Selectionof Donors 1) Economicallyproduc{iveand healthy cows should be chosen Selectionof donors is very important.

Healthy cows with normal cyclesobserved leasttwice shouldbe chosen. Cowswith at estrous disorders, inegular estrous rycle, endome[itis, reproductive postparhm anestrus other problemsare not suitableas donors. or When the donor has reproductive disorders, its responseto the treatmentis low and the numberof recoveredeggs superovulation and embryosis small. Table 2-1. shows superovulationand ova recoveryof normal cows and those with reproductivedisordersas (1n, and Hasler et al. (1983). In this reportedby Elsden et ^1. such as cystic ovary show low chart, cows with ovarian diseases ovulation rates even though they have received superovulation such as treatments. On the other hand, cows with uterine diseases but endometritisdo superovulate, the numberof normal embryosis treateda donor for very small. Elsdenet al. (1979),for example, reproductivedisordersand then observedmore than two normal estrouscycles,but the result was still poor.

45

Table2-1 Results superovulation embryo of and recovery normal on cowsand co'ivs with reproductividid;J @tro .t ur. 1e7e)
Type Normal cow Treated cows Average corpoa lutea Average Average recovery rate fetilization rate

26 (666) Cows with reproductive (318) disorders Endomebitis 11 Adhesion 3 't6 Ovarium Cystoma Unknown Et

11.3

9.5 (10.3) (6.1)

6.8 (6.4) Q.4)

30

2r

10.1 7.6 4.1 8.0

1.9 3.3 4.0 0.3 1.0

t.2

Numbersin ( ) are by Hasler (1983)

2) Explanationto clients

some dairy farmersexperience probremsof rong term infertility in theircowseventhoughtheypossess excellent genetic characteristics, desired physicalstature, a sturdyconformation.Somecowsare and too old to becomepregnanteven though they were productive earlier. Thesefarmersoccasionally want embryofiansferfor their problem cows, sometimes taking them to severalembryotransfer facilities. Technical specialists engaged in .ru.yo transfer (practitioners)must have not only sufficient knowledge and experience the field of clinical reproduction, must also be in but well awarethat good resultscan only be achievedwith hearthy, fertile cows. Practitioners shouldconvincefarmersof this fact. In canadaandAmerica,oneway of preventing probremcowsfrom being brought into embryo transfer facilities is to charge a 46

differentiatedrate. This shifts the financial burden to the clienrc (tabte 2-2). For embryo transfers,practitionersmust thoroughryexplain to the farmerany problemsassociated with the cow and at the sametime, clarify with whom the responsibility lies. It is advisable to exchange written contract,as shown in Table 2-3, betweenthe a client and the practitioner.

Table2-2 pample of_glagas embryo fransfer on problem oows for - (Cana-tla U.S.)or

Ftom 1 yeu to 2 year 100 Morc than 2 m ' When the charge for a nomal ow is g1O00 (about y100,0)), ro orG is requircf in advance. nrls fee U not UererueO *i" d,*gh P"::_1",1 is unsuccessful. the Eansfer

Table 2-3 Items confirmed betweenthe client and the operator

-fye Donc Contents Ovmership lncxpected rcident (during rhe Eeatment) frnbryo tramfer tedmique responsbilityand charge(ovariar reactori,r@very falure, discontinuaonof ernbryotansfe4 D(rreasc of milk otprt Spam (ourcc, anounL rctivit times of fertilizatior charge) Ownershp(mcthod of transfer,handling of extra embro.s) anbryos (ornaship, perird of scage, charge,resale, f'rozen insurance) Ownership Unepeaed accidert(durirg famport) tcst (examiner,method,charge) I:gto,ty Miscaniage (rcsponsibility) Rcgistsation procedure(personin chargeof regisfatior charge)

Aft ilicial iscmination Embryo

Rccipient

Calf bom by nansfer

Superovulation Treatments of Donors A cow is a uniparous animal,thus it usually ovurates singleegg a in an estrous cycle. An effective application of embryo transfer technologyis to induce multiple-ovulation, recover and transferthc embryosand produce many calves. To achieve this, many kinds or' gonadotrophic hormones(GHS) are given to the donor to induce thc production of many eggs ctabre z-4). This treatment is called superovulation induction,or multiple-ovulation induction Fig. 2-1. Table2-4 Multiple-ovulation method Treatment GT}I GTH + PGFza None None Ovulation Superovulation Suierovulation 1 None(followins the numbeir foilile) of gonadohophichormone 9Jltr rr?a: prostaglandinFza Method of recovery Uterineflushine Uterine!ur!,1" Uterine.flushin[ every 21 davs utenne ilushing every21
by punalring the

Asiration

Fig.2-t

Bovineovary aftersuperovulation treatment Many newlyformed orpora tutei 1in br.r.n. "un 48

hnrrvulation Treatment

SfnceCasidaet al. (1940, 1943),there have beenmany reporte inductiontreatment process cows(Foote Itdloson the superovulation in OnumaL970;Gordon1975;Betteidge,1977). This sectionreport l 11 two methods, one utilizing natural estrous cycles and the othe illllzlng PGFza (prostaglandinFza). For superovulationteatmens, blllclo-stimulating hormone(FSII) or gonadohophichormones (GTH) treh us pregnantmare'sserumgonadotrophin(pMsc)Cfable 2-5) and tll,?r aremainly used. Table 2-5 Major gonadohophichormones
1) Folliclc-Stinnrlatig Horrrcc (FS[)

cMsc) I Mcthodwhich utilizes natural estous cycles

In the conventionalsuperovulationinduction treatrnentfor cows, FSH (which is effective for stimulating follicles) or pMSG is administered from the 16th day of an estrouscycle (the previous cstrusday being set as the zeroday). On the 3rd and 4th daysafter thestartof administration, follicle hormone the eshadiolis injected two times (once a day). On the day of estrus,the ovulation of follicles is aocelerated by intravenously injecting luteinizing hormone(LtI) or humanchorionic gonadonophic hormone(HCG).

This method is effective as a superovulationinduction treabnent. The defect of this method is that the estrouscycre of individuat cows sometimesdiffers by one or two days accordingto their own characteristics.when the specific date of estrusof a donor cannor be detected,the embryo ansfer, which requiresa combination of technique, becomes difficult and resultsin problemsregardinglabor management the techniciansinvolved. As a typical exampleof of this method,Sugie'sprocedure (Ig73) is shown Fig. Z-2. 2) Method using PGF* Sincethe discoveryof PGF*'s (Fig. 2-3) strong luteo-regressive effect, it has beenapplied to synchronizeestus as well as in other uses: in superovulation induction by using a combination of PGFrwith PMSG or FSH, the reafinent has becomeeasiersince there is not as much trouble with the donor'sestrouscycle. Since this method is helpful when making an operationschedule,it has beenwidely appliedin practice. An outline of this methodis shown in Fig. 2-4. When GTH and pGF2"are usedin combination,GTH can be administered anytime betweenthe 9th to l,4th day. pMsG is administered once at dosages 2000 IU for infertile cows and of 4,000 ru for agedcows. FSH is administered two dosesdaily for in pGF,' is concurrently 4-5 consecutive daysfor a total of 40-50mg. administered 25- 40rng at

50

15205 PMSGsmdiol HCC fcriliarjon eg,g recovery

!ndcr cycle

r il 0

csuus

= :
ovulation

I
7'

'/-----\ /\ /\

\\.

-_11
Plg,2-z

,/

-rpu"luteum

PMS estradol HCG 3 000 2-3ry 2 000 IX2M U 4 000 IU

unit (1 MU = 3 JI4 MU: mouse

superovulation treatment carriedout simultaneouslv with a naturalestrous cycle usingMPSG,estrogenic hormoneand HCG (Sugie,L913\

Fig. 2-3 Cheliriclstructureof pGF2"

previous eshus
No. of days

inducedestus

PMSG or FSH

artificial insemination PGF2.

tl ll t

Fig.2-4 superovulation treatment using GTH combined with pGF Repetitionof Superowlation Treatment 1) Decline of ovarian response causedby superovulationtreatrnent when treanentsusing PMSG and HCG have been repeated z-4 times, the ovarian responseof the donor declines. This fact has beenreportedby many researchers (willett et al., 1953;Hafezet al., 1965; Jainudeen al., 1966: Mariana et al., l97O; Laeteret al., et 1972;Betteridge& Mitchell, tg74). Among thesereports,however, the treafnent intervals and the pMsG dosage are inconsistent. when saumande al. (1978)canied out pMsG - pGF2,treatments et at intervalsof.7-9 weeksfor 14 months,they foundthat theovarian response declined until the fourth or fifth time, recoveringa little after that. Christie et al. (1979) canied out pMSG - pGF2" treatments at intervals of six weeks; however, they obtained satisfactoryresultsevenafter more than 10 repetitions. Hasleret al. (1983) reportedthat under repeatedFsH - pGF2, treatments two normal estrous cyclesfollowed eachteanent; therefore,9-13 eggs and 6-8 embryoshad beenrecovered through the fifth time, but up in the latter period (from the sixth to the tenth heatment) the number of embryos showed a tendency to decrease(deoeased

52

did fertilization rate) eventhoughthe numberof eggsrecovered not decrease. of 1l Development anti-GTH A report by Jainudeen et al. (1966) suggeststhat the ovarian an declinesunderrepeated PMSG usagebecause antibody rcsponse administration is formed. In general,it is believedthat a repeated protein hormone such as GTH results in the formation of a ' of a gradually declines,eventually GTH antibody; the ovarianresponse making ovulation induction impossible. On the other hand, and Chupin (1981) reportedthat when PMSG was Saumande administered with Freund's adjuvant, superovulation was not eventhough a PMSG antibodywas formed. And Schams hindered administrationof PMSG, a et al. (1978) reportedthat after repeated that PMSGantibody was not identified. From the above,it appears a decline or change in the ovarian responseis brought about by causes other than immune-relatedfactors (Gordon, 1982). > < Methodsto avoid decline of ovarianresponse Someresearchers have obtainedsatisfactoryresultsin avoiding the treatments inoeasing by by dcclineof ovarian reactioncaused repeated the PMSG dosagein the secondheatment(Gordon & Boland, 1.978; 1980;Kanagawa Ishikawa,1980). Newcomb(1980)also & Ncwcomb, used PMSG in the first treatment and HMG (human menopausal gonadohophin) the secondheatmentto obtain the sameresultswith in the increaseof dosage. The author'smethods are the following: 1) dosageintervals greater than 70 days, 2) utilization of vo different

origin(table2-6 and3) increase dosage to GTH (500-1,000 (Tatrr. IU) 2-7).

Table 2-6 Number of.superovulated eggs producedby the adminishationof GTH threer fbur timei (an'igawa, 198,
Order of Gonadotrophic administration hormone 1st Znd 3rd 4th PMSG PMSG FSH FSH Number of corpora lutea 1 case2 case3 case4 22 8 722

6 15

-J

' administration pMSG of

83' -15

Table 2-7 Increase of dosage at the administration pMSc of (beef catrle) (Kaagawa & ry9gnd 1.980) Ishikawa,
Amount of PMSG at the second administration(IJ) Number of cows

Average number of corpora lutea 1st dose 2nd dose

0 100 500
Total

54

400

13 30
108

15.6

12.0 6.8
10.9

11.3 8.3 8.5

9.7

n:i-lysc mcreased.

wasincreased 500IU, thenumber corpora over of lutea

Normalcyof EggsRecovered from SuperowlatedCows Elsdenet al. (1978)repofed that the fertilizationrates of embryos from superovulated naturally ovulatedcows were and 59vo andTLvo respectively, significantdifference. They (Elsden a et ar., rg76) also reportedthat the fertilization rate of recoveredeggs was gg70 in 54

cattle and 70Vo n superovulated cows. It has also been that the rate of abnormality of embryos recoveredfrom cowsincreases whenthe recovery delayed (Table2-8). is believethat the longer the recoveryis delayedafter resbarchers greaterthe numberof abnormaleggsbecause uterus ion, the the uterine tubes of superovulatedcows develop an abnormal nt for the growth of eggs(Newcombet al., L976; du Mesnil Buissonet al., 1977; Boland et al., 1978). However,judgment ing thenormalityor abnormality theseembryos of requires further because is based it only on subjective criteria. In addition,there with the delayed h no data on the rate of abnormalityassociated of cows,and also,eventhe embryos that FOovery eggsfrom untreated normal and are recovered the 3rd or 4th day poston I considered show a low fertilization rate (d. the section on transfer). The mustbe investigated further. Theresultsobtained Renard Heyman(L979)showthat there by and in L no increase the number of abnormaleggsafter the 6th day post(1983a) studiedthe embryos recovered from the 6th Htrus. Takahashi b 9th day and reportedthat the rate of normal eggsws not decreased et tom the 7th to fth day. Betteridge al. (1980)reportedthat normal evenon the 16thdaypost-estrus; ttbryoscouldbe recovered thus,the had Uterus no vital effect on the eggsafter the 7th or 8th day. Also, Spilmanet al. (1973)andSaumande (1978)havereported that cows,compared untreated to Superovulated ones,secrete more estradiol in the follicle growth period and more progesterone 17p after that period to affect the movementof spermand eggs.

mo

>. rr 0i
a c)

or

x L
6) (f

oo6'

I
F'
@6q sn96 !

()
o
!) .t) >r

RS
N

oa
=6

s
&
t

8s 3's$e
N

I sf

q)

ll
NTd' o

o0
o) ()
lr

o6

!i

c) 00 I N

G5
d-h6

p*

()

98"
EC E9 6'E

R.;
F
OF
H

;EE
56

9,8

FactorsWhich Hinder Superowlation Table2-9 shows,n" r"ooo which areconsidered adversely to affect superovulation.The complexinteractionof someof thesefactorsmakes it difficult to control the numberof superovulated eggs. If the numbers of ovulated eggs and transfenable eggs ould be predicted, hansfer would become much easier. At present,thereis difficulty in scheduling embryotransferbecause dateof nansfermust oincide with the days the post-estrusof the recipient. Table 2-9 Major factors affecting superovulation Hormone Factors drug manufacturer, number, lot amou.nt, titer, dosingperiod, repeltlve dosagg,storage oonoluon,nandrng age,b'reed, nutritional condition, weight, birth rec sem, weather,region, envronment

Individual characteristics of oows Others 1) GTH dosage period

Table 10 shows the number of recovered eggs when pMSG (sometimesFSII) was administeredfrom the 7th to the 14th day post-estns. The 7th and 8th days showedrather poor results. In general,the adminishation of pMSG or FSH starts in the period from the 9th to the 4th day. It has beenreportedthat administering PMSGin combinationwith KF, in the middle of the esfrouscycle (uteal phase) induces superovulation more effectively than administeringonly PMSG on the 16th day @sden et al., L974; Seidelet al., 1975a; and Nelsonet al., 1926). PMSGworla most 57

from the 8th to the 1.2thday of effectively when it is administered the estrousrycle (Betteridge,t977). PGF2, is administeredtogether with GTH (or the start of GTH administration)trvo or three days later. However, according to Elsdenet al. (1q78), the numberof corporalutea, the numberof eggs,ad the numberof embryotransferrecipientsof the recovered two or eggswere the samewhen PGF, was administered recovered three days after the stat of FSH administration (fable 2-L0). and 2) Quantityof PMSG to be administered productionlot It is generallyknown that when the quantity of PMSG administered becomesgreater. As shown in is increased, ovarian response the (1975) reported however,Sugie(193) and Kanagawa Table2-1.1. that more than 3,000 IU of PMSG does not increasefurther the ovulation rate (number of corporalutea). In general,for old cows superovulationtreaments for many or cows which have undergone times,a largerquantityof PMSG(2,000-3,000ItD is required. the Evenwlren the samequantityof PMSG is administered, results the because breed,age, and the differ accordingto eachresearcher environmentof the donors differ. Furthermore,other factors to be considered differencesin the quality of PMSG dependingon the are production lot number; Table 2-L shows the different results obtainedby using different lot numbers. There was no significant of correlationin the databecause the large variability in the dataof that, in additionto individuallots. Newcombet al. (L979)reported the differencesin the titer within eachlot, the titer ratio of FSH to to levelsseemed be LH differedfrom lot to lot; also,lower response

causedby lots which showed a low overall titer rather than by differencesin the titer ratio of FSH to LH,

Table2-10 Relationbetween PMSG (FSID administration reriodand ovulationand embryorecovery

PMSG ndnrinistration rriod Sreenaner al. (1978) Ovulation Rate Kanagawa Grcve et al. (19?9) 0975) No. of No' o[ No. of corpon rccovcrcd normal lur embryos embryos Ilasler'et d. 0983) No. of recovercd cmbryos No. of normal cmbryos

7th 8rh 9th lorh lJ r2rh l3rh 14

'

s.0 s.2 t3.2 13.9 16j 7.6 t6.2

(s) (10) 9.0 (18) l4.z (45) 12.3 et) tz.e (6) 12.s 13.6 (4) ll.e

A) 5.1 0t (3s) 6.5 (47) 6.6 (40) 7.9 (34) (20) : 1

3.0 (17) e.6 4.0 (20) ro.o 2^8 Q6) e.s

J.l

7.8

(r2) tt.z (8) u.

s.2 6.1 6.s s.4 6.7 7.s

(47) (si) 002) t1o8) ils <lsr1

FSH administered The numbe in parcnthesis e number of donors. ae

Table2-11 PMSG doseand ovulationrate

I'MSG Dce (fU) (193) Sugic Owlaonnte 1,000 2,000 2,500 3,000 4,000 5,000 6000 7,000 -10,000
8.6 17.7 \4.7 1 1 .1 76.7 10.2

(195) Kuagawa Owlation nte

Srenan& Behu (1976) Owlation nte

115

Saunude cr al. (r978) Orulationnre z5 5.2 8.8

Owlation rate
8.0 14.7 9.4

u00

:
13.8 13.9 77.7 3.0 7.5 6.6

lo.

l'rnluclion lt A, ftoduction lt B, 1,@0 ru, 3,120 Iru

Table2-12 Relationship between ovulationrate and PMSG lot number

PMSG lot Gordon Boland et al. Sreenan & Beehan Newcomb et

number A B

(1975)' 17.9

(1976\ 5.7 7.4

(19?63) 8.0 11.5 9.4 16.8

al. (1979) 6.28 7.95 9.41

n.4

1,500 ru l.,0l.0IU

2,500 IU (FSIVry ratio0.91)

850 IU (FSII/LH ratio 1.16) 1,,W0 IU (FSII/LH ratio 1..))

Thesefiguresrepresent ovulationnumber 3) Dosage frequency FSH administration and of ' PMSG is administered only once,but FSH is usuallyadministered twice a day for threeto five consecutive days. Table 2-L3 shows the results of superovulation from different administration (Chupin & Procereur, frequencies 1983). Compared oncea day to or everyotherday treatment, administering FSH 2 or 3 timesa day increases number of corporalutea and the numberof recovered the eggs. Concerningadministrationtwice a My, some researchers supportthe method(Laster,t972; Godkeet al., L978),but Looney et al. (1981)reportsthat differences the numberof corporalutea in cannot be observed between once a day and trvice a day adminishation. Researchers trying to maintainthe FSH blood are concenfration a longer time by slowing down its absorptionwith for the addition of LVo Na carboxymethyl cellulose or 30Vo polyvinylpyrrolidone, but satisfactory results have yet to be obtained.

60

tlblc Z-fg

Frequency of FSH administration and results of superovulation treatment (Chupin & procureur,1983)
32 AU
lwe r y3 2 l othr dav lZ 9.3 5.6 4,1 12 9.3 5.7 4,3 12 7.6 4.4 2.3 10 25 1.0 0.6
50 AII

No. nll-total dosage of *rc. pcr day

wery other dav

Numbcrof @ws beated ( lrpus luteum (1.{o.) lavcred embryos(No.) Itruvered normal embryos (No)

L2 10.6 8.2

12 ll.2 7.7 5.9

1l 7.1 2.6 1.2

l0 5.5 LO 0.6

FSH is usually administered with a total dosageof Z-50AU. Nelson al. (1979)reported when48AU was administered, et that the number of corpora lutea increasedbut the number of normal embryos was the sameaswhen 32AU was administered. Halley et al. (1979)studieddosage quantities from 36 to 48AU and reporred that levels between40 and 43AU were appropriate.Garciaet al. (1982)caniedout an experiment usingdecreasing dosage levels:5, 4,3, and3AU doses twice a day for threedaysQ4AU in total); 5, 4, 3, and 2AU twice a day for four days (28AU in total); and 5, 4, 3,2, and2AU twice a day for five days(3ZAU in total)(pGF, was administeredon the 4th day). They also reported that the administration FSH for threeor four consecutive of daysresultedin large numbers of recovered eggs and normal oggs, so the administrationperiod of FSH could be shortenedby one or two days. There are three methods of administering FSH: constant dosage level throughout period,day by day decrease dosage the in (Elsden al., 1978)andgradualincrease dosage et in with a decrease on the last day (Crister et al., 1980). Bellows et al. (1969), Lubbadch al. (1980),ChupinandProcureur et (1983)andElsdenet al. (1978) report that the best method is to decrease dosage the (Iable 2-L4\. However,further studies

establisha theoreticalbasisand clarify whetherthe methodis really effectiveor not. Seidelet al. (198) andElsdenet al. (1976,1.978) report good results from a treatmentusing a combination of FSH of with LH in a 5:1.ratio, but the effectiveness LH is not clearly known. Table 2-L4 FSH administrationmethod and superovulation(Chupin & Procureur'1'983)
FSH-totaI dosagc Administration ctbod

4AU x2 tfuocspcr day r 4 days

65,3rAU (dcreasing daily &sage)x2 tas per

dv

6.25AU x 2 tirnes per day x 4 days

SJSSAU (deaeasog rlaly rbsagc) x2tirnccpcr day

Numbef of ows Aatrd Corporlea (!,o.) Recoracd cmbryos (No.) neovered pormalernbrvos No.l

14 &9 5.6 46

13 15.6 13.4 5.5

14 9'7 67 21

13 16.1 10.9
5.1

4) Administration of human chorionic gonadotrophin (IICG) or hormone(Gn - RH) gonadotrophin releasing HCG: Rowson(1951)and Hafezet al. (1963) reportedthat when HCG wds administeredat an estrus induced by supeovulation the treatrnent,it affectedthe owlation and decreased numhr of follicles which were left unovulated. Sugie (1.973) advocates with a subsequent PMSGon the 16thdaypost-estrus administering of continuousadministration follicle stimulating hormoneuntil the next estnswhen HCG is administered.Moore (1975) reported, was however,that when superovulation inducedby PMSG or an extract from equineanteriorpituitary, the adminishation of HCG during estrushad no effect on ovarianreasons. Newcomb (1980)

reportedthat when using PMSG-PGF,., HCG has no effect on the owlation of follicles, but it increases recoveryrate the of eggs. He, therefore'statesthat HcG might havesomeeffect on the movement and the maintenance the gggs of in the oviduct. Gn-RH: Cn_Rf is believed ro promote owlation. Newcomb (1990) reported the adminisration of Gn-RH to induce superovuration; however, onry oneadminis*ationauring-esrus had no effect' The author has beenstudying the effectiveness of IrIRH analogue (Des _ Gly 10 _ LH _ RH _ ethylamide,tII _ RH _ EA) on Horsteinheifers. After the induction of estus by 500 ru of PMsG, thefirst artificiar inseminationis canied out together with LH - RH - EA administered in trvo doses;the number of ovurated and recoveredeggs increased. when .MSG is increased to 3,000 IU' the administrationof LH - RM - EA hasno observabre etfect. Foster(197g)reportedthat when LH - RH was administered in two doses,it had remarkabre effectson heighteningthe LII rever. The most appropriate frequency and dosage of LH _ RH _ EA administrationshould be further examinedin the frture. 5) Breedof donor ovarian response superovuration to treatmenbdiffer depending on the breedof cow. Table 2-15 showsthe differences in the number of ovulated eggs by breed when tf* on," quantity of pMSG is administered' The differences in the number of ovurated eggs of each breed (sensitivity to pMsG) are indexed in Tabre 2-16 (saumande et ar' 1g7g); the breed shows rather low sensitivityto pMsG compared'reisian to other breeds. sheaet ar. (rg76) compared numbersof the ovulatedeggsof Simmental, Limousin-

chianina, and Maine - Anjou breeds(either was a beef or dualpurpose breed)obtaining 15.2, Li.6, !2.4, and9.9, respectively showing the differencesamongthe breeds. Table2-15

Number_ ovulated embryos by breed (at the sam of PMSG dosage)

No. of ovlatedggs Rcsearcber

PI\'ISG dosage (tIl) 1.600

z6 88) 62 (s2)
0t 6.2 (r7) 5.0 (16) r0.4 (14)
a<
<1 t.5

Mariana et aL (190)' Saumande aL(198) et Saunmnde al. (198) et r2.2 (8t 16.4 (3e) I&nagwa (19Q

(16) rL7 8' (31)

D.2 (m3)
32li

Scanloa (1969' Sreena

73.?
' Qrrtcd fiom Srecna & Bechau (1g6 a) ( ) numbcr of domr.

Table2-16 sensitivity to PMSG by breed'(saumande al., Lgzg) et

Breed Friesian Limorsin Normande Charglais B londe d'Aquitaine MaineOvulation index

1.00 2.r9 2.m 2.50

3.65 3.80

dairy cow beef cow dairy & beef (dual purpose)cow beef cow beef cow dairy & beef (dual purpose)cow

' pgqgpl mge by dosing several breeds simultaneously 1,000 wi -^ 4000 ru PMsc.

The numberof actually recovered embryos,the numberof ovulated eggs' and the numberof corporalutea were 6-g for beef breeds,but, in general,it was only 3-4 for milch breeds. However, there are repons of fewer embryosbeing recovered from beef breedsand as many as5-7
@

'I.-hesame quantlty ot rMsci deaily ]tbryos from milch breeds. to breed; therefore, the Foduces responsesthat differ from breed gpopriate quantity of PMSG for eachbreeddiffers dependingon the 1,000ru of PMSGto et hcd line. Saumande al. (1978)administered ft groups of the Charolais breed (the groups had semi-sibling Flationshipswith each other); he reportedthat the averagenumber of eggsvaried in the rangeof 3.6-1.5 :rmongthe groups. twulrted ) Age ofdonor

treatment Moore(195) canied out variouskinds of superovulation on heifers 14-18 monthsold and fertile cows of more than 8 years old. He found that the numbersof ovulated eggs and recovered embryoswere greaterin the heifers than in the older fertile cows. Therefore,he concludedthat the differencewas brought about by of the decrease oocytesin ovariescausedby aging. Although the of in relationbetweenthe decrease the effectiveness superowlation and the aging of cows has not beenclearly established treatments to the number of ovulated eggs is expec{ed decline in cows older than10 y@rs. Table2-L7 showsthe studymadeby Hasler(1983) the number of eggs recoveredfrom heifers or cows older than 15 years shows a tendenry to decline; the fertilization rate clearly (L975) in decline,s cows older than L0 years. In addition"Kanagawa reportedthe decline of embryore@veryin cowsmore than 10 years old. Newcomb(L978 b) reportedthat the numbersof ovulatedeggs from cows younger than 3.5 yearsand fertile cows 3.5-7 yearsold respectively.However,in older cows 1'0-16 were 11.3 and 1.0.0, declinedto 4.0. yearsold, the average

superovulationinduction treahnents on heifer carveswhich had no reached maturity were also attempted (Onumaet al., 1970). The ovariesof immature animarsrespond to the administrationof any foreign GTH, so effectiveovulation induction is possible. However the conditions of the uterus and oviducts have not sufficientr maturedto alrow the ovulatedeggsand the fert'izing spermto grow and function normally. As a result, the recovery of embryos is difficult. Thusfrrtherstudy is needed. Table 2-17
Age

Reration.between donorage of $d resurts superovuratio rrearment (Flasleret al., i93,

No. of donos No. of ecovered 6.1 8.0 No.

embryos
(ave)

Fertilization rate (Vo)

First Clvine Cow Multiparous-Corv

7 1r

_ _

10 L4

ry q

T?

i:; ':;

10.6

6.s s.3

6.8

67 s7

67

7) Superovulation heanentspost partum Resultd studies of ooncerning appropriate forsuperovuration the time treatmentsafter derivery are shown in Table 2-1g. There is no significant reration in the data between the numbers of recovered eggsand embryos,and the waiting period before heatrnent. In the past, it was generallyconsidered that normar *ur no, u, recoveredwhen freatment was "ruryo. initiated soon after parturition. However' eventhough treatmentwas conducted soonerthan g0-g0 days post-derivery,satisfactory resultswere obtained. Hahn and 6

schneider $n\ reported that embryos recovered by treatment cameafter the g'th day (with a surgical method, the former was 67Voandthelatterwas2gvowith a non-surgicar method, 43vo and 22Vo). However, treafinents 30_40 days post partum, *e not advisable because onsetof normaleshouscycres the is uncrear, thus, the possibility of fertilization is uncertain. c-oncerning milch cows, Darrow et ar. (1gg2) reported thar more embryoswere fecoveredfrom oows in ractation than from those which are not lactating (Table z-tg). The reration betweenda'y milk productionandthe numbers recoveredeggs of andembryosare shown n Tabre 2-20. Ava'abre data on rhis subject is limited; possibleeffectsshould be studiedin the fi.ture. Table2-18 Hmx
Rescrcer

of post-delivery waitingtime on superovulation

No of domra
Oeuldcd cmbryos

Daye aftcr po6t-dIivcry

Rovrd crkyos

Enbryos (t{o.) 4.8 5.0

f.orrnat cmbryos

ctvc d al (lttg)

3 0-d ) 6 l-s 9l -

t5()

n m n
t8 74 3l n9 252 113
9.1 5.7 7.4 7.4 43 43 10.5 t0.2 1O.7

s.2
7.8 4.6 3.2 zg 6.5 64 7.3

Drrow Q98,z)

ct

[.0

-135

136
Hsslrd al. (19&')

- no n kD <q) - 150 - 3oo

9t rst

67

Table 2-1.9 Difference in embryo recovery between lactating antl non-lactatingcows (Danow et al., L982) Type
Lactating cows Non-lactating cows Donors (No.) Corpora Recovered Embryos (No.) embryos lutea (No.) (No.)

r24 103

8.0 8.7

6.1 8.1

4.0 2.8

and 8) Seasons raising management Season: A cow is not a seasonallybreeding domestic animal; however,Tucker (L982) reportedthat high temperatures greater than27"C - hassomeeffect on the estrouscycles and the survival of embryos. The duration of daylight affectsthe secretionof hormones and conception. Gordon (1975) reported that some differenceswere observedamong cows in Finlan{ South Africa, and heland dependingon the season;Church et al. (Ly76) also in reporteddifferences North America. On the other hand,Table 221.containsdata which indicate that the number of ovulated eggs that effeas on doesnot depend the season.It is considered seasonal involve not only the weatherbut a combinationof other factors such as .raising and feeding conditions. Takahashi(1983 a), in his experiment on heifers in Hokkaido, reported that there was no difference betweenthe number of eggs recoveredin the pasture (winter). (summer)and in the barn season season that fasting right after Raisingand care: Imond (1970) reported response. causes declinein superovulation a the PMSG treatment also affects the growth of small follicles and Undernourishment cycle(Hill day largefollicles afterthe 1.5th of the estrous especially, is treatment given, the donor et al., L970). Whena superovulation 68

should be raised under favorable con<litions and rcd proPerly. wounds and advanced such as diseases, other aspects Furthermore, ageshould be taken into consideration.

'fable 2-20 Influence of mift production on embryo recovery

Milk production (id"y) Hasler et al. (1983) Donon (No.) Recovered embryos
fNo.)

Greve et al. (199) Pr o d u ctio n (tday)

Embryos (No)

Donors Ernbryos Normal (No) (No) embryos

(No.)

0 I il 2l tl 1l

- 10

10 5

9.5 r3.4
n5 11.3 11.0 9.0 10.2

-m
-30 - 40 - 50 50<

n
113 226 154

4.7 17.6 4.9 7.7 7.2

<5

10 15 m 25 30
35<

15 m 72 25 308
?<

74 10
1t 5

8.2 3.6 7.3 4.9 8.8 5.6

4.0 r.4 5.2 Z6 4.6 4.4

6.2

Table2-2L

Relationship between seasons and results of superowlaton teatments

ADRI Behan& Sreenan Ovulated eees lNo.) Sheaet al. (1976\ Ovulated eess lNo.) 12.7 14.9 12.5
Canada

Season Owlated esss lNo.) winter 9.5 9.7 spring Burnmer 8.8

Geve et al. (dng\ Embryos (No.) 6.1 6.3

Hasler et al. 11983) Recovered embrvoslNo.)

r0.2
9.9 8.4 8.6

r2.s (10s) s.2

11.1 (r71) 9.2

4 U.S.A.

tr.z (178)

0.0
Connfrv Canada

F,nsland

Denmark

winter: January- March, spring: April - June, autumn:October - December sunmer:July - September, Quoted by Animal Diseases Research Institute, Agriailture Cariada, Betteridge (1e77) ( No. of donors.

Segerson al. (1977) reportedthat when beef cows were treated et with PMSG,the addition of selenium(Se) andvitamin E to the feed under appropriatenutritional conditions increases fertilization the rate. Evenif thesenutritive elements produce noticeable no effects on ovarianresponse ovulation rate, they are considered or effective in increasingthe recoveryof normal embryos. In an intriguirg report by Kweon et al. (1983), the authorsfound that when they measuredthe total cholesterolin the serum of the donorbeforeadministrationof PMSG,the numbers ovulatedeggs of and recoveredembryosdiffered significantly beveen donorswith levelsgreaterthan 130mg/dland thosewith lower levels (Fig. Z-5).

aaaa a!

Total serum cholesterol(mg/dl)

Fig.2-5 Relationship between total serumcholesterol numberof and normal embryos- ryr_!\ superovulation treatment (dairy cow)(Kwoenet al, 1983)

70

< i-iorvto obtain many eggswithout superovulation treanent> ln general,the donor should be treatedwith hormonesto induce urierovulation,however, some other methods have also been tried Table4). - i Direct recoveryfrom follicles Bovine eggs are necessary researchmaterial, but obtaining an as ample supply is difficult. If the eggscould be obtainedfrom the numerouslarge follicles on the surfaceof the ovary, they could be utilized for studies on egg maturation, in vitro fertilization, and cryopreservation eggs. Removalof eggsfrom slaughtered of cows after deathwould also be possible. immediately :) Embryo recoveryat every estrus One embryois recoveredevery 3 weeksby a non-surgical method (cf. Chapter one,line t3). Estrus Detectionof Donors Signs of estrus: Estrus in cows is exhibitedthrough various signs rTable 2-22) which do not appearsimultaneously. In additiorU the combinationof the characteristicdifferencesbetweenindividual cows '*-ith the subjectivejudgmentsof observers createdifficulty in bringing rogethersimultaneous estrus betweenthe donor and recipient. In Cnada the United States, and only standingestrusis usedas a favorable :.strussign. Standingestnsis the position in which the cow standsstill ,"vhilebeing mounted. The author, also adopting this method, has 77

achieved excellent resultsin obtainingsimultaneous estrus. Table 2-22 Signs of estrus

No. signr
t. 2. 3. 4. t

unstable and excited b"h*iBloodshot eyes Moanins Decreas appetite in Decrease niilk production in

l. Congestdland swollenvulva v. .trstrous mucus

i. fittt,fff"

othei an cows sniffing vulva

Estrus inducedby exogenous hormones:when superovuration anc estrusare inducedby GTH hormones(PMSG or FSH) pGFr, more and than 68-807o of donors exhibit estrusand, in addition, most of thos not showingsignsalso ovurate Lg77).Tabrez-23contain @etteridge, dataon the rate of estrus@crrren@.Hasler et al. (1gg3) reportedthat when cowswith reproductivedisorders weretreatedas donors,the estns occurrence ratewas g'r,vo. However,the rate in other cows wrsgreate thanXJVo.

Most donor esfus - 7r - g1 o - were concenhated on the }nd day post-PGF, (rable z-24). Kanagawa (Lg76)reported donorswhich that had not reachedstandingestrus,even though they showed some other signs, respondedpoorly to artificial insemination and egg recovery treatments. Therefore,the post-artificial insemination scheduleshould be canceled. Greve et al. (1929) recoveredembryos from groups of donorswhich exhibitedestrus2-36 hours, 37-4ghours, 4g-60hours, and 6t-72 hours post-pcF2" administration. The embryo normalcy rates for these groups were 54.7Vo, 5S.0Vo,4l.OVo, and l6.3Vo.

72

ffiTtH r,;;l#ffiHIJ;;TH ffi;;"'Tff ;ffi,

Tabte2-23 Estrus appeuance during rate superovuration heatment
Researcher Treatedcows (No) ---.* wws ( lo.) Esuus appeaance

lspectively.Therefore, they reportedthat shich showed later than 4g hour rePorted thar in ".h", .,...:]i^:_*uo' **n

problemslay with groups Takahashi(1983g also

'l*,TtI"{',?rii Normat bws

I_rtrrra"ru fiAs

#iF:iif' $r';i"frsi['33

726 78 99 8s6

n.0 n.0
%5

%.2

Table2-24 Daily eshusappearan t@rate post_superowlation heatnent

Researcher

^*.ffirff*%
56 72

l*q*l & Behan luge dt Souma ll

8.8 &t.s 9.6 1.1 77.8 l5.3 r.4 4.2

Artillcial Insemination of Donons

-" artinciar insemination ;:,::ffii;T*: of fertilization expected : 1 *r: ,r**lar, is rn

Dya superovuration teatnent Difference shourd io*

Best period for insemination: kl

.Z_6).An estusinduced to this method.

fifi ,j:J

betrve sperm *tT,o,T is used *:fft"'i:ffir:*-ilffi"T::;

,ru

and frozen spermreportedbetter perfotmanceftom fresh sperm (tablc e.g.,donoryounger had thesestudies someproblems; 2-25). However, (Onumaet al., 1970)or the concenhation spermvaried of than 1,7weeks (Mckenzie& Kenny, 1.973; & Sreenan Beehan,1976). time during standingestrus Fig.2-6 Insemination
lapseof time insemination before ' -12 too early stafdins estrus 0 appropriatetime 12 best time after (time) 36 U time too late appropriate

Frequencyof insemination:In general,when frozen sperm is used with a superovulationtreatnent, two straws or ampulesof sperm are .applied several times every 8-12 hours. Table 2-26 comparesthe inseminated rate of recovered eggs with the frequency of sperm insemination. According to Crister et al. (1980), even an insemination of an ampule 1.2 hours after confirmation of estns resulted in a reportedthat rate satisfactory of normal eggs;however,otherresearchers the rate. Newcomb (1980) insemination at several times increases reported that an additional insemination after the estrus subsidesis important. Fertilization without fail is securedby a first insemination h the moming upon esfus confirrration, a secondone of sperm ".dy that evening and the third one the next moming. Dependingon the estrous condition, the first insemination is sometimes omitted. that Theoretically,however,it is considered asingle inseminationof one straw is sufficiently effective, even in the case of superovulation treafinentsif it is applied in the optimum period.

74

tr
c
.H

s8 g
cts

al
e, B /t
)f

SH
g
F

ss
s
(^
6

GE .

Ur OF

(! q

I
a

a
a

i: rlsi: 3 aE $ssrs
- --

- !r,!i'31. *ol

B u =lqlF -IH Fr f' *gEFEEgC F3gE g

N

$ 3 -lr H
l

I el

Id g

u
CT o N I t.J
tJl

EQ
: fo -at

9,

9"
6t
et

O\

F'

(o
C"

**Ei,EF
? tD
(D o U' E F'

EP

.!

g -a

EP o

'l
Table 2-26 Number of inseminations .and the fertilized embryo recoveryrate (normal egg rate)
Researcher rate and/or nonrlal epg,rate (Vo\ No. of inseminations Note

estrus
Cristeret al. (1980) 58.9 60.0 4 times: 8, 16, U, and 32 hr. post-estnrs l-ampule inseminaton:12 hr, post-estns 3 times: 1 ampule each at estrus and 72 arld ?A hr. posr-estns 3-ampule insemination: 1.2 hr. post-estrus 1.tirp9: a8 hr. post-pGF, administration 2 times:48 and 60 hr. postPGF. adminisrration 3_times: 48, ffi hr. postPGF, adminishation

57.2 Newcomb(1980) 70.1, 80.8 87.7

Performance differencedepending the bull used:Newcombet al. on (1978b)reportedthat when spenn was inseminatedseveraltimes in the optimum period, results differed dependingon the bull used: the normalcyratesof eggsusing frozen spermfrom A, B, and c bulls were 75vo, 86vo, and 33.3vo,respectively bun's performance (c was nor). sperm from bulls with artificial inseminationperformances (fertilieation rate) above- and below-averageaffected the inseminationrate of superovulated (rable 2-27). Therefore, quality offrozcn sperm eggs the is anotherimportantfactor.

76

Table2-27 sire bull fertil.ization rate differencesand superovuration freatnent fertilization rate
Sire bull fenilization rate Newcomb(1980) fertili?ti-on
-{bove average <r r

postd 32

fertilization

rate

:12
at

A hr. :12
?4

,p o st -

r et al. in the t the iwere poor). ation te of perm

77

suPerovulanlon lnouclon reatmen$

on neller calves wilcn

nao nol

reachedmaturity were also attempted(Onuma et al., 1970). The ovariesof immature animals respondto the administrationof any foreign GTTI,so effective ovulation induction is possible. However, the conditions of the uterus and oviducts have not sufficiently maturedto allow the ovulatedeggsandthe fertilizing spermto grow and function normally. As a result, the recovery of embryos is difficult. Thus further study is needed. Table2-L7
Age

donorageandresultsof superovulation Relationbetween teanent (Hasleret al., 1983)

No. of donos No. of recovered embryos 8.0
No. of

embryos
(ave)

Fertilization rate (vo)

First Calving Corv

26

5.3 6.8 6.9 5.3 2.6

67 67 67 57 50

Cov Multiparous 3-6282 7-10224 11-t4g

15< 9 Donon are Holsteins

10. 10.6 9.7

5.6

7) Superowlation heanentspost parhm

time for superovulation the Resultsof studiesconcerning appropriate delivery are shown in Table 2-18. There is no treatmentsafter significant relation in the data betweenthe numbersof recovered eggsand embryos,and the waiting period before teatnent. In the past, it was generallyconsidered that nomtal embryoscould not be recovered when treatment was initiated soon after parturition. soonerthan 80-90 However,eventhough fieatmentwas conduc{ed