Superovulation, Ovum Collection, Culture

and Transfer. A Review ' ' :

R. H. FOOTE
Department of Animal Science, Cornell University
Ithaca, New York 14850
and
HIDEO ONUMA
College of Veterinary and Animal Sciences, Kitasato University
Towada-shi, Japan

Introduction of tubal ooeytes or young embryos for study.

I n cattle (39), as in most other mammals, Since the ovary of the immature animal con-
the fetal or neonatal female produces thousands tains an abundance of oocytes, including many
of ooeytes which are never fertilized. Develop- in tertiary follicles (39), the opportunity exists
ment of ways to harvest and fertilize these effi- for removal of oocytes from females of all ages.
ciently and to culture and transfer them would Oocytes have been removed (26, 37, 38, 86, 120)
offer many potential advantages (1, 3, 4, 7, 28, by extirpating the ovaries and mechanically
32, 42, 50, 60, 70, 75, 77, 78~ 80, 89, 107, 110, rupturing the follicles, or aspirating the folli-
123). These include the possibility of a) de- cles in vivo. However, more efficient in vitro
creasing the generation interval, b) progeny fertilization must be developed before these
testing females, c) using superior females as ooeytes can be used effectively.
donors, d) increasing the number of progeny S u p e r o v u l a t i o n in cows. The ultimate objec-
per female through controlled multiple births, tive of superovulation usually is to increase the
e) transporting embryos with selected genetic number of normal fertile eggs or embryos
characteristics to distant places, f) conducting per donor. This should be the major criterion
special genetic experiments, as the formation of the success of a treatment, but values given
of chimeras, g) isolating prenatal maternal in the literature frequently are based only on
effects, h) investigating requirements for opti- animals responding normally.
mum fertility and normal development and The superovulatory response has been studied
i) studying effects of a variety of stresses on in many laboratory animals (28, 68, 80) and in
initiation and continuation of pregnancy. pigs and sheep (5, 34, 94). The first super-
E g g transfer probably has the greatest eco- ovulation in cattle was performed by Casida
nomic potential in cattle, but studies with cattle et al. (22). Since then many hormonal prepa-
have been limited by the high cost of experi- rations and injection schedules have been tried
mentation. Although this review will focus on (9, 14, 20, 22, 31, 35, 40, 49, 50, 52, 53, 72,
cattle, reference will be made to laboratory 97, 104, 105, 109, 112, 115, 116, 120, 127, 128).
animals (18, 28) and several farm animals (5, The basic principle is to stimulate extensive
34) where important concepts have been devel- follicular development through intramuscular
oped. W o r k published in Japanese on cattle or subcutaneous administration of a p r e p a r a -
and goats has been reviewed in more detail tion with follicle-stimulating hormone ( F S I t )
because this literature has been less available activity at levels in excess of normal endogenous
to many English-speaking scientists. levels. The most commonly used sources of this
activity are swine pituitary extracts or preg-
Source of Oocytes nant mares' serum, although early work often
was done with sheep or horse pituitary prepa-
Ovarian oocytes. I n the cow and many other rations. Many animals so treated will come into
species it is difficult to obtain large numbers estrus and ovulate within 5 days (109) pre-
sumably through release of endogenous lutein-
Received for publication May 22, 1970. izing hormone ( L I t ) . However to help assure
1 For simplicity the authors have followed the ovulation a preparation with luteal activity
majority of the literature in using "egg" and should be injected intravenously or intramuscu-
"ovum" as general terms to represent stages larly. Usually pituitary luteinizing hormone or
ranging from the unfertilized oocyte to cleavage hmnan chorionie gonadotropin (HCG) has been
stages in the young embryo. used.
2 Studies by the authors were supported in part E a r l y work on the duration of follicle-stimu-
by N I I t Grant HD03471, USPIIS. lating hormone treatment and the interval be-
1681
1682 F O O T E AND ONUMA

tween that hormone and luteinizing hormone active corpus luteum at ovulation usually can
injections (20, 22, 31, 52, 53, 97, 127) indicated be avoided by initiating treatment on Day 15
that pituitary follicular hormone preparations or 16 of the normal estrous cycle, by manual
should be given subcutaneously daily or twice removal of that gland or by giving a progesta-
daily for about 5 days, whereas pregnant gen sequence or o t h e r t r e a t m e n t to regress the
mares' serum could be given as a single sub- corpus luteum. Avery et al. (9) and Hafez
cutaneous injection. On the sixth day the ovu- et al. (49) obtained a better response with
lating hormone is given intravenously. This enucleation of the corpus luteum than without,
schedule has been confirmed and followed by and pretreatment with progesterone (9) was
many workers (9, 11, 50, 103, 112). A summary particularly successful. Superovulatory treat-
of several of the more successful schedules re- ment may induce an initial ovulation, and the
cently employed is shown in Table 1. corpus luteum formed interferes with fertiliza-
An active corpus luteum or the administra- tion of the eggs ovulated a few days later.
tion of progesterone at the time of ovulation The administration of 20mg of 17fl-estradioI
may increase the number of ovulations but on Days 19 and 20, following treatment with
greatly reduces fertility and hastens egg trans- pregnant mares' serum, increased the number
port (11, 22, 31, 35, 50, 72, 97, 127). An of developed follicles and the number of fer-

TABLE 1. Superovulation in cattle.

Ova
Animals
Refer- Gonadotropin Folli- Ovula- Reeov-
ence Age No. treatment cles tions ered Cleaved
(no.) (no.) (%) (%)
9, 11 Sexually 32 Days 1-4 a : 20, 10, 10, 10rag .... 21 53 61
mature F S H -4- 5rag L I t each day (4-55) b
Day 5 : 100rag L I t
50 " 14 Day 16 : 3,000 IU PMS 59 33 50 54
Days 19, 20 : 20rag 17 fl- (14-104)
estradiol
Day 21 : 2,000 IU t t C G
50 c " Same as in Re~:. 50 but 4,000- .... ~12 ~47
7,000 IU PMS on 16th or (2 ~ 28)
17th day
103 " 89 Day 16 : 3,000 IU PMS 15 9 71 68
Day 21 or 1st day in estrus (0-55)
2,000 I U H C G
14 " 7 Synchronized (see text) and 27 15 63 84
2.5rag F S H 2 × daily, (1-32)
days 8-12
9, 11 65-173 43 20, 10, 5, 0 and 10rag F S H on .... 34 33 24a
days Days 1-5; Day 6 : 100rag LIt (1-88)
63 4--24 13 Day 1 : 2,000 IU PMS 28 16 27-60 6
weeks Day 6 : 10rag N I H - L H (0-55)
89 8-17 55 Day 1 : 1,500-2,000 IU PMS 53 43 8-75 72~
weeks Day 6 : 50rag P L H
a Day 1 was the 16th day of the cycle for cows not receiving progesterone.
b Numbers in parentheses are ranges.
e Method currently employed. High pregnant mares' serum dosage possibly represents a dif-
ference in potency. Number of ovulation points was estimated by rectal palpation and ova col-
lected nonsurgically.
d Included only older calves where rectal examination was possible.
e When inseminated 2-3 times with liquid semen.
JOURNAL OF DAIRY SCIENCE V0L, 53, N0. ]2
 A P~EVIEW 1683

tilized eggs recovered (50). Treatment with 16, 22, 59, 63, 74), but are improving (89).
17fl-estradiol before administration with preg- Examples of treatments followed and results
nant mares' sermn gave a highly variable re- obtained are summarized in Table 1 for calves
sponse, but tended to reduce the number of of various ages. Different levels of nutrient
developed follicles. Avery and Graham (11) intake did not influence the response (88), and
previously had shown that 10 to ]5rag of di- individual differences persisted when all ani-
ethylstilbestrol administered to cows with func- mals were reared under standard conditions
tional corpora lutea increased the proportion of (89, 90).
fertilized ova. However superovulation was The most successful hormone treatment for
more successful without the corpus Iuteum. young calves appears to be 1,500 to 2,000IU
Table i reveals that there is great individual of pregnant mares' serum intramuscularly, fol-
variation in follicular development and ovula- lowed by luteinizing hormone given intrave-
tion rate, ranging from no response to over- nously 6 days later. Follicles stimulated to
stimulation. I f the dose of pregnant mares' develop with follicle-stimulating hormone are
serum is too high, many unovulatcd follicles difficult to rupture (35, 90). The apparent
result (31, 40, 127) and with 5,000IU follicles superiority of pregnant mares' serum over
tended to be hemorrhagic (49). The time of follicle-stimulating hormone may result from a
ovulation is variable, and the synchronization more favorable balance of follicle-stimulating
of the multiple ovulations and the proportion hormone and luteinizing hormone activity in
of follicles rupturing may be improved con- pregnant mares' serum, or due to its longer
siderably by intravenous injection of pituitary biological half-life.
luteinizing hormone or human chorionie gonado- Pretreatment with progesterone (16, 59, 89)
tropin. did not enhance ovulation in the calf, although
In a study to give a controlled increase in in earlier reports (22, 74, 97) prior luteal ac-
multiple pregnancies, Bellows et al. (14) ob- tivity seemed to do so. Progesterone depressed
tained a superovulatory response when animals fertility and hastened egg transport (89).
were given a total of 25mg of follicle-stimulat- Treatment with estradiol suppressed follicular
ing hormone. Beef heifers were synchronized growth (59, 89) and decreased ovulation rate
by feeding 180rag of medroxyprogesterone (89). This is opposite to the effect observed
daily for ]1 days, 5mg of estradio] valerate in cows (50).
on Day 2 (Day 0 ~ first day on medroxypro- Low fertility has been attributed to poor
gesterone) to regress the corpus luteum and sperm transport (74) in the infantile tract.
2.5rag of follicle-stimulating hormone twice However Howe and Black (58) found motile
daily on Days 8 to 12. The average number sperm in the oviducts of calves 13 minutes after
of ovulations was 14.6, with 84% of the ova vaginal insemination. Onuma et al. (89) ob-
fertilized. tained higher fertility when sperm were depos-
Ovum recovery rate often is low. I n one of ited through the cervix of calves with the aid
the more successful experiments (103) only an of cervical forceps and a duck-bill type of
average of 4.4 cleaved ova were recovered per speculum instead of in the vagina. Avery and
COW. Graham (11) obtained higher fertilization rates
Superovulatory response declines with re- in calves old enough to allow use of the rectal
peated hormone injections, due in p a r t to the method of insemination than in younger calves.
development of antigonadotropin titers (35, 36, The proportion of cleaved eggs was signifi-
45, 47, 49, 62, 126). This inhibition cannot be cantly increased from 33% for frozen semen
effectively overcome by increasing the dosage of to 72% by the use of liquid semen, and insemi-
hormone, thus limiting repeated use of donors. nation 3 times over a 4S-hour period following
Superovulation in calves. The calf provides luteinizing hormone injection tended to yield
an ovary with an abundance of oocytes. I t also higher fertility results when compared with
offers an econonfic advantage; there is oppor- 2 inseminations (89).
tunity to reduce the generation interval and an Calves also show refractoriness to repeated
opportunity to use an animal in which one pre- injections of gonadotropins (59, 89). However
sumably does not have to contend with cyclic the greatest problem is one of low recovery
variation or a corpus lutemn. One limiting rates of ova from calves. The fimhria and
factor is that the evaluation of ovarian condi- associated pickup mechanisms for ova seem
tion by rectal palpation is not possible in small to be unable to collect ova from the enlarged
calves. ovaries. The recovery rate tends to be low, but
Superovulatory response has been variable the actual number of ova recovered is greater
(9, 63). Fertilization rates have been low (11, when there is a large nmnber of ovulations.
JOURNAL OF DAIRY SCIENCE V0L. 53, NO. 12
1684 FOOTE AND ONUMA

Superovulation in goats. Studies with goats ful fertilization of cattle ooeytes in vitro has
(81, 82, 84, 85) may provide insight into pro- not been reported.
cedures which are useful in cattle. The pro-
cedure generally used at the National Institute Egg Recovery
of Animal Industry (Japan) is to inject 1,000 Embryos are found in the oviduct until about
to 1,500IU of pregnant mares' serum sub- the fourth day after ovulation. The best time
cutaneously on the 17th day of the cycle, give to recover eggs depends upon the objectives of
1,000IU of human chorionic gonadotropin in- the study, but usually ranges from 2 to 6 days
travenously on the first day of estrus and after estrus or the administration of LH.
inseminate at the same time human gonado- Slaughter or hysterectomy. The simplest pro-
tropin is given. The number of developed fol- cedure is to remove the organs at slaughter,
licles averaged 26 with an ovulation rate of clamp the horns of the uterus near the body,
53% (81, 84, 85). sever the oviducts near the tubouterine junction
and aseptically flush the oviducts and uterine
Fertilization horns. The oviducts may be flushed by insert-
Either efficient recovery of fertilized eggs ing a blunt needle attached to a syringe into
from a donor or in vitro fertilization is critical the infundibulum and flushing the ova with
in most embryo culture and transfer studies. 10 to 15ml of uledium into a dish or tube. Each
Several problems following superevulation have uterine horn is flushed aseptically by inserting
been alluded to. Excessive follicular stimula- a needle near the body of the uterus and flush-
tion (31, 120, 123), the presence of an active ing 20 to 30ml toward the opposite end. Or
corpus luteum and addition of progesterone both ends of the uterine horn may be clamped,
(11, 22, 31, 35, 50, 72, 97, 127) are associated 20 to 30ml of medium injected into either end,
with either no or tow fertility and abnormal a 14-gauge needle with attached tubing inserted
rate of egg transport. Progesterone interfer- into the opposite end and the fluid massaged
ence with sperm transport also may be a factor. into a collection tube.
Willett et al. (127) obtained no fertilized ova An alternative procedure is to leave the tract
during the luteal phase when sperm were depos- intact and flush the oviducts from the ovarian
ited into the uterus. Multiple ovulations may end into each uterine horn. A stab wound is
not be well-synchronized; several inseminations made with a 14-gauge needle near the tip of
over a 48 hour period following luteinizing the uterine horn to collect eggs which have been
hormone injection are desirable. I n the more flushed from the oviduct or which were in the
successful reports over one-half of the ova proximal part of the uterine horn. The uterine
recovered from cows were cleaved (Table 1) horn is flushed with an additional 20 to 30ml
and Dowling (31) recovered 48 ova from 10 of fluid injected near the body.
cows of which 92% were cleaved. Reported recovery, of ova from superovulated
Cleavage is not a completely reliable indica- cows ranges from about 25 to 75% of the num-
tor of normal fertilization. Careful morphologi- ber of ovulations (11, 31, 49, 101), with the
cal examination by phase microscopy coupled upper limit representing optimum conditions.
with stained preparations to show nuclear be- I n cows with single ovulations 85% of the eggs
havior should be utilized until routine transfer have been recovered (11). I n superovulated
techniques are available to permit actual devel- calves recovery rates usually are from about
opment. 20 to 35% (11, 63, 88-90) ; the higher responses
Problems with calves are similar, with the tend to occur in older calves.
addition of more difficult insemination. When ~Iost ova are found in the first flushings,
liquid semen was inseminated 0, 24 and 48 particularly from the oviduct. However subse-
hours after injecting luteinizing hormone into quent flushings occasionally dislodge more ova,
calves Onuma et al. (89) reported that up to and repeated flushing and examination are fea-
82% of the ova collected showed normal cleav- sible when organs are removed.
age following collection and culture. Surgical. Although the slaughter procedure
I n vitro fertilization following collection of has been used most commonly for collecting
recently ovulated oocytes or oocytes removed eggs, the donor should remain intact if egg
from the ovary offers another potential means transfer is to be practical. With the surgical
of fertihzation. Brinster and Biggers (19) approach the donor is anesthetized, laparoto-
fertilized mouse ova "in vitro" by placing them mized and the ovary, oviduct and uterine horns
within an oviduct in culture. The problems brought through the incision (102) for exami-
associated with in vitro fertilization have been nation and egg recovery. Flushing can be per-
discussed (27, 37, 38, 119), and so far success- formed similar to that described for the intact
JOIIRNAI~ OF DAIRY SOIEI~CE ~rOL. 53, N0. 12
 A REVIEW 1685

tract removed at slaughter or by hysterectomy. to inject fluid and wash any embryos out. This
Also the oviducts may be flushed toward the approach is applicable to recovering embryos
ovary by injecting medium through the tubo- which have advanced in development and space
uterine junction and out through a cannula to the uterus. Dracy and Petersen (33) devel-
snugly held in the infundibulum. The organs oped a cannula-tubing arrangement through
are returned to the abdominal cavity and the which 1,000ml of saline were flushed into the
incision closed. The animal may be reused at uterus. Ova were collected from 12 cows out of
a later date. Various modifications have been 37 attempts. Willett (123) reported that this
described (12, 31, 102, 106, 120). method caused considerable trauma. Rowson
Ovaries have been transplanted to the neck and DoMing (99) described an apparatus with
and translocated to the area of the paralumbar an inflatable cuff to hold the instrument in place
fossa or to the vagina, but complications arose and to prevent loss of flushing medium, but
(33). Also exteriorizing the resected uterine data on the efficiency were limited (31). Modi-
horn was unsuccessful (33). fications of this early equipment (33, 99) have
Nonsurgical. The nonsurgical approach of- been described (30, 35) which minimize the
fel~ the possibility of repeated collections of amount of flushing medium required, simplify
embryos from the same donor with minimum the number of tubes involved and retain the
trauma. It is simpler and has the potential of inflatable cuff. Varying degrees of success
field application. Nonsurgical procedures and were reported (Table 2); none of the proce-
results in cattle reported by several workers dures gave as consistent recovery as surgical
are summarized in Table 2. Tubes are passed procedures. Yamauchi and Nakahara (128)
through the cervix as in artificial insemina- recovered ova from 5 out of 6 cows surgically
tion. A cervical relaxant nmy be used to facili- or at slaughter but recovered none by uterine
tate passage (43). Dracy and Petcrsen (33) douche.
inserted a rubber tube into the uterus butted Recently, excellent recovery from superovu-
against the oviduct. Uterine contractions tended luted cattle has been reported by Sugie (112)
to dislodge the tube. One cow became pregnant with a two-cannula device along with 2,000 to
and no eggs were recovered, so this approach 3,000ml of flushing medium. Out of 32 cows,
was abandoned. 1 to 4 ova were collected from 10 cows, 5 to
Most procedures have involved irrigation of 8 ova from 11 cows and 10 to 16 ova from
the uterus with varying numbers of eannulae 5 cows. The entire uterine horn was flushed

TABLE 2. Nonsurgical ovum collections in cattle.

Ova collecteda
Maxi-
mum
Refer- Successful Avg per per
ence Year Collection method Attempts attempts attempt cow
(no.) (no.) (%)
99 1949 Rubber 3-lumen tube . . . .
31 ]949 Rubber 3-lumen tube 6 2 33
33 1950 Catheter to oviduct 7 0 0 0 0
33 1950 Cannula and inner tube 37 12 32 1.1 20
35 1958 Cannula, inner tube and
retaining balloon 26 0 0 0 0
35 1958 Cannula or tubing with
retaining balloon 10 2 20 0.8 7
35 1958 Tubing with holes and
retaining balloon 39 10 26 0.7 6
128 1961 Uterine douche unit 3 0 0 0 0
112 1970 Modified from Ref. 99 23 10 43 1.1 6
112 1970 Two-tube polyethylene cannula
with retaining balloon or
2-lumen catheter 32 26 81 5.1 16
" Time of collection, when given, usually was about 4 to 6 days after ovulation.
JOURNAL O~~ DAIRY SCIEI~CE VOL. 53, NO, 12
1686 FOOTE AND ONUMA

with this equipment rather than just the ante- negative results. Onuma and Foote (87) found
rior portion; this may have contributed to the heated bovine or rabbit serum to be as good
high recovery. I f this recovery rate can be as follicular fluid for culturing calf ova. Ham's
attained regularly and embryos obtained un- F10 synthetic medium (51) or Xrebs-Ringer-
damaged, the procedure could provide an accept- bicarbonate gave similar results, and Brinster
able means for routine recovery of embryos in (18) cultured two bovine ova in synthetic
cattle. media. Development seldom exceeded one or
Egg recovery i~ goats. Eggs are recovered two cleavage divisions, excepting after transfer
by flushing oviducts and uteri separately, either to a rabbit (2, 109).
after slaughter or by way of laparotomy, as in Pincus (95) stored bovine ova at 10C, and
cattle. Recovery rates range from 60 to 80% found limited cleavage after storage for 1 day
(82, 83, 93). Approximately 80% of the ova similar to freshly incubated ones. No trans-
are cleaved. fers of cultured ova were made. Sreenan and
Scanlon (107, 108) obtained some development
Media for Recovery and Storage of bovine ova in rabbits after culture in fol-
Cattle. Media optimal for embryo survival licular fluid at 10 and 26.7C.
in vitro would facilitate embryo storage and I t is clear that media or other aspects of the
transport, and possibly synchrony of donor and culture environment previously used for cul-
recipient by delaying cleavage. Chang (23) turing cattle ova were far from ideal. Consid-
showed that rabbit embryos could be stored erable reliance has been placed on blood serum
in homologous serum at 10C, with some em- alone or in combination with salt solutions
bryos remaining viable for 6 to 7 days. Fur- both for culture and for collection and transfer
ther studies (24) showed that serum of several of ova (Table 4). Rowson et al. (~02) obtained
species including cattle and sheep, but not no pregnancies when 33 ova in homologous
swine, contained a thermolabile factor which serum were transferred to recipients. But when
was lethal to rabbit ova after 10 minutes of a synthetic medium, TC199, was used 9 out of
exposure. Heating serum to 55C for 30 min- 11 heifers were pregnant following surgical
utes destroyed the factor. recovery and transfer of ova.
Little is known about the requirements for Sheep and goats. Sheep ova cultured in
culturing cattle ova. When ova were placed heated (24) sheep serum have survived trans-
in homologous serum usually not more than fer after culture for up to 72 ho~rs at 8C (7),
one cleavage occurred (20, 49, 95, 109). Unless after 48 hours at room temperature in serum,
heated serum was used an ovieidal factor for and after 24 hours at 37C in Whitten's medium
cattle ova might have been present. Sreenan (54). Other studies (21, 66, 67) also indicate
et al. (109) obtained better results with follicu- that sheep ova can be stored satisfactorily.
lar fluid than with serum, and Thibault (119) Successful culture and storage of goat ova, as
obtained embryos up to the 16- to 24-cell stage a prelude to studies with cattle, have been
in follicular fluid. I n a previous study by reported (83, 93, 118). A portion of this work
Brock and Rowson (20) follicular fluid yielded is summarized in Table 3.

TABLE 3. Culture and transfer of goat ova.

Recipients Ova transferred
Storage Cell stages Con- Resulting
Temp Time Before After No. eeived No. in kids
(c) (hr) (%) (%)
7-10 ~ 22-27 4-16 ........ 13 54 25 37
48-97 8 ........ 5 0 15 0
37h 24 4-8 8-16 4 75 9 56
48 8-16 16-32 4 50 9 44
72 8-32 M-B c 4 25 14 14
96 4-16 16-B c 6 0 20 0
Results by Otsuki et al. (93) with serum plus Ringer's solution or these plus "Seminan".
b Cultured in Carrel flasks containing serum plus Ringer's or Locke's solution by Sugie et al.
(118).
c M, morula; B, blastocyst.
JOU]~NAL OF DAIRY SCIENCE VOL. 53, ~TO. 12
 A REW~W 1687

Laboratory animals. Rapid advances have may be higher (20, 72, 127). However, the
been made in determining cultural requirements technique does require slaughter of or surgery
for mouse, rat and rabbit ova (6~ 18, 19, 41, on the donor mid usually a laparotomy of the
44, 56, 57, 64, 65, 69, 76, 80, 91, 122). Mouse recipient. One nonsurgieal technique by way
(122) and rabbit ova (64) can be cultured of the cervix and through the uterine wall to
in a completely defined nledium from the one- the ovarian ligament, where ova encapsulated
cell to the expanding blastocyst stage. Devel- in gelatin could be attached by barbs, has been
opment is superior to that obtained in natural described (72). The efficiency of the method
fluids such as blood sermn, and avoids the un- has not been reported. The same workers re-
known variables encountered with these fluids. ported 4 unsuccessful transfers by way of the
I n addition, the development of a simple cul- cervix, although this approach was used unsuc-
ture system (17, 18) has assisted in establish- cessfully in the rabbit (29).
ing nutrient and other requirements for young The uterus to uterus transfer offers the pos-
embryos in vitro. Maintenance of aseptic con- sibility of a combination of surgical or non-
ditions, sterilization of media at the time of surgical approaches, and would appear to hold
use, and adding antibiotics to the culture sys- the most promise for practical egg transfer.
tem are important in preventing bacterial con- A summary of surgical and nonsurgical trans-
tamination (18, 87). W i t h sufficient effort it is fers in cattle is in Table 4. The first transfer
likely that the requirements for culturing and resulting in a live calf was reported by Willett
storing cattle ova also can be determined. et al. in 1951 (124), and was based on the
surgical approach. A nonsurgical transfer in
Inovulation or Egg Transfer swine the same year was successful (71), but
The first transfers in f a r m animals were by in cattle was first successful in 1964 (79). I n
Warwick and Berry (121). Factors of major 1964 Otsuki and Soma (92) also reported suc-
importance in egg transfer include a) media cessful transfer of an egg by way of the cervix
to use, b) synchrony of donor and recipient, in goats. One transfer out of 7 goats receiving
c) stage of embryo development and site of hyosine-~c-butylbromide was successful, whereas
transfer, d) method of reaching the site of zero out of 7 receiving cocaine hydrochloride
deposition and e) number of embryos to trans- intraeervically and zero out of 11 controls
fer to a recipient. Media to use have been delivered kids. The use of these drugs was an
discussed previously. The importance of having attempt to minimize possible stimulation of the
the donor and recipient synchronized was first cervix with consequent expulsion of the egg.
carefully documented by Chang (25), and since Previously it had been reported that "artificial
confirmed in other studies (28, 100). Success- ova" inserted nonsurgically into the uterus were
ful transfers have been reported in cattle when expelled (15, 55), but this was not prevented
the asynchrony was as much as ± 2 days (102). by various drugs (98). Ha fez and Sugie (48)
I n a large population of cyclic cattle a con- devised a technique of bypassing the cervix
tinuous supply of estrous females would be and inflating the uterus with carbon dioxide.
available. I f the group of potential recipients Sugie (111) obtained two pregnancies with
is smM1 some form of estrous cycle synchroni- this method, and one resulted in a live calf.
zation is required (8, 34, 96). This usually Also, Rowson and Moor (101) reported success
involves the feeding of a progestational agent with carbon dioxide inflation (Table 4).
for up to 20 days, with estrus following 2 to The most successful series of transfers in
4 days later. Human chorionic gonadotropin cattle (102) was accomplished by surgical
may be administered. Fertility is somewhat transfer with TCM199 as a recovery and trans-
lower than normal at the first estrus, so the fer medium (Table 4). No pregnancies were
probability of a successful transfer may in- achieved when homologous blood serum was
crease by planning to transfer ova after the used. All transfers were made within 170 min-
second estrus. utes of collection. The longest period of stor-
Transfers should be from oviduct to oviduct age which was followed by- a successful transfer
or uterus to uterus. The relative hardiness of was 150 minutes. Sugie (113) stored cow eggs
the different early cleavage stages of the bovine for 11 to 14 hours. One calf was born from
egg to any unfavorable treatment during trans- 7 recipients.
fer is unknown. The oviduct possesses the The most successful transfers in goats were
potential advantages of efficiency of egg recov- by surgical procedures. When 56 ova were
ery, minimal probability of loss after transfer, transferred to 22 recipients 17 became preg-
and transfer near estrus while the egg is in the nant and produced 35 kids (117).
oviduct at a time when resistance to infection I n the report by Rowson et al. (102) most
JOIYR~AL OF DAIRY SCIENCE VOL. 53, NO. 12
]6~8 FOOTE AND ONU~C[A

TABLE 4. Surgical and nonsurgical ovum transfers in cattle.

Refer- Recipients Media Cell

ence Year Transfer method Total Calved Aborted used stages
By laparotomy (no.) - -
120 1949 Flank, mid-ventral 3 0 3 Seruma ]-8
124 1951 Mid-ventral 3 ~l 0 Serum 8
125 1953 Mid-ventral 5 3 0 Serum 8-12
10 1962 Paralumbar fossa 4 1 0 Serum Morula
102 1969 Mid-ventral 9 0 0 Serum 8-32
102 1969 Mid-ventral 20 135 0 TCM 199 8-32
NonsurgicaF
31 1949 Cervical pipette 8 0 0 Serum 2-16
97 1951 Cervieally 12 0 0 Serum or 4-16
loll. fluid
32 1953 Cervieally, in gelatin ? 0 2 ? ?
36 1954 Cervical pipette ? 0 0 Serum ?
35 1958 Cervical pipette 9 0 1 Serum- 1-
Ringer blastoeys~
10 1962 Cervical instrument 11 0 0 Serum 3-morula
10 ]962 Rectalneedle 3 0 0 Serum 4-6
10 1962 Peritoneal cavity 12 0 17 Serum 2-morula
79 1964 Cervical pipette Several 1 0 Saline 16d
116 1965 Vaginal and uterine 2 1 1 Serum- 16-morula
puncture; CO~ gas Ringer
101 1966 Cervical pipette 14 2 1 Serum 6-morula
and C02 gase
73 ]967 Cervix 4 O 1 ? 8-32
102 1969 See Ref. 101 9 0 O Serum 8-32
102 1969 See Ref. 101 20 4 0 T C M 199 8-32
113 1969 See Ref. 116 7 1 0 Serum- 8-16
Ringer
114 1970 See Ref. 116 Several 1 1 Serum- 16-morula
Ringer
a Bovine serum
b Diagnosed pregnant; 10 of the pregnancies resulted from 11 cows receiving eggs collected
surgically.
c. Many modified forms of equipment were used; consult original references for details.
The one that developed into a calf.
The two calves were from 6 cows synchronized with the donor within +---24hours.

recipients received two eggs, but no twins re- nique for increasing the number of progeny
sulted. Previous studies with superovulated per cow per year, a factor of considerable eco-
cattle (42, 45, 46, 52, 53, 105) have shown that nomic importance in beef cattle.
multiple pregnancies may be produced but At the present time embryo transfer in cattle
embryonic mortality is very high. This is espe- is a technique sufficiently developed to be uti-
cially true when excessive numbers of eggs are lized experimentally in expanding knowledge of
fertilized, and is also true in polytocous ani- reproduction. Before it can be used on a prac-
mals which are snperovulated or are recipients tical ]eve] repeated nonsm'gical recovery of ova,
of extra embryos (13, 28, 61). Thus, while storage methods, and pregnancy rate following
embryo transfer provides a more uniform means nonsurgical transfer need to be improved.
of inducing multiple pregnancy than superovu- References
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