 6ariation in cell si2e. 4olor and te-ture of the cytoplasm. ?verall diameter of the embryo. Eresence of e-truded cells. .egularity of the 2ona pellucid. Eresence of vesicles. Csing these sub5ective criteria embryos are classified as8 Tab/# ,. C/a!!'"'&at'o o" #mbryo ' $'""#r# t )ra$#!
Gra$# Frade ( Frade 2 Frade / Frade * R#mar9! E-cellent or Food 7air Eoor =ead or degenerating

So%r&#8 70? (200*) Embryos also are evaluated for their stage of development without regard to !uality. These stages are also numbered8

Tab/# -. C/a!!'"'&at'o o" #mbryo ' a&&or$' ) to $'""#r# t !ta)#!

Sta)# @tage ( R#mar9! Cnfertili2ed

11

@tage 2 @tage / @tage * @tage , @tage 6 @tage $ @tage < @tage )

2 to (2 cell Early morula :orula Early Blastocyst Blastocyst E-panded Blastocyst 9atched Blastocyst E-panding 9atched Blastocyst

So%r&#: 70? (200*) There is apparently no difference in pregnancy rates of fertili2ed cells in different stages of development" assuming that they are transferred to a recipient female in the appropriate stage of the estrous cycle. @tage *" ," and 6 embryos endure the free2ing and thawing procedures with the greatest viability. Embryo !uality is also of utmost importance in the survival of the free2ing and thawing stress. Frade ( embryos are generally considered the only ones to free2e. Frade 2 embryos can be fro2en and thawed" yet pregnancy rates typically are reduced. 1.(.7.- Embryo *a $/' ) 7ollowing collection and filtration of the medium" embryos are located under 6 (0- magnification with a stereoscope. 0lthough embryos are usually transferred to recipients as soon as possible after collection" it is possible to maintain embryos for (2 to (< hours at room temperature in holding medium (9asler" 200/). %t is possible to cool bovine embryos in holding medium and to maintain them in the refrigerator for 2 to / days. 7or long term storage" embryos must be cryo preserved. Embryos are normally held in a medium similar to that in which they were collected. :edia must be buffered to maintain a p9 of $.2 to $.6 and have an osmolarity around 2$,m?s. =ulbeccoMs EB@ or commercially available media with 9EEEs buffer" B@0 and antibiotics are normally used in the field. :ore comple- media with a bicarbonate buffer generally yield superior results for long term culture of bovine embryos" but they must be maintained in an incubator with an elevated 4? 2 atmosphere. 0s indicated earlier" embryo collection" holding and free2ing media which are free of animal products have recently become available" avoiding the need for refrigerated storage and increasing biosecurity. 1.(.7.5 Embryo "r##2' )4 #mbryo &ryo:+r#!#r0at'o Embryo cryopreservation is the preservation of embryos by free2ing (the prefi- LcryoL refers to free2ing). The first successful cryopreservation of mammalian embryos was achieved in ()$( by researchers wor'ing with mouse embryos (&hittingham et al. ()$23 &ilmut" ()$2)" and that brea'through was soon followed by successful cryopreservation of bovine embryos (&ilmut and .owson" ()$/). :uch of the initial impetus for research on cryopreservation of embryos stemmed from the desire to move Le-oticL breeds of cattle from one part of the world to another. Transporting germplasm resources as embryos rather than as live animals is cheaper" poses less ris' for disease transmission" and enables embryo transfer calves to develop in a recipient female whose colostrums provides maternal antibody protection against diseases endemic in that area. There are a number of other reasons why embryo cryopreservation may be employed. %t prevents wasting of LsurplusL embryos when donors produce a greater than e-pected number of embryos or when recipients do not e-hibit

12

estrus synchronously with the donors. 4ryopreservation may be utili2ed when recipient females are unavailable" inade!uate" or in instances when it may simply be more convenient to perform embryo transfer at a later time (e.g." when embryos from a particular donor are being stoc'piled). Embryo cryopreservation is used by many purebred breeders as a secondary avenue for mar'eting of their breeding stoc'" as embryos from a genetically valuable donor can be harvested and sold without having any change in ownership of the donor. .ecently" embryo cryopreservation also has become an integral part of germplasm preservation efforts. The primary obstacle to overcome during embryo cryopreservation is the high water content within the embryo. &hen water free2es" ice crystals form. These ice crystals have sharp" 5agged edges capable of cutting cell membranes and1or cell organelles. %f the membranes or organelles of a substantial number of cells in an embryo are damaged" death of the embryo will occur. 9ence" the primary challenge in embryo cryopreservation is the LdehydrationL of the embryo prior to free2ing. This is accomplished by placing embryos into a hypertonic solution containing a cryo protective agent" which forces water out of the cells of the embryo (Deibo" ())2). 9istorically" glycerol was the predominant cryo protective agent used for embryo cryopreservation. 9owever" glycerol is now being rapidly replaced by ethylene glycol. The process of free2ing embryos resembles the entire ET se!uence in that numerous steps are involved. 0ll preparations and procedures for free2ing must strive to minimi2e the time span between collection and free2ing" with highest pregnancy rates achieved when embryos are fro2en within two hours of collection. 1.(.7.6 Embryo !#/#&t'o ; 1a!*' ) a $ "r##2' ) @elect embryos to be fro2en according to stage of development" !uality" and integrity of 2ona pellucida. Eass embryos through (0 se!uential baths to LwashL pathogens from each embryoMs 2ona pellucida. @ome importing countries may re!uire two additional trypsin baths. &hen the embryo wash se!uence is complete" verify the integrity of each 2ona pellucida at ,0magnification. =ecide how many embryos will be pac'aged per straw" and then label straws according to %ET@ guidelines. Eartition active free2e media into three labeled dishes. 0ctivate the free2er" allowing it to stabili2e at ?N 4. Elace embryos into active free2e media. Doad embryos into rinsed" labeled straws. .ecord pertinent information on certificate 4. @eal the unlabelled straw ends and then insert straws into free2e chamber slots. @eed straws at ?N 4. Dabel goblets and canes while embryos free2e. &hen free2e run ends" plunge straws into a nitrogen filled goblet attached to a labeled cane. @ecure labeled straw ends to the cane with an inverted goblet and then plunge the cane into its assigned canister for storage. 1.(.7.7 C#rt'"'&at'o ; '$# t'"'&at'o a $ /ab#/' ) &hen embryos are collected" complete certificate 0. &hen embryos are transferred" complete certificate B.

13

&hen embryos are fro2en" complete certificate 4. &hen embryos are e-ported" complete certificate =. 4opies of all donor health test results should be attached to the embryo free2ing certificate. 4ertificates 0 and 4 must accompany fro2en embryos when they are moved between locations. @traws must be labeled" at a minimum" with the code assigned to the free2ing party" the breed code" donor registration number" free2e date" straw number" and number of embryos per straw. Foblets and canes must be labeled" at a minimum" with cane number" free2ing party code" free2e date" breed code" and registered name and number of donor plus the bull that the donor was bred to. The identification of any embryo in a straw must correlate with information on an 0 4 certificate.

1.(.7.< Embryo !+/'tt' ) (3#m':#mbryo) Embryo of blastocyst stage is differentiated into two regions" tropho ectoderm and inner cell mass (%4:). Trophoectoderm is a single layer of trophoblast cells lining the inner side of 2one surrounding the blastocele (a fluid filled cavity). Oona becomes the fetal membrane of placenta. The embryos of blastocyst stage can be split into e!ual two halves and transferred to females to produce identical twins. Thus" embryo splitting technology has increased the rate of pregnancy. To carry out embryo splitting" the blastocyst stage embryos are transferred for a few minutes into a cell culture medium consisting of hypertonic sucrose and bovine serum albumin. The medium of high osmotic strength enters into cell membrane of the embryo (2ona pellucida) and cells contract due to e-osmosis. The bovine albumin serum attaches to 2ona pellucida and provides negative charge to the embryo. Then it is transferred into a plastic petridish containing standard cell culture medium. The outer membrane of embryo is negatively charged and petridish positively. Embryo stic's to the surface of Eetri dish due to development of electrostatic interaction of two charges. Eetridish is 'ept on stage of an inverted microscope. 0 micromanipulator e!uipped with fine surgical blade is used to cut the embryo roughly into two halves with minimal damage to the cells. The hemispherical mass of half embryo" demi embryo or semi embryo reforms spheres. 9owever" it is not necessary for successful implantation of demi embryo that it should be enclosed in a 2ona pellucida. The demi embryos are transferred into oviduct of synchroni2ed recipients as described for normal embryo transfer. 0t this stage it is very necessary to 'now the situation for successful embryo implantation that the wall of uterus of synchroni2ed female recipient is waiting for embryo" and embryo is prepared to send chemical signals from the trophoblast. To increase the pregnancy rate" embryo can be cut into e!ual three or four pieces" and transplanted in synchroni2ed females. But too small trophoblast will not induce the uterus for pregnancy. Embryo biopsy (removal of small number of cells for genetic analysis) should be combined with splitting so that the twins which will be produced should be identical and of 'nown genotype. Biopsy is very necessary in breeding so that se- and genetic diseases could be detected. %n case the embryo has any genetic disease" it can be prevented from implantation in recipient females.

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F')%r# 58 0n outline of embryo splitting. 1.(.7.= Embryo !#>' ) @e-ing of embryos before transfer and implanting has great potential for the livestoc' industry. :anipulating the se- of offspring has been a dream of the cattle industry for decades. 0 number of approaches to the se-ing of semen have been attempted" and several have been reported as successful. Embryo se-ing has been attempted by a variety of methods" including cytogenetic analysis" assays for P lin'ed en2yme activity" analysis of differential development rates" detection of male specific antigens" and the use of # specific =I0 probes. @ince the development of =I0 amplification techni!ues" particularly the polymerase chain reaction (E4.)" these techni!ues have been applied to numerous situations in which the analysis of rare se!uences is desired. the use of polymerase chain reaction (E4.) machine in se- detection. E4. amplifies =I0 se!uence of # chromosome and reaction products can be seen directly. %t is true that the E4. was first commercially implemented for embryo se-ing of livestoc' Iow the se-ed embryos are available commercially but these are very costly (Ohu et al. 20(2). 1.,. Embryo tra !"#r %n cattle" embryos are routinely transferred to the uterine horn. This is because nearly all embryos are recovered non surgically from the uterus" and therefore should be returned to this site. 7urthermore" it is much easier to transfer embryos to the uterus than to the oviduct. Both surgical and non surgical methods of embryo transfer can be made to wor' well. Cnder most circumstances" non surgical transfer is greatly preferred" although surgical transfer can be done !uite rapidly" even in rather primitive circumstances. 1.,.1 S%r)'&a/ tra !"#r 0lthough thousands of embryos have been transferred via mid line abdominal incision to cows under general anesthesia" in most circumstances a flan' incision is far more practical. .ecipients are placed in s!uee2e chutes that give access to either flan'. The 4D is located by rectal palpation and the flan' ipsilateral to the 4D is clipped" washed with soap and water" and sterili2ed with iodine and alcohol.

15

0bout 60 ml of 2 percent procaine is given along the line of the planned incision. %n everyday practice this seems more reliable than using a paravertebral bloc'. 9aving scrubbed" the surgeon ma'es a s'in incision about (, cm long" high on the flan'" 5ust anterior to the hip. :uscle layers are separated" and the peritoneum is cut. The surgeon inserts a hand and forearm into the incision" locates the ovary" usually about 2, cm posterior to the incision" and visuali2es or palpates the 4D. The uterine horn is e-teriori2ed by grasping and stretching with the thumb and forefinger the broad ligament of the uterus" which is located medial to the uterine horn. The uterine horn itself is very fragile. 0 puncture wound is made with a blunted needle through the wall of the cranial one third of the e-posed uterine horn. Csing about 0.( ml of medium in a small glass pipette (Q(., mm outside diameter)" an assistant draws up the embryo from the storage container. The pipette is then inserted into the lumen of the uterus" and the embryo is e-pelled. %t ta'es some e-perience to be confident that the embryo has been deposited in the lumen. The incision is then closed" using two layers of sutures. &ith practice" the surgery ta'es about (, minutes. 1.,.( No :!%r)'&a/ tra !"#r The big problem with non surgical transfer is the difficulty in becoming proficient in this techni!ue. 7irst" it is necessary to be able to palpate ovaries accurately in order to select the side of ovulation. Eregnancy rates are mar'edly lowered if embryos are transferred to the uterine horn contra lateral to the corpus luteum (@eidel" ()<(). 0lso" recipients should be re5ected if no corpus luteum is present or pathology of the reproductive tract is noted. Even very e-perienced palpators ma'e some errors in palpating corpora lutea. The ne-t step is to pass the embryo transfer device through the cervi-. This is more challenging during the luteal phase" which is when embryos are transferred" than during estrus" when artificial insemination is done and the cervi- is more open. 9eifers present a special challenge because of the small cervi-3 some breeds of cattle are more difficult than others" e.g. certain Bos indicus breeds re!uire greater s'ill. The best training prior to underta'ing non surgical embryo transfer is e-perience in artificial insemination. The third step with non surgical transfer is to be able to insert the tip of the instrument into the desired uterine horn !uic'ly" smoothly and atraumatically. @ome people never master this techni!ue" and others re!uire hundreds of transfers to become proficient. This is not surprising since pregnancy rates from artificial insemination are usually mar'edly lower for the first ,0R (00 cows bred by a newly trained inseminator than after he or she has become proficient. &ell trained inseminators generally re!uire (00 200 non surgical transfers until their pregnancy rates plateau3 others usually re!uire more. :ost technicians who are successful with non surgical transfer had low pregnancy rates for their first (00 non surgical transfers. ?ne approach for people starting an embryo transfer program is to begin with surgical procedures until acceptable pregnancy rates are achieved. %f one begins with non surgical transfer and pregnancy rates are low" it is difficult to distinguish among problems such as identifying usable embryos" problems with media" problems in storing embryos from collection to transfer" poor non surgical embryo transfer techni!ue" recipient problems" etc. Eregnancy rates are fre!uently low with surgical embryo transfer also" but one of the problems 5ust mentioned is usually the cause" not the surgical transfer. ?nce the entire se!uence of superovulation" embryo recovery" surgical embryo transfer" etc." is wor'ing well" it is advisable to switch to non surgical transfer until proficiency is achieved. 0 fre!uent error is to have a number of embryo transfer teams wor' in a given province or country" none of which become proficient because of insufficient opportunities to gain e-perience. The result is that proficiency is attained slowly or not at all" and the program is abandoned because of poor results. @ome people believe that there is a , (0 percent advantage in pregnancy rates with surgical transfer" even when very proficient technicians are doing the non surgical transfer. Even if this is true" in most circumstances nonsurgical transfer is still preferred because it is less e-pensive3 it is !uic'er and

16

does not involve surgical procedures. This may also obviate the need for veterinary supervision" which is re!uired for surgery in many countries. 1.- .r#) a &y $'a) o!'! The first good indicator of pregnancy is failure of the recipients to show estrus (<R2* days after the pre transfer estrus3 obviously" the converse" showing estrus" indicates non pregnancy" although a small percentage of pregnant animals are in behavioral estrus about three wee's after the previous estrus. Erogesterone assay of mil' or blood samples 22R2* days after the pre transfer estrus is ), percent accurate in diagnosing non pregnancy and about <0 percent accurate for pregnancy. 9owever" with good estrus detection" one gets about the same information as with progesterone tests. %n other words" if the accuracy of estrus detection is so poor that progesterone tests provide much additional information" the embryo transfer program is li'ely to fail because of poor estrus detection. 0nother point is that neither estrus detection nor progesterone assay gives sufficiently accurate information for definitive pregnancy diagnosis on individual recipients. The information from returns to estrus is very useful on a population basis" however" because it gives early information concerning success or problems. 0t about day 26 of pregnancy in heifers and day 2< in cows" pregnancy can be diagnosed accurately under field conditions by ultrasonography or even earlier in very s'illed hands. %n most embryo transfer program" ultrasonography e!uipment is not 5ustified" although it is very useful for a variety of purposes. &hen costs of ultrasonography e!uipment decline to half current prices" such e!uipment will probably be considered indispensable and will come into general use. 0 serious problem with early pregnancy diagnoses is that about (0 percent of 2< day pregnancies will not go to term. %t has been found that ), percent of two month and )$ percent of three month embryo transfer pregnancies go to term. Eregnancy diagnosis can usually be diagnosed definitively by palpation per rectum after day /, of pregnancy. ?f course" ultrasound can also be used at these later stages. &e do not recommend palpation prior to day *," both because the conceptus is more fragile at early stages and because the information is not definitive anyway due to occurrence of spontaneous abortion even in the absence of palpation. Thus" our recommendation is to palpate per rectum at *, 60 days of gestation and confirm this with another palpation one month later. 4ows that show estrus may be chec'ed earlier than *, days by palpation or ultrasonography. %f they are" in fact" non pregnant they can be recycled for use as recipients. %t is often useful to distinguish between an embryo transfer pregnancy and one from artificial insemination or natural breeding during the ne-t cycle. The following steps will ma'e it possible for most non pregnant recipients to be bred or reused as soon as possible. 7irst" it is imperative not to breed recipients that show estrus earlier than normal" that is artificial insemination or e-posure to bulls should be delayed ($R(< days from pre embryo transfer estrus. This ma'es the )0 ), percent of non pregnant recipients without short cycles eligible for breeding. Eregnancy diagnosis is then done in a window of time that permits une!uivocal distinction between the embryo transfer pregnancy and a possible subse!uent one" which will be at least ($ days younger. &ith rectal palpation" this window may e-tend to about 6, days after pre transfer estrus for s'illed palpators" and perhaps to 60 days for less s'illed ones. Iote that a good portion of this window is needed if embryo transfer was done over a seven to ten day period" if all recipients are to be e-amined at once" and recipients are not palpated prior to a *, day pregnancy. ?ne other point is that it is common to diagnose pregnancy per rectum by Aslipping membranesB. This method should not be used" at least not prior to day ,0" because it leads to abortion in a significant" though small" percentage of cases. Ealpation should be done on the basis of fluids" tone and

17

si2e of the uterine horn. The small percentage of false positives due to undiagnosed uterine pathology is lower than the abortion rate caused by slipping membranes.

F')%r# 6: Sy o+!'! o" bo0' # #mbryo tra !"#r +ro&#$%r#

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1.5 A++/'&at'o ! o" #mbryo tra !"#r ?ver the years" techni!ues associated with embryo transfer have had many uses" especially in research. The widespread use of this technology in animal breeding schemes" however" is relatively recent. Fenetic engineering and related technologies will only increase its utili2ation. %n fact" many laboratories are presently using in vitro fertili2ation techni!ues to study the fertili2ing capacity of sperm (Brac'ett" ()<(3()</3 &right and Bondioli" ()<(). 0 few of the more common uses of embryo transfer technology in animal production follow. 1.5.1 G# #t'& 'm+ro0#m# t Fenetic progress is considered to be slower through the use of embryo transfer than it is using conventional artificial insemination (0%)" especially on a national herd basis (Bradford and +ennedy" ()<0). 9owever" with increased selection intensity and shortened generation intervals" i.e. transferring female offspring" genetic gain can be made on a within herd basis (:cdaniel and 4assell" ()<(). %t was possible to genetically test a bull in /., years as opposed to ,., years using traditional progeny testing schemes" which also resulted in shortened generation intervals (:apletoft" 20(/). The production of about si- offspring per donor cow could double selection intensity and the rate of response to genetic selection for traits such as growth that can be measured in both se-es (Dand and 9ill" ()$,3 4hurch and @hea" ()$$3 &ilmut" ()<2). This would be especially worthwhile in improving elite herds" the genetics of which could be spread over a large population through the use of 0%. 0lthough the rate of genetic improvement in dairy cattle may range from 2., <K" @eidel suggested that through the use of embryo transfer it could be increased three to four times if dairy replacements were selected from the top (0K of the herd (@eidel" ()<(). %n addition" new genomic techni!ues are being used increasingly to select embryo donors3 genomic analysis has become essential for the selection of bull dams to be used in embryo transfer (@eidel" 20(0). Embryo transfer is now commonly used to produce 0% sires from the very best proven cows and bulls available. Economics would not at this time support the use of embryo transfer techni!ues for anything but seed stoc' production. %t allows a tremendous shortening of the generation interval considering the slow maturity and the long interval between birthing in these species. This techni!ue can also be used as a method to maintain superior females in production that may have ac!uired pathologies that reduce their ability to maintain a pregnancy to term. 1.5.( ./a #$ mat' ) By far the most common use of embryo transfer in animal production program is the proliferation of so called desirable genotypes (@eidel and @eidel" ()<(). 0% has permitted the widespread dissemination of a maleMs genetic potential. Embryo transfer provides the opportunity of disseminating the genetics of proven elite females (Eowell" ()<(). Embryo transfer also permits the development of herds of genetically valuable females" most of which may be sibs if not full sibs. 0s 0% has led to the very valuable bull" now embryo transfer has resulted in the very valuable female. :any breeders have identified individual females whose offspring are most saleable and used them e-clusively in embryo transfer. Embryo transfer has also been used to rapidly e-pand a limited gene pool. 1.5., G# #t'& t#!t' ) 0 common use of embryo transfer procedures is to genetically test 0% sires for deleterious heredity traits (>ohnson et al. ()<0). @ome 0% organi2ations 'eep 'nown carriers of certain genetic defects on hand to

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serve as donors in testing new sires. Embryos are transferred into unrelated recipients and pregnancy may be terminated at various stages to e-amine fetuses for presence or absence of the defect. =epending on the heritability of the defect" generally eight to ten non affected fetuses are sufficient to declare a bull free of that trait. 0nother alternative is to mate the bull in !uestion to seven or eight of his superovulated daughters. ?ffspring will then represent all recessive traits that a bull may carry. 0 less attractive alternative would be to naturally mate the bull in !uestion to *0 ,0 of his daughters. 1.5.- T1' ' ) ' &att/# 4ompared to other domestic animals" beef production is somewhat less efficient because not all cows produce a calf each year. 9owever" it has been estimated that unit beef production can be increased by 60K in intensively managed herds through twinning (@reenam" ()$$). 0ppro-imately $0K of nutrient inta'e by a beef cow is utili2ed for her own maintenance" whereas only about /0K is for growth and maintenance of the calf during pregnancy and lactation (@eidel" ()<(). Thus" it seems desirable to ta'e advantage of the efficiency of gestation and lactation. Fenetic selection for twinning in cattle has been largely unsuccessful (@reenam" ()$$) and Fonadotropin treatments to induce twinning have also been unreliable (Bellows and @hort" ()$2). Embryo transfer does provide a very real alternative in the production of twins (.owson et al." ()<(). The most limiting factor at this time is the cost of transfer which continues to e-ceed the average price for calves to be raised for meat (@eidel and @eidel" ()<(). The nonsurgical transfer of a previously fro2en embryo to a recipient that has already been serviced may become an economical method of producing twins. ?ther alternatives include the nonsurgical transfer of two or three fro2en embryos to commercial beef cows (0nderson" ()$<). %t must be remembered that recipients carrying twins will re!uire e-tra nutrition and management" especially around calving. 7urthermore" recipients must be sufficiently large to carry twins and produce enough mil' to feed twins (Fordon and Boland" ()$)). 7reemartins" ma'e twinning unattractive for pure breeding purposes. 9owever" the incidence is relatively low and se-ing of embryos would eliminate the problem entirely. @imilarly" the production of identical twins by microsurgery would ma'e twinning very feasible for embryo transfer program in purebred herds (@eidel" ()<2). 1.5.5 3'!#a!# &o tro/ @everal large studies have now shown that in vivo produced bovine embryos do not transmit infectious diseases (:apletoft" 20(/3 @ingh and 9are" ()<,). 4onse!uently" it has been suggested that embryo transfer be used to salvage genetics in the face of a disease outbrea' (Eaglesome et al." ()<0). This may be a useful alternative in the establishment of dairy herds that are free of bovine leu'osis as this virus was not transmitted with embryos (@ingh et al." ()<*). %n fact" the %ET@ has categori2ed disease agents based on the ris' of transmission by a bovine embryo (@tringfellow and Fivens" 20003 :apletoft and 9asler" 200,). 4ategory ( diseases include disease agents for which sufficient evidence has accrued to show that the ris' of transmission is negligible" provided that embryos are properly handled between collection and transfer. This includes inspection of the 2ona pellucida at more than ,0P magnification and washing1trypsin treatment procedures. 4ategory ( diseases include En2ootic bovine leu'osis" 7oot and mouth disease (cattle)" Bluetongue (cattle)" Brucella abortus (cattle)" %nfectious bovine rhinotracheitis" Eseudorabies in swine and Bovine spongiform encephalopathy. 4ategory 2" /" and * diseases are those for which less research information has been generated. 9owever" it is noteworthy that none of the infectious diseases studied have been transmitted by in vivo produced bovine embryos. 4onse!uently" it has been suggested that embryo transfer be used to salvage genetics in the face of a disease outbrea' (&rathall et al." 200*). 1.5.6 Im+ort a $ #>+ort

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The ability to utili2e embryos in preventing the transmission of infectious disease ma'es them ideal for the international movement of animal germplasm (:apletoft" 20(/). The intercontinental transport of a live animal may cost S("000 or more" whereas an entire herd can be transported" in the form of fro2en embryos" for less than the price of a single plane fare. This may be the single most important potential application of embryo transfer. 0dditional benefits of the e-port of embryos over that of live animals include a wider genetic base from which to select" the retention of genetics within the e-porting country and adaptation. This is particularly true of tropical and subtropical climates where the embryo would have the opportunity to adapt both in the uterus and then suc'ling a recipient indigenous to the area. There are several potential problems which must be overcome in order to ma'e the international movement of embryos commonplace. 7irstly" this use is dependent on the successful free2ing of embryos. @econdly" the inadvertent introduction of disease into a herd and1or country with or within the embryo presents some very difficult regulatory problems. &ell defined methods of collection" handling and washing embryos must be followed to ensure that disease transmission is avoided. 7inally" the international movement of embryos is heavily dependent on technology transfer as personnel within the importing country must be able to successfully thaw and transfer embryos. The 2ona pellucida of in vitro produced bovine embryos differs from that of in vivo derived embryos (@tringfellow and Fivens" 2000)" and it has been shown that pathogens are more li'ely to remain associated with in vitro produced embryos following washing than with in vivo derived embryos. This has potentially serious ramifications for international movement" and protocols must be revised accordingly. 1.5.7 Sa/0a)# o" r#+ro$%&t'0# "% &t'o Embryo transfer procedures have been useful in the diagnosis" treatment and salvage of reproductive function in so called infertile cows (Bowen et al. ()$<3 Elsden et al. ()$)3 :apleloft et al. ()<0). 0lthough it is recommended that the cause of the infertility not be of genetic origin" this is often difficult to determine. The mar'etplace should sort this out if a genetic problem were inadvertently propagated through embryo transfer. 0nother very important use of embryo transfer is to salvage the genetics of terminally ill animals. %t may be possible to produce an additional two or three offspring through embryo transfer before the animal dies. The fact that the animal may not be cycling can be overcome through the use of progestational steroids given by in5ections or implant (:apletoft et al. ()<03 :apletoft" ()<2). 1.5.< R#!#ar&* Embryo transfer techni!ues have proven to be a very useful research tool. %n fact" many technical developments in embryo transfer prior to ()$0 were directed toward research purposes rather than for the propagation of superior livestoc'. These studies included natural limitations to twin pregnancies" uterine capacity" and endocrine control of uterine environment" maternal recognition of pregnancy" embryo endometrial interactions" and the endocrinology of pregnancy (@reenam" ()</). @tudies that were originally planned to answer basic physiologic !uestions are now being used to improve and increase the utili2ation of embryo transfer. Iewer techni!ues have added an entirely new perspective to the utili2ation of embryo transfer for research purposes. The production of identical twins" clones" chimeras" to mention a few" will certainly advance many of these sciences (@eidel" ()</). 1.5.= 3'!a$0a ta)#! o" #mbryo tra !"#r %t is important to note that pregnancy rate may be less than that e-pected in natural mating. .egulatory aspects will need to be developed as to what health re!uirements should be established for semen and embryo donors in order to avoid venereal transmission of disease.

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7inally from the economics aspects" value of animals may be reduced" particularly if the number of offspring is not regulated. ?ther important aspects that should be addressed concern ethical and animal welfare issues. %nterspecific ET" genetic testing and gender selection before transfer" use of poor fertility animals" superovulation effects on the female and animal suffering if the techni!ues are used by untrained persons" are 5ust a few of the aspects that should be considered in drafting any recommendations for the use of these biotechnologies. %ncreased e-penses and higher brea'even costs for calves. .e!uires a higher level of management. %ncreased potential for spread of certain diseases. Iot all potential donors respond positively to treatment (Frimes" 200<).

1.6 S%&&#!! rat# o" #mbryo tra !"#r @uccess rates in terms of embryo production per superovulation attempt" and pregnancy rates following transfer have changed little over the last several years" e-cept that we are now more able to control ovarian function and collect embryos more fre!uently. This has doubled embryo production in many donor cows. Eregnancy rates on a national basis are around 60 K with fresh embryos and ,, 60 K with fro2en1thawed embryos. The factors affecting success of embryo transfer are8 0ge and !uality of embryos @ite of transfer and no. of embryos transferred. =egree of estrus synchroni2ation between the donor and recipients %n vitro culture conditions @'ill of personnel and management techni!ues @uccess rates decline after the third or fourth superovulation with some donors. #oung cows may be more fertile as donors and recipients than heifers or old cows. 7ewer pregnancies will result per half embryo" but more per embryo collected" if embryos are split. Eregnancy rates are lower with non surgical transfer for some technicians. 7ewer pregnancies will result from fro2en embryos. 7resh semen is superior to fro2en semen from certain bulls. %nfertile donors will have lower responses than normal ones. 1.7 Co!t o" #mbryo tra !"#r The costs associated with ET can be significant and are highly variable. There are a wide range of services offered by ET technicians or organi2ations. 0 breeder can choose to have the ET process done entirely on the farm where the donor and recipients are located. 0nother option is to send the donor to a boarding facility and custom hire the entire process. .ecipients can be hauled in for implanting embryos or pregnant recipients can be purchased. The cost of collecting and free2ing embryos will vary depending on the number of donors being collected and the fee schedule of the ET practitioner. The most significant e-pense associated with ET will be the cost of owning and maintaining recipient cows. The recipient cow issue can be handled several different ways. Breeders who choose to own their own recipient cows can purchase them or use e-isting cows that are less desirable in terms of phenotype. The desire to have high mil'ing recipient cows has led to the popularity of dairy beef cross females. The annual cow maintenance cost is highly variable due to feed costs and contributes to the cost of raising an

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ET calf. Dabor costs will be significant for an operation considering ET. E-tra time must be devoted to the administration of drugs" heat detection" and artificial insemination. +eep in mind that for the average ET procedure" a donor cow will go through the chute (2 times and a recipient 2 times prior to flush day. 7acility costs will be similar to any operation using artificial insemination. Tuality of the facilities will be dictated by the number of donors and recipients as well as the time of year that ET wor' is being completed. 0dditional costs may include drugs or products for synchroni2ation" e-tra registration fees" 0.%. certificates" or blood typing or =I0 testing fees for donors or calves (Frimes" 200<). 1.< A$o+t'o o" #1 t#&* o/o)'#! a $ "%t%r# o" #mbryo tra !"#r 1.<.1 A$o+t'o o" #1 t#&* o/o)'#! Erenatal determination of se- potentially has great economic impact (@eidel" 200/) and the polymerase chain reaction (E4.) to determine the se- of bovine embryos is a service offered by many embryo transfer practitioners (Thibier and Iibart" ()),). 9owever" embryo biopsy re!uires a high level of operator s'ill" and is an invasive techni!ue resulting in disruption of the integrity of the zona pellucida and some reduction in the viability of the embryo" especially after cryopreservation. %n the near future" E4. assays to identify other traits of economic importance will no doubt become available (Bishop et al." ()),). :ar'er assisted selection (:0@)" based on identifying genetic mar'ers for un'nown alleles of valuable traits" probably has a similar future (Feorges and :assey" ())(). Di'e genotyping of specific alleles" :0@ can potentially be applied to embryo biopsies if sufficiently valuable mar'ers can be identified. 0 E4. assay currently e-ists for simultaneous detection of the bovine leu'ocyte adhesion deficiency gene and the se- of embryo biopsies (9asler" 200/). %t is probable that E4. techni!ues will be developed that permit the analysis of a large number of mar'ers from one biopsy leading to the concept of Aembryo diagnosticsB. %t is also li'ely that genomic testing of embryos with single nucleotide polymorphism (@IE) technology will occur in the near future" again utili2ing embryo biopsies and E4. technology (@eidel" 20(0). The flow cytometric technology used to separate P and # bearing sperm into live fractions has been improved over the last (, years (>ohnson et al." ())*3 >ohnson" 2000). 0ppro-imately (0 million live sperm of each se- can be sorted per hour (@eidel" 200/)" with a resulting purity rate of U)0K. %n 0% field trials" pregnancy rates following insemination with ( million se-ed" fro2en sperm were reported to be $0 to )0K that of unse-ed controls inseminated with 20 to *0 million sperm (@eidel et al." ()))). 0 recent study which compared ,$* calves produced from se- sorted sperm with /<, control calves concluded that there were no differences in gestation" neonatal deaths" ease of calving" birth weight or survival rate to weaning (Tubman et al." 200/). The disadvantages of flow cytometry are the slow speed of sorting" the decreased sperm viability (pregnancy rates)" especially in superovulated donor cows" the cost of the semen" and the availability of semen from specific bulls (0mann" ()))). %t is li'ely that se-ed semen will have the greatest use in bovine embryos in the near future. 1.<.( F%t%r# o" #mbryo tra !"#r The dimensions of development in the embryo transfer field are multiple and e-citing. The commercial reali2ation of the techni!ue is very li'ely to have a greater impact on research. The present technology indicates a trend of improvement and refinement. ?ne important area which is actively researched by many investigators" including the writers" is the storage of embryos by free2ing" similar to sperm preservation. 6ery promising success rates have recently been obtained with fro2en mouse embryos (&hittingham et al." ()$2). 0lso" a live calf has been born from the transfer into a recipient cow" by researchers in 4ambridge" England" of an embryo that had been fro2en. The future in this area loo's

23

promising. ?nce bovine embryos are successfully fro2en" ban's can be established and the world can e-perience a new era of animal trade between countries. %f embryos can be stored in a fro2en state their immediate transfer becomes unnecessary. They can be transferred to the desired host animal anywhere and at any time. This could tremendously reduce present costs and ma'e the practice accessible to many farmers. %n the meantime" it is possible that embryos can be shipped to other countries in a non fro2en state" as it has been shown by the writers and others that they can survive under relatively simple laboratory conditions for as long as four days. In vitro fertili2ation could be another area of development. The advantage here lies in the fact that the ovaries contain many more potentially viable eggs than can be utili2ed by the present system of in vivo fertili2ation. &hen abundant supplies of embryos become available from superior animals" even after slaughter" two could be transferred into one host cow" thus inducing artificial twinning which could have a great impact on meat production. 0nother possibility is the se-ing of these embryos before transfer" so that the se- of the calf to be born can be predetermined. This has obvious implications in the dairy sector. %t has also been shown that superovulation of immature females can be achieved3 the resulting embryos when transferred to mature animals develop and grow into normal calves. 0 considerable shortening of the generation interval and the early progeny testing of females are indicated here. These and possibly other developments in embryo transfer are e-pected to play a significant role in world animal production in the coming years" with substantial benefits for both developed and developing countries. 1.= Embryo tra !"#r t#&* o/o)y (ETT) ' N#+a/ Embryo transfer (ET) which is advanced in reproduction technology initiated in the country in cattle using >ersey fro2en embryos imported from Iew Oealand for the (st time in Iepal in 200/.This was initiated in 5oint collaboration of I0.4 and =D@ with some support from &inroc' %nternational. 4attle from Bovine .esearch Erogram" I0.4 and the farmersM herds in the +athmandu valley were used for transferring embryos. Dater" more animals both from farmersM herds and from government farms in different parts of the country were transferred with embryos by =D@ and I0.4. Iational Divestoc' Breeding 4enter in collaboration with I0.4 initiated Embryo transfer in 4attle in the country with the ob5ectives of producing superior bulls and bull mothers. The >ersey cattle embryos imported from Iew Oealand were transferred in government and private livestoc' farms in +athmandu" Dalitpur" Bha'tapur" For'ha" .upandehi" =ola'ha" Bara" +as'i" 4hitwan and @unsari districts. The overall conception rate was around ).**K. Three of the male calves thus born are now being used as semen donor for 0% purpose. The female calves were supposed to be purchased by I0.4 for further wor' on using these pure >ersey females as potential donors for embryo production however3 the program could not be reali2ed due to financial and technical insufficiencies. IDB4" 0B=" 09.= and I0.4 possesses the facilities for evaluation and grading of embryos. 0lthough transfer of embryos imported from abroad has been done in Iepal in the past" complete process from multiple ovulation up to the transfer has not been practiced till now (.ana et al. 200)). Tab/# 6: Stat%! o" ET ' &att/#
S.N. ( No. (2$ Co &#'0#$ (2 3#/'0#r#$ $ Ca/0#! , CR? ).** R#mar9! =D@" Erivate sectors

@ource8 (Eaudel et al." 200$)

24

%n the 0nimal Breeding =ivision" I0.4 a gene ban' has been established in the past years. The aim of the ban' is to preserve the genetic materials of valuable" endangered indigenous and e-otic animals" and breeds at ris'. 0t present the gene ban' has the reserve of semen and embryos of different species and breeds. Tab/# 7: Sto&9 o" #mbryo ' G# # Ba 9; NARC
S.N. ( S+#&'#! 4attle Br##$ IO >ersey 3o!# ' !to&9 (< R#mar9! =D@

@ource8 (Eaudel et al." 200$)

(. Ob@#&t'0#! To review the current status of embryo transfer technology in cattle. To compare the situation of embryo transfer worldwide with Iepal. To get ac!uainted about past" present and prospectus of embryo transfer technology. ,. M#t*o$o/o)y =ata and relevant information for the review were collected from the secondary sources. The main sources of these informationMs were8 ,.1 I t#r #t !%r"' )

25

The published and un published information regarding embryo transfer from the governmental and non governmental agencies were obtained from the internet in the form of the files" documents" proceedings of the meetings and conferences. ,.( I "orma/ ' t#r0'#1! %n order to obtain deeper and richer understanding with the sub5ect matter" interviews were ta'en with the e-perts of I0.4 and =D@ informally. ,., V'!'t to t*# /'brary The information related to the embryo transfer were also collected from the library of the I0.4. @everal boo's" 5ournals and research papers were consulted. -. R#!%/t! a $ 3'!&%!!'o ! -.1 C%rr# t !tat%! o" #mbryo tra !"#r t#&* o/o)y ' &att/# The history and current status of ET have been reviewed by Betteridge (200/) and 9asler (200*) describing how it has evolved through VVthree generations;; the first with embryos derived from donors (in vivo)" the second with embryos produced in vitro" and the third including further in vitro techni!ues" notably somatic cell nucleus transfer and transgenesis. :ore than half a million (,<*"$62) bovine embryos were reported to have been transferred in 200/" *0K of them after free2ing and thawing and (<K having been produced in vitro (Thibier" 200*). There is general agreement that a severe limitation to the more widespread use of ET is the problem of reliably inducing superovulation in selected donors. Transvaginal ultrasonically guided VV?vum Eic' Cp;; (?EC) at fre!uent intervals" in combination with in vitro fertili2ation (%67)" is proving a more efficient route for producing embryos from individual donors where facilities and s'ills permit (Falli et al." 200/3 6an" 200,). Embryo se-ing is !uite widely practiced3 in 4anada in 200/" almost (0K of embryos transferred were se-ed" close to 2000 of them after free2ing and thawing (Thibier" 200*). The great ma5ority of commercial embryo transfer is with cattle. This is mainly because ET is relatively easier in cattle than the other species and also because it is more economical in cattle li'e cattle are worth more. 0dditionally" the low reproductive rate and the long generation interval of cattle ma'e ET much more advantageous in the species. Embryo transfer in the other domestic species is less widely used. Thibier;s summary of the most recent data available (Thibier" 200*) records /$00 transfers in small ruminants (which is considered an underestimate) and (("$$, in horses. =ata from pigs have been particularly difficult to obtain but it is estimated that about 20"000 embryos were transferred in 200/ (Thibier" 200*). There has also been ET activity in buffaloes (especially in 6ietnam) and in rabbits in Taiwan (Thibier" 200*). .ecent reviews of the techni!ues and applications in these domestic species include those of @!uires et al. in horses (@!uires et al. 200/)" 4ognieW et al. in sheep" goats and deer (4ognieW et al. 200*) and 9a2eleger and +emp in pigs (9a2eleger and +emp" 200*). By and large" then" ET has been put to good use in agriculture in developed countries and is undoubtedly contributing to the genetic improvement of dairy cattle as far as mil' is concerned" though this has been at the e-pense of fertility (Bous!uet et al. 200*). Tab/# <. .o!!'b/# a++/'&at'o ! o" b'ot#&* o/o)y to t*# !o/%t'o o" +rob/#m! o" /'0#!to&9 +ro$%&t'o ' $#0#/o+' ) &o% tr'#!.
.rob/#m! .o!!'b/# !o/%t'o S&a/# o" 'm+a&t #&o om'& .robab/# t'm# to &omm#r&'a/ %!#;

26

y#ar! =ifficulty of implementing selection program @election in nucleus herds" using 0l" ET" embryo se-ing Cse of %67" ET and embryo se-ing Darge (0 (,

=ifficulty of maintaining dairy cattle performance after 7( cross

Darge

U(0

4ost and environmental challenge to imported cattle

@election among local breeds using 0%" :?ET Cse of ET to import embryos

:edium1large @mall

U(0 Q,

So%r&#!8 0dapted (with additions) from 4unningham (())0)3 =oyle and @pradbrow (())0). The e-pected pregnancy rate from embryo transfer is highly variable. The vast variability can be attributed to breed of donor and recipient" parity" stage of lactation or post partum interval" body condition" age" environmental inputs such as nutrition and temperature. Eregnancy rates achieved following the transfer of both fresh and fro2en %6E embryos are significantly lower than the rates observed with in vivo embryos (9asler" ())<). The %6E morula e-hibit very poor survival after free2ing and thawing processes than in vivo. %t may also depend upon the e-perience of embryologist who evaluate the embryo morphologically (7arin et al." ()))). The misclassification of embryo grade might also be the reason for the differences in pregnancy rates between embryo transfer.

F')%r# 7: @uccess rate of ET according to different grades -.( T*# #mbryo tra !"#r ' $%!try y#!t#r$ay a $ to$ay 4ommercial ET in cattle started in the early ()$0s" primarily as a result of the high prices being paid for various breeds of so called Ae-otic that had been imported in small numbers. %nitially" all embryo

27

recoveries and transfers were performed surgically. 0lthough some researchers have described nonsurgical embryo recovery techni!ues" these wor'ed rather poorly and few embryos were recovered. 0s a conse!uence" the first commercial ET programs during the early ()$0s relied on mid ventral surgical e-posure of the uterus and ovaries with the donor under general anesthesia. 4oncurrent with the escalating value of e-otic cattle" the newly available prostaglandin 72 made synchroni2ation of donors and recipients practical. The necessity for anesthesia and surgery limited most ET to in house programs with suitable facilities. %n those times" another reason that ET was not widely utili2ed by the owners of dairy cattle was due to the fact that the udder of dairy cows hindered mid ventral access to the reproductive tract. The introduction of nonsurgical embryo recovery (flushing) in the mid ()$0s and nonsurgical transfer techni!ues in the late ()$0s allowed ET to be practiced on the farm and made it especially attractive to dairy farmers. The ET industry grew rapidly in the late ()$0s" both in terms of the number of practitioners and in the number of donors flushed. Erior to the early ()<0s" however" the inability to free2e and thaw embryos made it necessary to maintain sufficient numbers of estrous synchroni2ed recipients so that on any given day all embryos could be transferred. 0fter the development of effective methods of free2ing embryos" utili2ing either =imethyl sulfo-ide or glycerol as cryoprotectants" ET became a much more efficient technology that was no longer dependent on the immediate availability of suitable recipient cattle. 0fter the introduction of embryo free2ing" there were no really significant additional improvements in ET technology during the ()<0s. The ()<6 change in the federal ta- regulations" eliminating the taadvantages of owning cattle as investments" reduced the sale value of many donor cows. This temporarily reduced ET activity for many ET businesses. =evelopment of new technology related to in vitro embryo production has allowed for the commercial use of ET in reproduction. %67 procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of 'nown se- are needed within a short period of time (Bos!uet et al."()))). The research for assessment of embryonic respiration rates using embryo respirometer in association with other viability parameters allows for a more accurate embryo evaluation by identifying the embryos with higher developmental competence (Dopes et al. 200$) has been done. %n appro-imately *0 years" commercial embryo transfer in cattle has become a well established industry with more than ,00"000 embryos being transferred on an annual basis throughout the world. %n 200(" *,0"000 embryos were transferred globally" mainly in dairy cattle" with 62K being transferred in Iorth 0merica and Europe" (6K in @outh 0merica and ((K in 0sia. 0lthough this results in a very small number of offspring on an annual basis" its impact is large because of the !uality of animals being produced. Embryo transfer is now being used for real genetic improvement" especially in the dairy industry" and most semen used today comes from bulls produced by embryo transfer. 9owever" the real benefit to embryo transfer is that in vivo produced bovine embryos can be made specified pathogen free by washing procedures" ma'ing this an ideal procedure for disease control programs or in the international movement of animal genetics. Techni!ues have improved over the past *0 years so that fro2en thawed embryos can be transferred to suitable recipients as easily and simply as artificial insemination. %n vitro embryo production and embryo and semen se-ing are also successful" but time and cost limit their widespread use. 0 combination of embryo transfer using proven cows inseminated with semen from proven bulls" followed by industry wide artificial insemination appears to be the most common use of bovine embryo transfer in the near future.

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F')%r# <8 4omparison of the annual number of in vivo derived and in vitro embryo produced embryos transferred internationally since 2000 through 20((. (4ompliments of the %nternational Embryo Transfer @ociety.) Iumerous differences between in vitro and in vivo derived embryos have been described (9asler" ())<). %n vitro embryos are characteri2ed by morula undergoing less compaction" a smaller peri vitelline space" a lower number of inner cell mass cells" slower development in early cell cycles" and faster development of male embryos" resulting in a s'ewed se- ratio.