SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
AND GENETIC IMPROVEMENT
Current Status and Potential of Embryo Transfer
and Reproductive Technology in Dairy Cattle
ABSTRACT
Significant use of embryo transfer in
dairy cattle commenced with the in-
troduction of nonsurgical embryo recov-
ery in the mid-1970s and developed with
the use of nonsurgical transfers in the
late I970s. Numbers of registered Hol-
stein calves from embryo transfer dou-
bled yearly through 1980, after which the
rate of increase slowed; the total reached
nearly 19,000 calves in 1990. However,
the efficacy of superovulation procedures
and commercial success rates of trans-
ferred fresh embryos have not improved
the past 10 to 15 yr. Fertilization rates in
superovulated donors remain low.
Although embryo-splitting techniques
were perfected in the early 1980s, they
are not used widely. A practical, com-
mercial embryo-sexing procedure re-
mains unavailable. Recent significant
improvement is apparent in the technol-
ogy of ultrasound-guided oocyte collec-
tion and in vitro oocyte maturation, fer-
tilization, and embryo culture. In the
future, this technology may be used in
conjunction with sperm separated by sex
with a flow cytometer. Modest numbers
of embryo clones have been produced in
several commercial programs via nuclear
transfer techniques. However, the effi-
ciency of gene transfer experiments in-
volving ova of cattle and other domestic
species has been low. Recently, DNA
probe technology has begun to provide
genotype information for cattle and will
ultimately be applied to embryos.
Received October I, 1991.
Accepted March 16, 1992.
1992 J Dairy Sci 75:2857-2879
JOHN F. HASLER
Em Tran, Inc.
Elizabethtown, PA 17022
(Key words: embryo transfer, reproduc-
tive technology, cloning, in vitro)
Abbreviation key: ES = embryonic stern, ET
= embryo transfer, IVC = in vitro culture, IVF
= in vitro fertilization, IVM = in vitro matura-
tion, PMSG = pregnant mare serum
gonadotropin.
INTRODUCTION
Commercial embryo transfer (ET) in cattle
began in North America during the early 1970s
largely because of the availability of PGF2a.
and the high prices and demand for several
breeds of "exotic" beef cattle. During the early
years of the ET industry, embryo recoveries
were performed via a midventral surgical tech-
nique; the donor was under general anesthesia
in a surgical facility. During this period, ET
was not widely utilized in the dairy industry
because the udder of dairy cows hindered mid-
ventral access to the reproductive tract. In ad-
dition, in some donors, the formation of adhe-
sions following surgery sometimes led to loss
or impairment of fertility (49). Consequently,
few reproductively healthy dairy cows were
transported to surgical ET facilities.
Early attempts at nonsurgical recovery of
bovine embryos did not prove successful for
various reasons (43, 45, 184, 208). However,
in 1976, several groups (44, 50, 183) reported
an efficient, nontraumatic, nonsurgical tech-
nique utilizing Foley catheters (C. R. Bard
Inc., Covington, GA). Following the introduc-
tion of the nonsurgical "flushing" technique,
the application of ET in the dairy industry
grew rapidly. As shown in Table I, the first
Holstein calves produced by ET were
registered in 1974. Nonsurgical transfer tech-
niques were developed and refined by numer-
2857
2858 HASLER

TABLE 1. Holstein registrations from embryo transfer 11,756 cattle registered. The application of ET
(ET) for 1974 to 1990.
to genetically superior dairy cows is reflected
Birth year Males Females Total to some degree by the number of sires
1974 1 1 2
produced by ET. The percentage of bulls
1975 5 10 15 produced by ET represented as much as 44%
1976 41 93 134 of the highest index bulls in 1990 to 1991
1977 101 164 265 among three dairy breeds (Table 2).
1978 293 332 625
1979 705 782 1487
1980 1598 1968 3566 SUPEROVULATION
1981 2419 3403 5822
1982 3166 5132 8298 There have been no significant improve-
1983 3807 6399 10,206 ments in techniques for the superovulation of
1984 4359 8452 12,811 cattle in the last 15 yr. Much of the early
1985 4616 9952 14,568
1986 4957 10,393 15,350 research on superovulation involved the use of
1987 5187 9975 15,162 pregnant mare serum gonadotropin (PMSG).
1988 6252 11,387 17,639 Elsden et al. (51) showed that a higher ovula-
1989 6783 11,138 17,921 tory response was obtained with the use of
1990 1 7188 11,539 18.727
Total 51,478 91,120 142,598
FSH than with PMSG. In addition, there is no
commercially available source of PMSG ap-
1990 Non-ET
total -30,000 -365,000 -395,000
proved by the FDA for use in cattle in the US.
Consequently, most research has utilized FSH
INot completed. as the gonadotropin for superovulation. A good
deal of attention has centered on the amount of
LH contained within FSH preparations. All
commercially available FSH are pituitary ex-
ous ET practitioners during the late 1970s. The tracts from domestic species and contain varia-
adoption of both nonsurgical embryo recovery ble amounts of LH. The FSH:LH ratio in one
and ET techniques enabled ET to become an product varied over a 20-fold range among
on-farm procedure, and the number of ET different production lots (126). Laboratory rats
calves increased by more than 100% annually superovulated better when low LH concentra-
through 1980. The rate of growth slowed dur- tions were added to FSH than with pure FSH
ing the 1980s, reaching a total in 1990 of (4). Above a certain concentration, however,
18,727 Holstein ET registered. The ET calves addition of more LH resulted in lower su-
constituted 3% of all females and 19% of all perovulation responses. In cattle, however, this
bulls registered in 1990. Assuming that ap- detrimental effect of LH is not as clear-cut;
proximately 51% of all ET calves were bulls some studies failed to find an effect of the
(103), between 40 and 50% of ET bulls variation in FSH:LH ratios in commercially
produced over the last 5 yr were not registered, available FSH on superovulation (126, 234),
and the actual number of ET Holstein calves whereas, in other studies with controlled ratios
for 1990 was nearly 23,000. Baker's (7) data of FSH:LH, there was a detrimental effect with
covering 1973 through 1988 are the latest
available on ET calves of all breeds in North
America. We adjusted Baker's (7) totals on ET
calves by estimating the number of un- TABLE 2. Top bulls of three dairy breeds for 1990 to
registered male and female ET calves. Hol- 1991.
steins constituted 95% of the total ET calves Percentage
from 7 dairy breeds in 1988 and 37% of all ET Breed Evaluation Number by ETI
calves born, including those from 26 beef Holstein TPI! 100 44
breeds. By comparison, in the Jersey breed, a Jersey PTA-Protein 1 50 22
total of 566 ET were registered in 1990, which Brown Swiss PTII 33 27
represents 1.1 % of the 49,569 total registra- lET = Embryo transfers; TPI = Type-Production In-
tions for the breed. In the Brown Swiss breed, dex; PTA-protein = PTA for percentage and pounds of
365 ET (3.1 %) were registered out of a total of protein; PTI = Production-Type Index.

Journal of Dairy Science Vol. 75, No. 10, 1992

 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
TABLE 3. Variation in superovulation among 5 Holstein cows.
Super- Mean
Cow ovulations total ova
(no.)
A 13 12.2
B 13 5.4
C 11 18.6
D 9 10.3
E 15 8.7
high LH concentration (28, 41, 88). In studies
utilizing commercial FSH preparations in
which the LH content was assumed to be
higher in one product than another, there were
no differences among superovulatory responses
because of the use of different products or
different batch lot numbers of the same
product (15, 33, 242).
In 1983, the first superovulation of 984
Holsteins at Em Tran (Elizabethtown, PAl
yielded an average of 5.1 embryos (81), and
this average has not improved during the inter-
vening years. With a range of 0 to 101 ova and
o to 50 embryos, the average, during a
13-yr period at Em Tran, with over 6000
different Holstein females superovulated one
or more times, was approximately 4.5 to 5.0
usable embryos per superovulated Holstein
donor. Enormous variation is evident in the
responses of individual cattle to superovula-
tion. Table 3 shows very large differences (P <
.001, by ANOVA) in fertilization rates and
embryo numbers among 5 Holstein cows su-
perovulated 9 to 15 times each. On the low
end, cow B averaged only 5.4 ova per su-
perovulation compared with 18.6 for cow C.
Simply increasing the amount of FSH ad-
ministered to a cow has not resulted in the
production of more embryos (36, 153).
Some other variables related to superovula-
tion are relatively well understood. For exam-
ple, initiating superovulation early in the es-
trous cycle resulted in reduced responses (70,
125, 205). Age, to at least 10 yr (37, 81, 124),
and repeated superovulations (40, 42, 81) had,
at best, only a small effect on the number of
embryos produced. However, older cows
selected for superovulation and females under-
going repeated superovulations represent
highly selected individuals. Neither the use of
2859
Mean ova
Fertilized Degenerate Freezable
(%) (no.)
74 3.8 5.2
64 .8 2.6
55 1.3 8.9
93 .1 9.2
90 1.2 6.3
GnRH or analogs (61, 163, 164) nor the use of
human chorionic gonadotropin (108) in con-
junction with superovulation regimens has in-
creased the number of embryos.
The number of small follicles observed via
ultrasound prior to superovulation was posi-
tively correlated with the size of the response
and the number of embryos collected (181).
There have been a number of attempts to in-
crease the number of follicles likely to respond
to superovulation by injecting one or two small
doses of FSH or PSMG early in the estrous
cycle. This treatment resulted in increased em-
bryo production in some studies (155, 215,
224) but not in others (71, 73, 171, 176).
Ultrasound also was used to increase the un-
derstanding of follicular wave cycles during
the bovine estrous cycle (62, 106). This work
indicated that a dominant follicle develops in
each follicular wave and inhibits the matura-
tion of other follicles in that wave. Some re-
cent work (72, 77) suggests that initiating su-
perovulation in the presence of a dominant
follicle reduces ovarian responses. However,
several studies failed to demonstrate a correla-
tion between the presence of a dominant folli-
cle and the size of the superovulatory response
(71, 238). The mere presence of a dominant
follicle may be less important than its suppres-
sive capability (189). More research in
methods of assessing the functional charac-
teristics of dominant follicles may lead to im-
provements in superovulation of cattle.
Recently, bovine FSH has been produced
by recombinant DNA technology (27). Initial
tests of this product for superovulation of cat-
tle indicated a remarkably high production of
embryos because of both a high level of su-
perovulation and a high fertilization rate (239).
In a subsequent study, the degree of superovu-
Journal of Dairy Science Vol. 75, No. 10, 1992
2860
lation was moderately large in Hereford heifers
and very high in Brahman heifers with high
fertilization rates for both (12). This recom-
binant FSH has not yet been approved for use
by the FDA.
FERTILIZATION
Fertilization of the ova produced by su-
perovulation is a component of ET with sig-
nificant room for improvement. In a 1984
study of 984 Holstein cows and heifers su-
perovulated one time each, 61 % of the 8771
recovered ova were fertilized (81). In a current
study of 1337 repeat superovulations of ap-
proximately 150 Holstein cows at Em Tran,
64% of the 11,537 ova were fertilized. Another
large commercial ET company reported a fer-
tilization rate of 63% in 476 superovulated
Holsteins (124). Cows in these studies were
inseminated with 2 to 4 straws of frozen semen
from a variety of commercially popular bulls.
Fertilization was higher (70 to 80%) for su-
perovulated beef cattle in some commercial ET
programs (23, 127, 191). In single-ovulating
cows that were not superovulated, fertilization
rates often range from 85 to nearly 100% (84,
180). In direct comparisons between superovu-
lated and nonsuperovulated cattle, fertilization
rates were inevitably higher in the single-
ovulating females (84, 187, 196). It has not
been proven that sperm transport is inhibited in
superovulated versus single-ovulating cattle.
However, accessory sperm counts have aver-
aged fewer than 1 per zona from superovulated
cows versus over 40 per zona of ova from
single-ovulating cows in some studies (84,
187). A histological study demonstrated
bilateral spenn reservoirs in the uterotubal
junctions and isthmus of single-ovulating cat-
tle, whereas superovulated cattle exhibited
only unilateral or a lack of reservoirs (91). In
some studies (129, 198), but not in others (84,
139), fertilization rates in single-ovulating cat-
tle were higher following semen placement in
the uterine hom versus insemination in the
body of the uterus. Fertilization rates in su-
perovulated cattle were not improved by in-
seminating more than twice (12 and 24 h
postestrus) in one study (225) or once (at 12,
15, or 24 h) versus three or four times in other
studies (130, 190).
The cost of semen in a commercial ET
program is often an important consideration. It
Journal of Dairy Science Vol. 75. No. 10. 1992
HASLER
is tempting to use more semen from bulls
suspected or known to be less than average in
fertility. However, fertilization rates in Hol-
stein cows at Em Tran did not increase when 4
or 5 versus 3 straws of semen were used.
Hawk et al. (83) increased the fertilization rate
from 53% in superovulated cows inseminated
with 70 million frozen spenn to 93% when 4.4
billion fresh sperm were used. Even with 93%
fertilization, however, only 32% of the ferti-
lized ova exhibited accessory sperm.
Fresh and frozen semen from bulls selected
for high and low nonretum rates was used to
inseminate 160 superovulated beef heifers, and
ovulated ova were recovered from the heifers
at slaughter (25). The 89 and 70% fertilization
rates of superovulated ova for the two treat-
ment groups correlated highly with the non-
return rates of the sires. Analysis of some
currently popular Holstein sires used on su-
perovulated cows at Em Tran indicated signifi-
cant differences (P < .001, by ANOVA) in
fertilization rate and in the number of embryos
per flush (Table 4). The differences in fertility
of the 9 bulls listed in Table 4 are representa-
tive of the variation seen in the total data set of
inseminations using 96 bulls, resulting in 1210
embryo recoveries. The differences in fertiliza-
tion rate among bulls were demonstrated wi-
thin in vitro studies (201) in which significant
differences existed among bulls in the propor-
tion of fertilized ova that continued to develop
to the blastocyst stage. In another in vitro
study, fertilization and first cleavage rates were
similar among bulls, but neither was correlated
with the percentage of development to
blastocysts (55). Similarly, in Table 4, which
represents an in vivo comparison, differences
were significant among bulls in the percentage
of degenerate embryos. These data demon-
strate differences among bulls not only in fer-
tility, but also in the viability of preimplanta-
tion embryos. This concept is further supported
by the report from a commercial ET program
that the pregnancy rate of transferred embryos
was significantly affected by the Holstein sire
used to inseminate the donor (29). Recent data
demonstrated a positive relationship in the
number of accessory sperm and the quality of
embryos in single-ovulating Holstein cows
(32). These data appear to indicate that in-
creasing the number of spenn contacting su-
perovulated ova might improve not only fertili-
zation rate, but also embryo quality.
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY 2861
TABLE 4. Fertility of AI sires used on embryo donors.

Degenerate Frozen Mean

Bull flushes UFOl ova ova frozen oval
(no.) (%) (no.)
Ambrose 46 40 10 49 4.9
Blackstar 206 38 13 48 4.8
Cubby 39 35 5 60 4.1
Leadrnan 178 32 16 49 4.6
Michael 74 20 18 61 4.7
Southwind 61 31 15 62 4.3
Tesk 51 39 8 51 3.8
Wister 37 55 10 35 2.5
Laban 20 53 19 27 2.2
TotaI2 1210 35 13 51 4.4
1UFO = Unfertilized ova; mean frozen = mean number of embryos frozen per flush.
2Total values for all 96 bulls used in 1210 flushes.

RECIPIENTS (63%) in this study became pregnant following

Any ET procedure ultimately depends on
the availability of a suitable recipient. Numer-
ous factors related to bovine females have been
studied in order to maximize the rate of preg-
nancy. A classic concern is the importance of
the synchrony of estrus between the recipient
and the donor of the embryo. In 1969, Rowson
et al. (186) showed that a deviation of ± 2 d
could result in pregnancy, but a later study
(185) indicated a large drop in pregnancy rate
with a deviation of only 1 d. However, the data
from the second study (185), which form an
almost perfect bell-shaped curve, do not apply
in all situations. The effect of asynchrony on
pregnancy rate in that study (185) may have
been magnified by the young age of the em-
ET than either beef cows (57%) or heifers
(49%). Synchrony may become less important
with a higher average pregnancy rate. In four
of the five programs, it was advantageous that
the recipient was in estrus before the donor.
Heifers sychronized with PGF2a exhibited a
higher pregnancy rate following ET than
recipient heifers in natural estrus in one study
(80), but there was no difference in another
":
10

~
bryos. The graphs in Figure 1 represent preg- ....0
nancy rates as they relate to donor versus .., 60
..•.•.....•..•...
recipient estrous synchrony from five large
commercial ET programs. The bottom curve "..,
Q,
. ., .
~
represents over 13,000 transfers, primarily into .....,Z 50
'<'il
beef cows that came into estrus naturally.
U
Similarly, the two curves in the middle "
. ., 40
Q,
represent transfers primarily into beef cows.
The top two, very similar curves represent a
total of more than 8000 embryos transferred 30 + - ~ _ ~ ~ _ ~ ~ _ ~ ~ _ ~ ~ _ - - i
-60 -48 ·36 -24 .12 0 -12 -24 -36 --48 -60
into Holstein heifers, most of which had been
HOURS ± SYNCHRONY
synchronized with PGF2a. It is not clear why
there are such differences among some of the Figure 1. Effect of donor-recipient synchrony of estrus
commercial programs represented. The curve on the pregnancy rate of recipients. Recipient in estrus
before donor (+) and recipient in estrus after donor (-).
from Putney et al. (168) is based on more than Key to references and sample sizes: (0) (29) (n = 1202);
13,000 transfers, of which 1320 were dairy (.) (80) (n = 6996); (e) (168) (n = 13,205); (ll) (38) (n =
cattle. A higher percentage of dairy heifers 13,164); and (0) (199) (n = 2016).

Journal of Dairy Science Vol. 75, No. 10, 1992

2862
study (29). The quality of the corpus luteum of
recipients was not correlated with pregnancy
rate (29, 39, 80, 207). Determination of serum
progesterone concentrations in recipients on
the day of ET has been investigated by numer-
ous workers as a possible means of predicting
the success of ET. In most studies, there was
no difference in average blood progesterone on
the day of ET in recipients that became preg-
nant after ET and those that did not (79, 147,
154, 207). However, in some reports (3, 147,
148, 174, 207), pregnancy rates were lower
among recipients for which progesterone con-
centrations were below 1 to 2 ng/ml of serum.
Pregnancy rate in recipients injected with
progesterone at the time of ET was not higher
than that in control females (223). However, in
another study (188), recipients that received
progesterone either by injection or by in-
travaginal sponge and that were in estrus 12 to
48 h after the donor sustained a pregnancy rate
of 53 versus 37% in untreated controls. Injec-
tion of progesterone on d 1 to 5 in recipients
reduced the effect of a 3-d asynchrony between
d-8 embryos and d-5 recipients (64). The preg-
nancy rate in progesterone-treated females was
47 versus 5% in untreated controls. In attempts
to increase progesterone secretion from the
corpus luteum, human chorionic gonadotropin
was injected into ET recipients without im-
proving embryo survival (76, 128, 188). Simi-
larly, treatment of recipients with a GnRH
analog, either at the time of ET or 4 to 7 d
later, did not increase pregnancy rate (47). The
f3-mimetic agent clenbuterol, utilized in order
to maintain uterine quiescence, did not in-
crease pregnancy rate when it was injected
prior to nonsurgical ET (1, 9, 132, 223) or
surgical ET in one study (9), but pregnancy
rate increased when clenbuterol was used prior
to surgical ET in other studies (30, 132).
EMBRYO FREEZING
During the 19705, commercially recovered
bovine embryos in most cases were either
transferred to recipients within hours or dis-
carded. The unpredictability of embryo
production from a given donor and the neces-
sity for donor-recipient synchronization of es-
trus frequently created discrepancies between
the number of embryos and the number of
suitable recipients available. Although it is
Journal of Dairy Science Vol. 75, No. 10, 1992
HASLER
possible to culture bovine embryos with excel-
lent survival at ambient temperature (80) or
O·C (121) for at least 24 h. suitably syn-
chronized recipients still must be available.
Only by long-term storage, necessitating freez-
ing, can surplus embryos be preserved for
transfer at a later date to suitably synchronized
recipients.
In 1972, the survival of cryopreserved
mammalian embryos was proven for the first
time with the birth of live mice that had been
frozen as 2- to 8-cell embryos, stored at
-196·C, and transferred to recipients (227).
Amazingly, this study reported a 65% post-
thaw survival rate for almost 1000 mouse em-
bryos. Although the birth of the first calf
resulting from ET of a frozen embryo was
announced in 1973 (237), it was some years
before the reliability of freezing cow embryos
approached that of mice. Improved techniques
for the freezing of bovine embryos resulted
from the investigation of a number of factors,
including comparisons of types and concentra-
tions of cryoprotectants, optimal rates of freez-
ing and plunge temperature, influence of the
stage and quality of embryos. methods of
cryoprotectant removal, thaw temperature, and
cryoscopic examination of cytoplasmic
responses during freezing and thawing (18. 52,
102, Il2, 113, 145, 146, 231). By the early
19805, some commercial ET companies
reported pregnancy rates from frozen bovine
embryos exceeding 50% (157, 200, 241).
Reliable freezing technology led to the rapid
development of an international trade in frozen
embryos; pregnancy rates exceeded 60% in
some cases (143, 214, 219).
Pathogens have not been demonstrated in-
side the zona of cattle. It is now generally
accepted that the intact zona pellucida is an
effective barrier to the passage of microbes.
including viruses (202). A number of both
viral and bacterial pathogens are effectively
removed from the outside of the bovine zona
pellucida either by rinsing the embryos 10
times in culture medium or, for some patho-
gens. by exposing the embryos briefly to tryp-
sin (202). The pregnancy rate of embryos ex-
posed to trypsin was not compromised (82).
These findings have led to the adoption by
many countries of specific import regulations
that recognize that it is safer to import properly
handled preimplantation embryos than live
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
animals. For example, between 1983 and 1986,
France imported more than 1000 Holstein em-
bryos from donors tested only for brucellosis
and tuberculosis (214). The embryos were
rinsed 10 times prior to freezing, and none of
the resulting calves was clinically positive for
any diseases. Although a significant number of
the embryo donors were positive for bovine
leukemia virus and infectious bovine
rhinotracheitis virus, all of the recipients re-
mained serologicaIly negative. The Interna-
tional Embryo Transfer Society (Champaign,
IL) recognizes a yearly updated list of patho-
gens ranked according to scientifically
documented risk of transmission by ET. Table
5 lists the pathogens for which the risk was
considered to be the lowest as of 1992. These
findings facilitated the export from North
America of frozen embryos from Holstein
donors. The Holstein Association (Brattleboro,
VT) reported that 3200 export certificates for
frozen embryos were processed in 1990, which
may represent 10,000 to 12,000 embryos.
Several additional approaches to embryo
freezing may have the potential to impact the
ET industry in the future. In 1982, Leibo et al.
(120) first described a system in which an
embryo is frozen in a straw along with a
separate volume of liquid necessary to rehy-
drate the embryo. After thawing a one-step
straw, the freeze solution, containing glycerol
and the embryo, and the rehydration solution,
containing sucrose, are mixed by shaking the
straw. Within a few minutes, the embryo can
be transferred nonsurgically directly from the
straw. The patented one-step system (115)
eliminates the need for using a microscope,
unloading thawed embryos, rehydrating them,
and then reloading them again into straws.
Consequently, it eliminates the need for
specialized technical expertise, reduces equip-
ment needs, and saves time. This technique has
not been widely adopted by the ET industry,
probably because the pregnancy rate was be-
tween 26 and 42% (116, 117), which is lower
than that rate achieved with traditional technol-
ogy in the ET industry. However, this may be
a reflection of the conditions under which the
field trials were conducted rather than of the
potential of the freezing system. Pregnancy
rates approximated 50% for other, similar,
single-step systems (134, 175).
Fahy et al. (58) first described different
approach to preservation of embryos during
2863
TABLE 5. Category 1 diseases recognized by the Interna-
tional Embryo Transfer Society in 1992. 1
Trypsin
Diseases required
Leukosis No
Foot and mouth disease No
Brucellosis No
Bluetongue No
Infectious bovine rhinotracheitis Yes
Pseudorabies (swine) Yes
lCategory I diseases are those for which sufficient
evidence has accrued to show that the risk of transmission
is negligible. provided that the embryos are properly han-
dled between collection and transfer.
freezing: vitrification, a phenomenon in which
highly concentrated solutions of cryoprotec-
tants fail to crystallize when rapidly cooled,
passing instead from the liquid state to an
unstructured glassy state. Vitrification involves
exposing embryos to a mixture of different
permeating cryoprotectants and abruptly
plunging them into liquid nitrogen (118).
Although relatively toxic cryoprotectants are
used in the vitrification procedure, 80% of
frozen thawed mouse embryos developed in in
vitro culture (IVC), and a 17% pregnancy rate
was produced by ET (173). There are a few
reports (35) of ET pregnancies produced from
vitrified embryos; one study (134) cited 9
pregnancies from 23 ET. Other than the ease
of the verification freezing procedure, advan-
tages over conventional freezing procedures
are not clear.
CLONING
The word "clone", derived from the Greek
word for twig, suggests the asexual production
of new plants by breaking off twigs (193). In
the context of animal breeding and ET, cloning
implies the production of a number of identical
copies of an individual animal. Unfortunately,
no one has been successful in making clones
from the somatic cells of adult mammals.
Clones in mammals have been produced by
several techniques only from preimplantation
embryos. Consequently, in the case of dairy
cattle, if predictable phenotypes and milk
production are desired, cloned female embryos
must be frozen and stored for approximately 3
yr while one clone reaches maturity after ET
and goes through progeny testing. The im-
Journal of Dairy Science Vol. 75, No. 10. 1992
2864
TABLE 6. A comparison of percentage of pregnancies obtained with split bovine embryos by different researchers.
Transfer Zona (105) (74)
Surgical With 60
Nonsurgical With 57 50
Nonsurgical Without 57
mediate advantage of cloning to cattle breeders
is as a tool for increasing the number of em-
bryos produced through superovulation. For
research purposes, identical animals provide a
large advantage over randomly bred animals
(14). Nicholas and Smith (144) described the
predicted rapid genetic advances of breeding
programs based on genetic selection of clones.
Embryo-Splitting
Embryo-splitting on a commercial basis
owes its roots to experimental studies of
separating the early embryonic blastomeres of
species such as the mouse (211), rabbit (141),
and pig (142). Willadsen and Polge (230) fIrst
described a technique in 1981 by which bovine
monozygotic twins were produced from the
bisection or splitting of one embryo. Following
this, the technology was refined, and successes
were achieved with different kinds of splitting
instruments and embryos of varying ages (149,
232). Application of embryo-splitting within
the ET industry began in the mid-1980s, and a
number of groups reported high success rates
after both surgical and nonsurgical transfer of
both zona-clad and zona-free demi-embryos
(Table 6). The pregnancy rates in this table,
which range from 50 to 76%, translate into 1.0
to 1.52 pregnancies per original embryo, an
obvious advantage over the transfer of intact
embryos. To date, cloning by embryo-splitting
in commercial ET has been limited in the
bovine to production to two demi-embryos per
parent embryo. Early research in the mouse
suggested that the embryological transition
from morula to blastocyst, occurred after a
genetically determined number of cleavage di-
visions, not after a certain number of cells
were present (213). As a consequence, the
number of segments into which an embryo
could be divided, with survival, is limited by
the minimum number of cells necessary to
Journal of Dairy Science Vol. 75, No. 10, 1992
HASLER
References
(8) (210) (3) (197) (119)
64 76
60 57 52
72
form a competent blastocyst. It was recently
shown that a high percentage of embryos der-
ived from single blastomeres, isolated from
4-cell bovine embryos developed in culture
(131). These embryos contained approximately
one-third the number of cells found in normal
control blastocysts. Although Willadsen and
Polge (230) reported a high initial survival rate
of bovine "quarter" derived from separating
8-cell embryos into four 2-cell embryos, the
pregnancy rate after ET was low. In addition to
low success, blastomere separation of 4- or
8-cell embryos is not commercially feasible
because it necessitates the surgical recovery of
the embryos from the oviducts and the use of
an intermediate recipient, such as a sheep, until
the embryos are old enough to be transferred
to the uterus of a bovine. Splitting of bovine
morulae would theoretically be even less suc-
cessful than blastomere separation because of
the inevitable loss of some cells by the split-
ting process (203).
At least one bull stud embarked on a pro-
gram of purchasing pairs of twin bulls and
evaluating them in a progeny test program
(19). The Holstein Association recently in-
itiated a study of performance and type differ-
ences between female twins produced by
embryo-splitting (1 to). An initial finding,
based on 40 pairs of twins maintained within
the same herds, was that 53% of the females
produced at the same level, within 909 kg
(2000 lb) of milk per lactation, as their twins.
The currently used mathematical model
predicted that only 37% of the pairs would be
within 909 kg. Continued studies of this type
may increase the understanding of heritability
versus environmental components of milk
production and other traits. In spite of the
usefulness of such studies, embryo-splitting
has not become widely adopted by the dairy
breeders utilizing ET. Table 7 shows the an-
nual registration of Holstein calves that were
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY 2865
TABLE 7. Registration of Holsteins resulting from split ing pronuclear zygotes as the recIpIent were
embryos for 1982 to 1990.
unsuccessful (136, 179). Additional research
Birth led to a clearer understanding of the impor-
year Males Females Total tance of synchrony between the donor and
1982 4 0 4 recipient eggs, involving various nuclear and
1983 19 25 44 cytoplasmic factors. Tsunoda et a1. (216) were
1984 39 51 90 able to produce mouse clones by fusing
1985 100 149 249
1986 64 158
8-cell nuclei to enucleated 2-cell blastomeres.
222
1987 34 73 107 Cloning technology was significantly im-
1988 3S 61 96 proved when Willadsen (228) showed that sin-
1989 39 73 112 gle blastomeres from 8- and 16-cell sheep
1990 36 81 117 embryos that were fused to enucleated, unferti-
Total 370 671 1041
lized sheep ova supported development to the
blastocyst stage. Lambs were born after ET of
clones from the 8-cell donors. The percentages
of cloned embryos that reached the blastocyst
produced by embryo-splitting. Clearly, after a stage were similar whether Sendai virus or an
peak in 1985 to 1986, the number produced electrofusion apparatus was used to achieve
yearly declined and reached a plateau at a fusion between the donor and recipient mem-
modest number. There are several possible rea- branes. The success in using unfertilized 00-
sons for this. The splitting equipment em- cytes as recipients led to the concept that the
ployed by most ET practitioners is not easily oocyte cytoplasm had the capacity to "repro-
utilized on the farm. Simplified splitting tech- gram" the mitotic clock of the donor nucleus,
niques (182, 233) that do not require the use of thereby turning it back to start the first cell
a micromanipulator have not been widely cycle [for discussion, see (204)].
adopted by the ET industry. Perhaps more The first cloned calves resulted from the
importantly, the inability to know the sex of electrofusion of blastomeres from 9 to
embryos to be split may discourage breeders IS-cell morulae to enucleated oocytes (165).
from increasing the number of calves produced The first report from a commercial bovine
by this technology. As seen in Table 7, only cloning program indicated a 23% pregnancy
approximately one-half of the bull calves rate in recipients receiving embryos cloned
resulting from splitting have been registered from S- to 6-d-old embryos composed of be-
since 1986. tween 16 and 64 cells. (17). Pregnancies were
obtained from clones derived from both fresh
Nuclear Transfer
and frozen donor embryos. In addition, preg-
nancies were obtained from second generation
Mammalian cloning evidenced by the birth clones, i.e., clones derived from clones. The
of three live mice was announced first in 1981 efficiency from the start of the procedure to the
(92). This achievement resulted from the trans- end was low. For example, using blastomeres
fer of nuclei from the inner cell mass of obtained from S-d-01d donor embryos, electro-
blastocysts into fertilized ova from which the fusions between donor blastomeres and
pronuclei were removed. These results have recipient oocytes were attempted 882 times,
not been repeated (136, 179). In 1983, the resulting in 636 fusions (72%), of which 196
success rate of cloning in mice was high, (22%) developed to at least the morula stage
resulting from an improved technique of after 6 d of culture in sheep oviducts. If all 196
removing the pronucleus from the recipient clones had been transferred, the quoted preg-
egg and from the use of Sendai virus to fuse nancy rate of 23% would have produced 45
the donor pronucleus and recipient egg (135). pregnancies, or 5.1 % of the original 882 donor
However, because single-cell pronuclear donor blastomeres. This involved the use of recipient
embryos were used, this technique only had oocytes surgically recovered just prior to ovu-
the potential to produce one copy of each lation from superovulated cattle. A subsequent,
donor embryo. Further attempts to produce much less expensive approach utilized in vitro
clones from multicellular mouse embryos us- matured oocytes from ovaries obtained from

Journal of Dairy Science Vol. 75. No. 10. 1992

2866
the slaughterhouse (10). The rate of cloning
and the pregnancy rate after ET of clones were
similar between recipient oocytes matured in
vitro and in vivo. The birth of the fIrst 100
calves resulting from ET of cloned embryos
was recently announced by another commer-
cial organization (229). The pregnancy rate in
that study (229) was 38% at 90 d of gestation.
No technology related to production of the
clones was presented, but it was noted that
some calves seemed exceptionally large at the
time of birth. Although over 30 clonal preg-
nancies have been produced from one original
donor embryo, a large degree of variation ex-
ists in the ease with which individual embryos
can be cloned (K. Bondioli, personal commu-
nication). As of April 1991, the Holstein As-
sociation and registered 64 calves resulting
from ET of cloned embryos.
CHIMERAS
According to Greek mythology, chimeras
were fIre-breathing monsters, with a lion's
head, a goat's body, and a serpent's tail. Em-
bryologically, chimeras represent an organism
composed of genetically different cell popula-
tions. Tarkowski (212) and Mintz (138) in-
dependently produced the fITSt mouse chimeras
by aggregating two early cleavage stage em-
bryos that combined into a single embryo. The
resulting offspring had tissues derived from
both embryos. Subsequently, mice with 6 or 8
parents have been produced (156). Mouse
chimeras have been extremely valuable in
studying germ cell distribution, sexual
differentiation, and immunological tolerance.
Manipulations to produce chimeras with
trophoblast and inner cell mass originating
from embryos of different species may provide
a means to overcome placental incompatibili-
ties between donors and recipients. For exam-
ple, neither domestic goats nor sheep carry the
embryo of the other species to term. However,
chimeras were constructed so that each
recipient carried young of the opposite species,
whereas the placental tissue was derived from
the same species as the recipient (59).
Chimeras were produced in cattle by aggregat-
ing morulae (22) and by injecting the cells of
one embryo into the blastocoelic cavity of
another (209). Because of the unpredictable
composition of chimeric offspring, it is not
likely that this technology will be applied to
Journal of Dairy Science Vol. 75. No. 10. 1992
HASLER
cattle commercially. However, it may be possi-
ble to produce transgenic cattle by inserting
the desired gene into embryonic stem (ES)
cells, which then would be injected into the
blastocoelic cavity of the host embryo, as has
been done in mice [for review, see (21)]. The
resulting chimera mayor may not express the
transferred gene in its germ line. This would
be a slow and expensive approach, requiring
two generations to produce the desired geno-
type, and then only if the gene is in the germ
line.
SEXING OF EMBRYOS AND SEMEN
Embryos
Sex determination of preimplantation cattle
embryos utilized in ET has economic impor-
tance. The sex ratio at birth in dairy cattle
produced by artifIcial insemination was
reported to be 50.8% males (60), which com-
pares closely with the 51.1 % recorded in ET
calves of mixed breeds (103). Male embryos
develop, on the average, faster than female
embryos, both in vivo (5) and in vitro (6).
However, this phenomenon cannot be utilized
accurately for sexing embryos on an individual
basis (5). Karyotyping is an invasive approach
to embryo-sexing that involves obtaining cells
from an embryo, preparing metaphase spreads,
and identifying the sex chromosomes. DefIni-
tive karyotypes were obtained in only 58% of
hatched blastocysts (78) and in 62% of d-7
embryos (172). In addition to its low effI-
ciency, this invasive technique is not suitable
for embryos requiring an intact zona in order
to be frozen or exported.
Two noninvasive approaches to embryo-
sexing have been investigated: 1) colorimetric
monitoring of X-linked enzyme activity prior
to X chromosome inactivation and 2) im-
munodetection of H-Y antigen with antibodies.
Both of these techniques were only moderately
accurate in sexing mouse embryos [for review,
see (218)). The H-Y antigen technique resulted
in correct sex determination in 84% of
8-cell to blastocyst stage bovine embryos (226)
but has not been adopted by the ET industry.
Seidel (194) reviewed the economics, advan-
tages, and disadvantages of producing ET
calves from sexed embryos based on a
hypothetical 80% rate of accuracy.
 SYMPOSruM: REPRODUCTIVE TECHNOLOGY
Development of probes specific for Y chro-
mosomes has led to an evolution of increas-
ingly accurate and rapid sexing techniques.
Embryo sex detennination with probes specific
for Y chromosomes required about 30 h using
an immunocytochemical technique (123) and 8
d with a radioactive isotope technique (16).
Utilization of the polymerase chain reaction
for amplification of DNA sequences specific
for Y chromosomes shortened the assay time
to a few hours (86, 159). In spite of the speed
and accuracy of this technique, it still requires
invasion of the zona and the use of a
micromanipulator. In addition, current poly-
merase chain reaction technology is not likely
to be usable in on-farm ET situations because
of risk of contamination (87). Consequently,
the primary use of this technology in the future
may be in conjunction with cloning.
Semen
In many cases, even if a group of bovine
embryos could be accurately and rapidly
sexed, some of them would be discarded. Use
of sex-selected semen on superovulated donors
would increase, if not double, the number of
embryos of the desired sex. Theoretically, se-
men can be sex selected either by physical
separation of the X- and Y-bearing sperm
populations or by selectively killing or inac-
tivating one or the other population. Over the
years, success has been claimed for many
different bovine semen-sexing techniques
[reviewed by Amann, (2)]. None of these tech-
niques has been credited with significantly
skewing fetal sex ratios in field trials.
Dead sperm from mice (Microtus) were
separated by flow cytometry into sex-
determining populations with an accuracy of
about 80% (162). Improvements in the flow
cytometric cell sorting equipment and in stain-
ing of the sperm led to 90% accuracy in sepa-
ration of bull sperm that retained the ability to
form pronuclei when injected into hamster 00-
cytes (98). Furthermore, the technology was
used to demonstrate that bovine semen sam-
ples purportedly enriched in X- or Y-bearing
populations by a number of different tech-
niques, were, in fact, unaltered in sex ratio
(161). Rabbits inseminated with sperm sorted
by flow cytometry produced 81 % males and
94% females (99). Because only approximately
350,000 spermlh can be sorted by flow cytom-
2867
etry, this technology is not currently useful in
cattle insemination programs. However, this
technique can possibly be used to provide ade-
quate numbers of sex-selected sperm for in
vitro fertilization (IVF) procedures.
GENE TRANSFER
Mammalian gene transfer was first
described in mice in 1980 (68). Shortly there-
after. Palmiter et al. received a great deal of
attention for the production of transgenic mice
carrying the structural gene for rat (150) and
human (151) growth hormone. Some of the
transgenic mice grew twice as large as con-
trols, and growth hormone concentrations were
as much as 800 times higher. Half of the
transgenics (151) transmitted the growth hor-
mone gene to their offspring, which also grew
faster than controls. These early successes in
mice catalyzed numerous attempts to produce
transgenic livestock; a major effort was
directed to pigs. In the previously mentioned
frrst success with growth hormone (150), 7
transgenic mice resulted from 170 injected ova
(4%). In contrast, Pursel et al. (167) reported
in a review paper that only 73 transgenic pigs
were produced out of a total of almost 11 ,000
(.7%) ova injected. In addition to the very low
efficiencies, there has been no expression to
date of a commercially useful transgene in
pigs. Although some pigs that were transgenic
for growth hormone exhibited enhanced
growth rates and feed efficiencies, they ex-
hibited a variety of problems, including lame-
ness, gastric ulcers, and lack of libido.
Progress in producing transgenic livestock
has lagged behind that in mice for several
reasons. Mouse ova and embryos are readily
available and inexpensive. The pronuclei of
mice are clearly visible within the cytoplasm,
whereas the ova of cattle (221) and pigs (222)
must be centrifuged to displace cytoplasmic
lipid granules, which pennits visualization and
micropuncture of the pronuclei. Also, a much
greater research effort has been directed to
mice and has resulted in production of hun-
dreds of different transgenics, including some
very valuable models for the study of human
disease [for review, see (90)]. Consequently, as
pointed out by Pursel et al. (166), much more
is known regarding the conditions favoring
gene transfer in mice than in any other species.
Journal of Dairy Science Vol. 75, No. 10. 1992
2868
There are only a few reports of transgenic
cattle. Four transgenic 60-d fetuses were
produced from 1325 centrifuged, fertilized ova,
of which 819 had been injected with a con-
struct of chloramphenicol acetyltransferase
structural gene (13). The same research group
(13) later reported the birth of one calf known
to be transgenic with a human estrogen recep-
tor gene linked to a skeletal actin promoter
(133). Still other pregnancies had not reached
term at the time of the report (133); however,
only 79 pregnancies were produced from a
starting total of 1704 (4.6%) injected ova. One
of the preceding studies (13) utilized the sheep
oviduct as an intermediate host for 6 d be-
tween the time of gene insertion into ova and
the nonsurgical transfer of embryos into bo-
vine recipients. Taking into account the failure
to recover 16% of the ova from sheep
oviducts, 17.6% of injected ova developed into
embryos that developed normally. When a
much less expensive system, NC, was utilized
for gene-injected ova, a similar percentage
(15%) developed into embryos (133). Another
laboratory (85) reported a higher proportion of
development (17 to 47%) when DNA-injected
bovine ova were cultured in ligated rabbit
oviducts for 7, 8, or 9 d.
Another approach to gene transfer, men-
tioned in the section on chimeras, involves the
use of ES cells. First described in 1981 [for
review, see (177)], they represent pluripotential
cells isolated from the inner cell mass of
blastocysts. Mouse ES cells can be maintained
indefinitely in vitro in an undifferentiated state.
They can be injected into the blastocoelic
cavity of mouse blastocysts, whereby they con-
tribute to the formation of chimeras. Conse-
quently, DNA can be inserted into ES cells,
and chimeric, transgenic mice can be produced
(178). Unfortunately, although ES-like cells
have been isolated from bovine blastocysts
(54, 206), their ability to contribute to the
germ layer after transfer to a blastocyst has not
been demonstrated. Wilmut et al. (235) sug-
gested a future strategy for more efficient
production of bovine clones if bovine ES were
to become available. By inserting DNA into
ES cells rather than into the pronuclei of early
embryos, only those ES cell lines in which the
DNA is incorporated would be selected for
use. The next unique step in the procedure
would be to produce clones by electrofusing
Journal of Dairy Science Vol. 75, No. 10, 1992
HASLER
single, transgenic ES cells to enucleated 00-
cytes. This would avoid the lengthy and expen-
sive process of producing two generations of
cattle, which would be necessary if the ES
cells were transferred into blastocysts that
would then develop into transgenic chimeras.
This system cannot be attempted at present in
either cattle, because of the lack of ES cells, or
in mice, because it has not proven possible to
produce clones with mouse inner cell-mass
cells.
Notwithstanding the low rates of success in
producing transgenic cattle, there is interest in
getting growth hormone gene constructs into
dairy cattle with the goal of increased milk
production. Because the metallothionein
promoter used in previous sheep and pig
projects causes continuous transcription of the
growth hormone gene, a more suitable
promoter must be found (220). At present, few
genes controlling traits of economic impor-
tance are known in cattle. However, a number
of applications for gene transfer in dairy cattle
directed at changes in milk composition have
been suggested (235). Suggested applications
are related to the protein composition of milk
because it "is controlled very directly by gene
expression" (235). Gene transfer related to
milk proteins falls into two general categories:
1) changes in concentrations of particular pro-
teins, which, depending on the protein, could
result in improvements in the response of milk
to processing, improvements in cheese produc-
tion, increases in the digestibility of milk by
humans, and increased resistance of the trans-
genic cow to mastitis and 2) production of
pharmaceutical proteins. The results of
research in mice and sheep that are relevant to
these potential applications in dairy cows were
reviewed by Wilmut et al. (235, 236). The
birth of a transgenic dairy bull calf carrying a
gene for the milk-specific production of human
lactoferrin was recently announced (107). This
calf was one of 2 transganics (the other of
which was a mosaic) out of 21 calves that
resulted from 129 embryos transferred to
recipients. The project started with the follicu-
lar aspiration and in vitro maturation (IVM) of
2297 oocytes, successful NF of 1358, gene
injection of 1154, and development to the
blastocyst stage in culture with subsequent ET
of 129. Two reports simultaneously published
detailed the production of several transgenic
 SYMPOSIUM: REPRODUCI1VE TECHNOLOGY
goats with human-type plasminogen activator
in their milk (46) and three transgenic ewes
with human antitrypsin in their milk (240).
The efficiency of producing the transgenic
goats and sheep in terms of the number of
embryos that were gene injected was some-
what higher than that described for the in vitro
system utilized to produce the transgenic bull.
Expensive projects such as these often have
strong financial inducements to seek patents
for transgenics. The status of patenting trans-
genics was reviewed by Raines in 1990 (170).
On June 13, 1991, US senator Mark Hatfield
introduced a bill to impose a 5-yr moratorium
on the granting of patents for genetically en-
gineered vertebrate or invertebrate animals (US
Senate bill number 1291).
IVF AND Ive
The advantages of mice versus cattle in
reproductive research have perhaps been most
evident in the development of IVM, NF, and
IVC systems. Mouse ova and embryos can be
inexpensively obtained in large numbers at any
stage of development. Until the recent de-
velopment of efficient bovine NF systems,
embryo culture experiments were dependent on
surgical collection of early embryos or nonsur-
gical collection of older embryos from su-
perovulated cattle. Seidel's literature review
(192) of bovine embryo IVC between 1970
and 1977 documented the widespread use of
small sample sizes. Of 47 experimental groups
in 15 papers, the embryo sample size was 20
or smaller in 22 groups. Much greater progress
was made during this period than was possible
with cattle or other livestock by the use of
mouse ovum culture systems, which utilized
large numbers of ova in multifactorial experi-
ments. However, mouse IVC systems do not
necessarily work well for other species, includ-
ing cattle. For example, the developmental
IVC block that occurs at 2 cells in mice occurs
at 8 to 16 cells in cattle, and the culture
conditions to overcome it differ for the two
species (11). The temperature of culture sys-
tems for mice is routinely 37·C, and most
early work in cattle was performed at 37·C in
spite of the fact that the body temperature of
bovines averages approximately 39°C. Lenz et
al. (122) showed that a significantly higher
percentage of ova were fertilized at 39 versus
2869
37°C. Only after large numbers of fertilized
bovine ova became available were IVC sys-
tems rapidly improved.
Bovine NF was first achieved in 1977 with
semen that was capacitated in the oviduct or
uterus of estrual cow or the uterus of a rabbit
(93). The first live calf resulting from IVF was
born in 1981 (20). During the evolution of
NF, fertilization rates remained modest when
media of high ionic strength were used for
capacitation of sperm (75, 101). The IVF pro-
tocols changed significantly when the effec-
tiveness of capacitation with calcium iono-
phore (24) and heparin (152) was reported.
The first studies of NF in cattle depended on
surgically recovering, via laparotomy or
laparoscope, the activated ova from superovu-
lated females (109). Improvements in culture
systems led to NF of IVM ova in numerous
laboratories (24). Consequently, NF and IVC
experiments were able to use ova obtained
from slaughterhouse ovaries. At present, high
rates of NF are possible with rather different
systems. An NF rate of 88% was achieved
with 24-h incubation of ova and sperm using
heparin capacitation a medium containing
HEPES and modified Tyrode's salt solution,
but only 35% fertilization resulted from
6-h incubation (243). In a defined medium
using calcium ionophore capacitation, 6-h in-
cubation resulted in 87% fertilization (97).
To be commercially useful, IVF embryos
must develop and remain viable for approxi-
mately 7 days. In the early 1970s, Lawson et
al. (111) showed that 1- to 8-cell bovine em-
bryo cleaved when cultured in the rabbit
oviduct, and pregnancy rate was high follow-
ing ET. Similarly, sheep, rabbit, and cow
oviducts were used to culture NF embryos
(114); however, various coculture IVC systems
have largely replaced in vivo systems. Bovine
trophoblastic vesicles significantly enhanced
the development of bovine embryos from the
I-cell to the morula stage (26). More recently,
bovine oviductal cells substantially improved
the IVC development of bovine embryos (56).
Improvement was similar with conditioned
medium, from which oviductal cells were re-
moved prior to IVC, and with frozen-thawed
conditioned medium (57).
Reported pregnancy rates from ET of NF
and IVC embryos are based on small numbers
but are apparently in the 50 to 70% range (56,
Journal of Dairy Science Vol. 75, No. 10, 1992
2870
95, 158), despite the report that blastocysts
produced by NF and IVC contained only one-
half the number of cells found in oviduct-
cultured embryos (94). Also, use of a double
staining technique demonstrated that the IYC
embryos contained only one-half the number
of inner cell-mass cells (95). However, d-5
IVF morulae were reported to have similar
numbers of cells whether they were produced
by IVC or cultured in the rabbit oviduct (48).
It was recently demonstrated that 31 % (63) or
47% (140) of IVM and NF embryos reached
the late morula or blastocyst stage in lYe
without coculture. Thus, in just a few years
bovine culture systems have evolved from the
necessity of oviductal culture in an intermedi-
ate host, to coculture systems, to very promis-
ing IYC successes without coculture.
With the goal of producing twin pregnan-
cies in beef cows, a large commercial project
was developed to provide NF embryos for ET
into previously inseminated cows (67). Be-
cause it is less critical for the IYF embryos in
this program to have genetic value than in
some other situations, the ova originate from
slaughterhouse ovaries, and IYF is by ap-
propriate beef sires. In contrast, application of
IVF and IYC in dairy cattle necessitates that
the ova come from genetically superior donors.
Laparoscopic collection was difficult (109) and
has not been widely adopted for use. In addi-
tion, timing of collection relative to onset of
ovulation is critical. Ultrasound-guided aspira-
tion of antral follicles appears to be a promis-
ing technique. Initial studies demonstrated that
an average of 5 (160) to 10 (217) ova per
attempt could be aspirated on a once or twice
weekly basis. There was no detectable,
detrimental damage to the ovaries after 17 to
27 aspiration attempts (217). Widespread use
of IYF embryos requires that they be frozen.
However, few data are available on the sur-
vival of frozen-thawed embryos resulting from
IVF.
Zona drilling, a technique that involves
chemical dissolution of a hole in the zona, may
have an application when sperm for IYF are in
low concentration. There may be problems
with polyspermy associated with zona drilling
in human NF [for review, see (100)].
However, a recent study showed enhanced NF
and lYe development with no increased poly-
spermy in bovine ova that had been zona
Journal of Dairy Science Vol. 75, No. 10, 1992
HASLER
drilled (104). Sperm injection involves insert-
ing single sperm into the ooplasm of activated
oocytes. In the bovine, exposure of sperm-
injected ova to a calcium ionophore greatly
enhanced activation of the ova (244). Further-
more, it was recently shown that sperm killed
by repeated freezing and thawing could ferti-
lize ova when injected into the ooplasm (69).
Four pregnancies resulted from ET of embryos
fertilized by injection of dead sperm. Another
variation of sperm injection, involving place-
ment of single sperm into the perivitelline
space, resulted in low (9 to 11 %) fertilization
rates (89). Zona drilling and sperm injection
are both techniques that might benefit from the
availability of semen that was sex selected by
flow cytometry (98). Finally, it was recently
shown that primary ovarian follicles from
12- to 16-d-old mice could be grown in Ive to
the preovulatory stage (169). Also, mice were
born after ET of embryos produced from an
NF and IVC system that utilized oocyte-
granulosa cell complexes taken from 12-d-old
mice and cultured in vitro for 10 d (53).
GENE PROBES
Complementary to specific chromosomal
sites, DNA probes represent one of many tools
developed by molecular geneticists. Applica-
tions to livestock have been a very small off-
shoot of the numerous and, in some cases, very
large genetic engineering projects involved
primarily with human medicine and plant
genetics. As mentioned in the section on sex-
ing of semen, specific DNA probes have been
developed that will hybridize with repetitive
DNA sequences found exclusively on the bo-
vine Y chromosome (16). In addition, several
commercial companies have developed probes
for the prolactin (31), K-casein (137), and ~­
lactoglobulin (65) loci, all of which are linked
to increased milk yield, protein yield, or both.
Recently, probes for the weaver trait in Brown
Swiss and for bovine leucocyte adhesion defi-
ciency were made commercially available (1.
M. Massey, personal communication). Markers
closely linked to the bovine polled trait and to
red color in Holsteins are being sought. The
development of DNA probes such as these is
based on the genetic mapping of DNA mar-
kers. Markers, which represent restriction frag-
ment length polymorphisms, are mapped by
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
the study of inheritance patterns in bovine
families, combined with DNA hybridization
techniques and the use of polymerase chain
reaction amplification. Several laboratories are
engaged in the development of bovine
genomic maps, and at least one commercial
laboratory reported that over 200 markers have
been identified (66).
Specific DNA probes available for cattle
owners are being used on blood samples from
cattle of any age and, in the case of the Y-
specific probe, on 4- to lO-cell embryo biop-
sies (86). Continued improvements in IVF and
IVC techniques and increasing availability of
probes make it highly likely that single blasto-
meres of embryos will be monitored in various
ET projects. The substitution of traditional
genetic selection based on phenotype for DNA
markers with probe technology has become
known as marker-assisted selection (66). Ge-
orges (66) suggested the use of the term
"velogenetics" for the selective utilization by
ET of embryos identified through gene probes.
Technology permitting, fetal oocytes would be
obtained and matured through IVF and IVC,
and superior embryos would again be selected
by gene probes and then transferred. This ap-
proach significantly decreases the number of
recipients needed to produce the best from a
potentially large group of IVC embryos. It also
decreases generation time from the use of fetal
oocytes. Obviously, this technology also syner-
gizes very well with cloning and gene transfer
techniques.
CONCLUSIONS
Within the current boundaries of the state of
the art, commercial bovine ET routinely results
in the production of 5 to 7 usable embryos per
superovulated donor and a pregnancy rate of
65 to 80% with fresh embryos and a 55 to 70%
pregnancy rate with frozen embryos transferred
into suitable recipients. Marked improvements
in these pregnancy rates are unlikely. Probably
5 to 10% of the females in any group of
recipients are unsuitable for use because of
undetected fertility problems. The system may
be close to its biological limits of success in
the ET of fresh embryos. Field trials involving
attempts to enhance pregnancy rate with vari-
ous hormones and drugs showed no improve-
ment over controls when the pregnancy rate in
2871
the untreated, control recipients was at the
industry average. There is apparently no sub-
stitute for good management of recipients.
There would appear to be more possibility
for improved pregnancy rates with frozen-
thawed embryos, which are currently 10 to 15
percentage points below those produced from
fresh embryos. Survival of embryos may be
enhanced by new procedures such as vitrifica-
tion. This procedure may have a genuine ad-
vantage over traditional procedures if the
problem of toxic cryoprotectants can be
reduced. There are some unpublished indica-
tions that vitrification may be the method of
choice for IVC embryos produced by IVF and
cloning, which are usually considered to be
more fragile than in vivo embryos. Also, the
delivery system for frozen embryos is cur-
rently a limiting factor in the efficient use of
frozen embryos in the field. Traditional thaw-
ing techniques require the use of a microscope
and the movement of thawed embryos through
two or three rehydration solutions, after which
the embryos are reloaded into straws for trans-
fer. Single-step systems eliminate these proce-
dures and make it practical for a technician to
transfer a single frozen embryo on-farm, much
as a single insemination is currently provided.
If only small increases are possible in preg-
nancy rates from transferred embryos, there
may be more opportunity to achieve greater
numbers of pregnancies per donor by increas-
ing the number of usable embryos per flush.
One approach is to learn how to know more
exactly when to initiate each superovulation
attempt for individual donors. Perhaps a com-
bination of endocrine and ultrasound monitor-
ing will permit the start of superovulation on
the optimal day for a given estrous cycle in a
donor. However, given the commercial neces-
sity of using sires with quite variable fertility
and the variation among donor females, it may
not be possible to push fertilization rates of
superovulated ova much higher.
The rate of progress within different areas
of the ET field is extremely variable. Present
knowledge of how to increase the chances of
making an ET heifer pregnant has not in-
creased in the past 10 yr, and the same number
of embryos are obtained now from superovu-
lated donors as 15 yr ago. As originally prac-
ticed, ET only provided a means to increase
the number of calves per unit of time from a
Journal of Dairy Science Vol. 75. No. 10. 1992
2872 HASLER
donor female. In the early 19808, specific
genetic selection schemes, such as multiple
ovulation embryo transfer (MOET) programs,
were developed to use ET as a means of
decreasing the time necessary to provide a
genetic evaluation on bulls. Now, as pointed
out by Seidel (195), we have recently started
using a number of new technologies, some of
which probably were not even thought of 20 yr
ago. The polymerase chain reaction, for exam-
ple, was perfected less than 10 yr ago and is
now being utilized in several areas of cattle
breeding and ET. Other less exotic but poten-
tially powerful techniques, such as separation
of X- and Y-bearing sperm, IVF and IVC, and
cloning are starting to make an impact within
traditional ET programs. Based on current
technology, it seems reasonable that, in the
near future, primary foJIicles from bovine fe-
tuses wilJ be cultured in vitro over a period of
many weeks to maturity. As proposed in a
velogenetics scheme, four generations of selec-
tion could be achieved in 28 mo by utilizing
ova from 7-mo-old fetuses, IVM, IVF and
IVC, gene probes, and ET. Thus, ET could
become part of a genetic selection program in
which there is no need for calves to be born.
However, there wilJ continue to be a need for
efficient ET techniques to produce on-farm
pregnancies from the results of this futuristic
selection and production technology.
ACKNOWLEDGMENTS
I thank G. E. Seidel, Jr. for reading the
manuscript and making helpful suggestions.
M. J. Hasler was very helpful in organizing the
references. I also want to thank the following
individuals for providing data on registration
of ET cattle: I. Robertson and T. J. Lawlor, Jr.
of the Holstein Association, M. Neymeyer of
the American Jersey Cattle Club, and R. Neit-
zel of the Brown Swiss Cattle Breeder's As-
sociation of the USA.
REFERENCES
1 Almeida, A. P. 1989. Nonsurgical embryo transfer in
cattle: the effect of clenbuterol on pregnancy rates.
Theriogenology 31:166.
2 Amann, R. P. 1989. Treatment of spenn to predeter-
mine sex. Tberiogenology 31:49.
3 Arave, C. W., T. D. Bunch, C. H. Mickelsen, and K.
Warnick. 1987. Factors affecting survivability of
transferred whole and demi-embryos in a commercial
dairy herd. Theriogenology 28:373.
Journal of Dairy Science Vol. 75, No. 10, 1992
4 Annstrong, D. T., A. Suida, M. A. Opavsky, and Y.
Chandrasekhar. 1989. BiomodaI effect of luteinizing
honnone and role of androgens in modifying su-
perovulatory responses of rats to infusion with puri-
fied porcine follicle-stimulating honnone. Bioi.
Reprod. 39:511.
5 Avery, B., A. Bak, and M. Schmidt. 1989. Differen-
tial cleavage rates and sex determination in bovine
embryos. Theriogenology 32:139.
6 Avery, B., V. Madison, and T. Greve. 1991. Sex and
development in bovine in-vitro fertilized embryos.
Theriogenology 35:953.
7 Baker, R. D. 1989. Embryo transfer calves produced
in the United States from 1973 to 1988. Page 108 in
Proc. Am. Embryo Transf. Assoc. Annu. Mtg., Hast-
ings, NE.
8 Baker, R. D., and B. F. Shea. 1985. Commercial
splitting of bovine embryos. Theriogenology 23:3.
9 Barnes, F., and N. L. First. 1985. The effect of
clenbuterol on producing pregnancies from bovine
embryo transfer. Theriogenology 23:174.
10 Barnes, F. L., M. E. Westhusin, and C. R. Looney.
1990. Embryo cloning; principles and practice. Proc.
4th World Congr. Genet. Appl. Livest. Prod. Edin-
burgh, Scotland XVI:323.
11 Bavister, B. D. 1988. Role of oviductal secretions in
embryonic growth in vivo and in vitro. Theriogenol-
ogy 29:143.
12 Bellows, R. A., R. B. Staigmiller, 1. M. Wilson, D.
A. Phelps, and A. Darling. 1991. Use of bovine FSH
for superovulation and embryo production in beef
heifers. Theriogenology 35:1069.
13 Biery, K. A., K. R. Bondiolo, and F. J. De Mayo.
1988. Gene transfer by pronuclear injection in the
bovine. Theriogenology 29:224.
14 Biggers, 1. D. 1986. The potential use of artificially
produced monozygotic twins for comparative pur-
poses. Theriogenology 26: I.
15 Boland, M. P., D. Goulding, and J. F. Roche. 1991.
Alternative gonadotrophins for superovulation in cat-
tle. Tberiogenology 35:5.
16 Bondioli, K. R., S. B. Ellis, J. H. Pryor, M. W.
Williams, and M. M. Harpold. 1989. The use of
male-specific chromosomal DNA fragments to deter-
mine the sex of bovine preimplantation embryos.
Theriogenology 31:95.
17 Bondioli, K. R., M..E Westhusin, and C. R. Looney.
1990. Production of identical bovine offspring by
nuclear transfer. Theriogenology 33:165.
18 Bouyssou, B., and D. Chupin. 1982. Two-step freez-
ing of cattle blastocysts with dimethylsulfoxyde
(DMSO) or glycerol. Theriogenology 17:159.
19 Boyke, D. W. 1988. Duplicate and divide-a step to
the future. Holstein World (August):29.
20 Brackett, B. G., D. Bousquet, M. L. Boice, W. 1.
Donawick, 1. F. Evans, and M. A. Dressel. 1982.
Normal development following in vitro fertilization
in the cow. BioI. Reprod. 27:147.
21 Bradley, A. 1987. Production and analysis of
chimaeric mice. Page 113 in Teratocarcinomas and
Embryonic Stem Cells-a Practical Approach. IRC
Press, Oxford, Eng!.
22 Bream, G., H. Tenhumberg, and H. Krilusslich. 1984.
Chimerism in cattle through microsurgical aggrega-
tion of morulae. Theriogenology 22:609.
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
23 Breul, K. F., R. D. Baker, R. L. Butcher, E. C.
Townsend, E. K. Inskeep, R. A Dairy, and S. P.
Lerner. 1991. Effects of breed, age of donor and
dosage of follicle stimulating hormone on the su-
perovulatory response of beef cows. Theriogenology
36:241.
24 Byrd, W., and D. P. Wolf. 1986. Acrosomal status in
fresh and capacitated human ejaculated sperm. BioI.
Reprod. 34:859.
25 Callaghan, B. D., and G. 1. King. 1980. Determina-
tion of the fertilization rate of A. I. sires. Therioge-
nology 14:403.
26 Camous, S., Y. Heyman, W. Meziou, and Y.
Menezo. 1984. Cleavage beyond the block stage and
survival after transfer of early bovine embryos cul-
tured with trophoblastic vesicles. J. Reprod. Fertil.
72:479.
27 Chappel, S., C. Looney, and K. Bondiolo. 1988.
Bovine FSH produced by recombinant DNA technol-
ogy. Theriogenology 29:235.
28 Chupin, D., Y. Cognie, Y. Combamous, R.
Procureur, and 1. Saumande. 1987. Effect of purified
LH and FSH on ovulation in the cow and ewe. Page
63 in Follicular Growth and Ovulation in Farm
Animals. Martinus Nijhoff Pub!., The Hague, Neth.
29 Coleman, D. A., R. A. Dailey, R. E. Leffel, and R.
D. Baker. 1987. Estrous synchronization and estab-
lishment of pregnancy in bovine embryo transfer
recipients. J. Dairy Sci. 70:858.
30 Coulthard, H. 1982. The use of clenbuterol in em-
bryo transfer recipients. Theriogenology 17:82.
31 Cowan, C. W., M. R. Dentine, R. L. Ax, and L. A.
Schuler. 1970 structural variation around prolactin
gene linked to quantitative traits in elite Holstein sire
family. Theor. Appl. Genet. 79:577.
32 De Jamette, 1. M., R. G. Saacke, 1. Bame, and C. J.
Vogler. 1992. Accessory sperm: their importance to
fertility and embryo quality and attempts to alter
their numbers in artificially inseminated cattle. J.
Anim. Sci. 70:484.
33 Del Campo, M. R., F. Becerro, M. Gonzalez, B. D.
Murphy, and R. J. Mapletoft. 1990. Superovulation
with three different commercial pituitary extracts in
the cow. Theriogenology 33:208.
34 de los Santos-Valadez, S., G. E. Siedel, Jr., and R. P.
Elsden. 1982. Effect of HCG on pregnancy rates in
bovine embryo transfer recipients. Theriogenology
17:85.
35 Dobrinsky, J. R., F. F. Hess, R. T. Duby, and J. M.
Robe. 1991. Cryopreservation of bovine embryos by
vitrification. Theriogenology 35:194.
36 Donaldson, L. E. 1984. Dose of FSH-P as a source
of variation in embryo production from superovu-
lated cows. Theriogenology 22:205.
37 Donaldson, L. E. 1984. Effect of age of donor cows
on embryo production. Theriogenology 21:963.
38 Donaldson, L. E. 1985. Matching of embryo stages
and grades with recipient oestrous synchrony in bo-
vine embryo transfer. Vet. Rec. 117:489.
39 Donaldson, L. E. 1985. Recipients as a source of
variation in cattle embryo transfer. Theriogenology
23:188.
40 Donaldson, L. E., and B. Perry. 1983. Embryo
production by repeated superovulation of commercial
donor cows. Theriogenology 20:163.
2873
41 Donaldson, L. E., and D. N. Ward. 1987. LH effects
on superovulation and fertilization rates. Therioge-
nology 27:225.
42 Dom, C. G., 1. F. Baker, D. K. Lunt, and D. C.
Kraemer. 1991. Repeated, short interval superovula-
tion in virgin heifers. Theriogenology 35:302.
43 Dracy, A. E., and W. E. Peterson. 1950. Isolation of
ova from the living bovine. J. Dairy Sci. 33:797.
44 Drost, M., A. Brand, and M. H. Aarts. 1976. A
device for nonsurgical recovery of bovine embryos.
Theriogenology 6:503.
45 Dziuk, P. J., J. D. Donker, 1. R. Nichols, and W. E.
Petersen. 1958. Page I in Problems associated with
the transfer of ova between cattle. Minnesota Agric.
Exp. Sin. Tech. Bull. 222, St. Paul, MN.
46 Ebert, K. M., J. P. Selgrath, P. Titullio, J. Denman,
T. E. Smith, M. A. Memon, J. E. Schindler, G. M.
Monastersky, J. A. Vitale, and K. Gordon. 1991.
Transgenic production of a variant of human-type
plasminogen activator in goat milk: generation of
transgenic goats and analysis of expression. BioI
Technology 9:835.
47 Ellington, J., R. Foote, J. Webb, J. Hasler, and A.
McGrath. 1990. The use of a GNRH analog in an
embryo transfer field trial. Theriogenology 33 :225.
48 Ellington, 1. E., P. B. Farrell, M. E. Simkin, R. H.
Foote, E. E. Goldman, and A. B. McGrath. 1990.
Development and survival after transfer of cow em-
bryos cultured from 1-2-cells to morulae or
blastocysts in rabbit oviducts or in a simple medium
with bovine oviduct epithelial cells. J. Reprod. Fertil.
89:293.
49 Elsden, R. P. 1977. Embryo collection by surgical
methods. Page 10 in Embryo Transfer in Farm
Animals. A Review of Techniques and Applications.
Monogr. No. 16. Agric. Canada, Ottawa, ON.
50 Elsden, R. P., 1. F. Hasler, and G. E. Seidel, Jr. 1976.
Non-surgical recovery of bovine eggs. Theriogenol-
ogy 6:523.
51 Elsden, R. P., L. D. Nelson, and G. E. Seidel, Jr.
1978. Superovulating cows with follicle stimulating
hormone and pregnant mare's serum gonadotrophin.
Theriogenology 9: 17.
52 Elsden, R. P., G. E. Seidel, Jr., T. Takeda, and G. D.
Farrand. 1982. Field experiments with frozen-thawed
bovine embryos transferred non-surgically. Therioge-
nology 17:1.
53 Eppig, J. J., and A. C. Schroeder. 1989. Capacity of
mouse oocytes from preantral follicles to undergo
embryogenesis and development to live young after
growth, maturation, and fertilization in vitro. BioI.
Reprod. 41:268.
54 Evans, M. J., E. Notarianni, S. Laurie, and R. M.
Moor. 1990. Derivation and preliminary characteri-
zation of pluripotent cell lines from porcine and
bovine blastocysts. Theriogenology 33:125.
55 Eyestone, W. H., and N. L. First. 1989. Variation in
bovine embryo development in vitro due to bulls.
Theriogenology 31: 191.
56 Eyestone, W. H., and N. L. First. 1989. Co-culture of
early cattle embryos to the blastocyst stage with
oviductal tissue or in conditioned medium. J. Reprod.
Fertil. 85:715.
57 Eyestone, W. H., J. M. Jones, and N. L. First. 1991.
Some factors affecting the efficacy of oviduct tissue-
Journal of Dairy Science Vol. 75, No. 10, 1992
2874
conditioned medium for the culture of early bovine
embryos. J. Reprod. Fertil. 92:59.
58 Fahy, G. M., D. R. MacFarlane, C. A. Angell, and H.
T. Meryman. 1984. Vitrification as an approach to
cryopreservation. Cryobiology 21:407.
59 Fehilly, C. B., S. M. Willadsen, and E. M. Tucker.
1984. Interspecific chimerism between sheep and
goat. Nature (Lond.) 307:634.
60 Foote, R. H. 1977. Sex ratios in dairy cattle under
various conditions. Theriogeno10gy 8:349.
61 Foote, R. H., S. E. Allen, and B. Henderson. 1989.
Buserelin in a superovu1atory regimen for Holstein
cows: II. Yield and quality of embryos in commer-
cial herds. Theriogenology 31:385.
62 Fortune, J. E., J. Sirois, and S. M. Quirk. 1988. The
growth and differentiation of ovarian follicles during
the bovine estrous cycle. Theriogenology 29:95.
63 Fukui, Y., L. T. McGowan, R. W. James, P. A. Pugh,
and H. R. Tervit. 1991. Factors affecting the in-vitro
development to blastocysts of bovine oocytes ma-
tured and fertilized in vitro. J. Reprod. Ferti!. 92: 125.
64 Geisert, R. D., T. C. Fox, G. L. Morgan, M. E.
Wells, R. P. Wetterman, and M. T. 'Zavy. 1991.
Survival of bovine embryos transferred to
progesterone-treated asynchronous recipients. J.
Reprod. Fertil. 92:475.
65 Geldermann, H., U. Pieper, and B. Roth. 1985. Ef-
fects of marked chromosome secretions on milk
performance in cattle. Theor. Appl. Genet. 70:138.
66 Georges, M. 1990. Perspectives for marker-assisted
selection and velogenetics in animal breeding. Page
285 in Animal Applications of Research in Mam-
malian Development. Gold Spring Harbor Labora-
tory Press, Plainview, NY.
67 Gordon, I., and K. H. Lu. 1990. Production of em-
bryos in vitro and its impact on livestock production.
Theriogeno10gy 33:77.
68 Gordon, J. W., G. A. Scangos, D. Plotkin, 1. A.
Barbosa, and F. H. Ruddle. 1980. Genetic transfor-
mation of mouse embryos by microinjection of puri-
fied DNA. Proc. Natl. Acad. Sci. USA 77:7380.
69 Goto, K., A. Kinoshita, Y. Takoma, and K. Ogawa.
1991. Birth of calves after the transfer of oocytes
fertilized by sperm injection. Theriogenology 35:205.
70 Goulding, D., D. H. Williams, P. Duffy, M. P.
Boland, and 1. F. Roche. 1990. Superovulation in
heifers given FSH initiated either at day 2 or day 10
of the estrous cycle. Theriogeno10gy 34:767.
71 Grasso, F., L. A. Guilbault, G. L. Roy, and J. G.
Lussier. 1989. Ultrasonographic determination of
ovarian follicular development in superovulated heif-
ers pretreated with FSH-P at the beginning of the
estrous cycle. Theriogeno10gy 31:1209.
72 Grasso, F., L. A. Guilbault, G. L. Roy, P. Matton,
and 1. G. Lussier. 1989. The influence of the
presence of a dominant follicle at the time of the
initiation of a superovu1atory treatment on su-
perovu1atory responses in cattle. Theriogeno10gy 31:
199.
73 Gray, B. W., R. E. Carter, D. A. Stringfellow, M. G.
Riddell, K. P. Riddell, and J. C. Wright. 1991. The
effects of dominant follicle regression and FSH
priming on the superovulatory response in cattle.
Theriogenology 35:207.
Journal of Dairy Science Vol. 75, No. 10, 1992
HASLER
74 Gray, K. R., K. R. Bondioli, and C. L. Betts. 1991.
The commercial application of embryo-splitting in
beef cattle. Theriogeno1ogy 35:37.
75 Greve, T., D. Bousquet, W. A. King, and K. 1.
Betteridge. 1984. In vitro fertilization and cleavage
of in vivo matured bovine oocytes. Theriogeno1ogy
22:151.
76 Greve, T., and H. Lehn-Jensen. 1982. The effect of
HCG administration on pregnancy rate following
non-surgical transfer of viable bovine embryos.
Theriogenology 17:91.
77 Guilbault, L. A., F. Grasson, J. G. Lussier, P. Rouil-
Iier, and P. Matton. 1991. Decreased superovulatory
responses in heifers superovulated in the presence of
a dominant follicle. J. Reprod. Fertil. 91:81.
78 Hare, W.C.D., D. Mitchell, K. J. Betteridge, M. D.
Eaglesome, and G.C.B. Randall. 1976. Sexing two-
week-old bovine embryos by chromosomal analysis
prior to surgical transfer: preliminary methods and
results. Theriogeno10gy 5:243.
79 Hasler, J. F., R. A. Bowen, L. D. Nelson, and G. E.
Seidel, Jr. 1980. Serum progesterone concentrations
in cows receiving embryo transfers. J. Reprod. Fertil.
58:71.
80 Hasler, J. F., A. D. McCauley, W. F. Lathrop, and R.
H. Foote. 1987. Effect of donor-embryo-recipient
interactions on pregnancy rate in a large-scale bovine
embryo transfer program. Theriogeno10gy 27:139.
81 Hasler, J. F., A. D. McCauley, E. C. Schermerhorn,
and R. H. Foote. 1983. Superovu1atory responses of
Holstein cows. Theriogenology 19:83.
82 Hasler, J. F., and A. N. Reinders. 1988. Pregnancy
rate following transfer of bovine embryos treated
with trypsin prior to freezing. Page 91 in Proc. Am.
Embryo Transf. Assoc. Annu. Mtg., Hastings, NE.
83 Hawk, H. W., H. H. Conley, R. 1. Wall, and R. O.
Whitaker. 1988. Fertilization rates in superovulating
cows after deposition of semen on the infundibulum,
near the uterotubal junction or after insemination
with high numbers of sperm. Theriogenology 29:
1131.
84 Hawk, H. W., and T. Y. Tanabe. 1986. Effect of
unilateral cornual insemination upon fertilization rate
in superovulating and single-ovulating cattle. 1.
Anim. Sci. 63:51.
85 Hawk, H. W., R. 1. Wall, and H. H. Conley. 1989.
Survival of DNA-injected cow embryos temporarily
cultured in rabbit oviducts. Theriogenology 32:243.
86 Herr, C. M., N. A. Holt, K. I. Matthaei, and K. C.
Reed. 1990. Sex of progeny from bovine embryos
sexed with a rapid Y-chromosome-detection assay.
Theriogeno10gy 33:247.
87 Herr, C. M., and K. C. Reed. 1991. Micromanipula-
tion of bovine embryos for sex determination. Theri-
ogeno10gy 35:45.
88 Herrler, A., F. Eisaesser, N. Parvizi, and H. Nie-
mann. 1991. Superovulation of dairy cows with puri-
fied FSH supplemented with defined amounts of LH.
Theriogenology 35:633.
89 Heuwieser, W., X. Yang, S. Jiang, and R. H. Foote.
1991. Fertilization of bovine oocytes by microinjec-
tion of spermatozoa into the perivitelline space.
Theriogenology 35:213.
90 Hooper, M. L. 1990. Genetically engineered animals:
implications for the understanding and treatment of
human disease. Biofutur 86:30.
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
91 Hyttel, P.• H. Callesen, T. Greve, and M. Schmidt.
1991. Oocyte maturation and sperm transport in su-
perovulated cattle. Theriogenology 35:91.
92Illrnense, K., and P. C. Hoppe. 1981. Nuclear trans-
plantation in Mus musculus: developmental potential
of nuclei from preimplantation embryos. Cell 23:9.
93 lritani, A.• and K. Niwa. 1977. Capacitation of bull
spennatozoa and fertilization in vitro of canle follic-
ular oocytes matured in culture. 1. Reprod. Fertil. 50:
119.
94 Iwasaki, S., and T. Nakahara. 1990. Cell number and
incidence of chromosomal anomalies in bovine
blastocysts fertilized in vitro followed by culture in
vitro or in vivo in rabbit oviducts. Theriogenology
33:669.
95 Iwasaki, S., N. Yoshiba, H. Ushijima, S. Watanabe,
and T. Nakahar. 1990. Morphology and proportion of
inner cell mass of bovine blastocysts fertilized in
vitro and in vivo. J. Reprod. Fertil. 90:279.
96 Jiang, H. S.• W. L. Wang, K. H. Lu, 1. Gordon, and
C. Polge. 1991. Roles of different cell monolayers in
the co-culture of IVF bovine embryos. Theriogenol-
ogy 35:216.
97 Jiang, S., X. Yang. S. Chang, W. Heuwieser, and R.
H. Foote. 1991. Effects of sperm capacitation and
oocyte maturation procedures on fertilization and
development of bovine oocytes in vitro. Theriogenol-
ogy 35:218.
98 Johnson, L. A., and R. N. Clarke. 1988. Row sorting
of X and Y chromosome-bearing mammalian sperm:
activation and pronuclear development of sorted bull.
boar, and ram sperm microinjected into hamster 00-
cytes. Gamete Res. 21:335.
99 Johnson, L. A., J. P. Rook. and H. W. Hawk. 1989.
Sex preselection in rabbits: live births from X and Y
sperm separated by DNA and cell sorting. BioI.
Reprod. 41:199.
100 Keefer, C. L. 1990. New techniques for assisted
fertilization. Theriogenology 33:101.
101 Keefer, C. L.. B. G. Bracken, and C. G. Troop. 1985.
Bovine fertilization after in vitro insemination with
motile sperm fractions. Theriogenology 23:198.
102 Kennedy, L. G., M. P. Boland, and 1. Gordon. 1983.
The effect of embryo quality at freezing on subse-
quent development of thawed cow embryos. Theri-
ogenology 19:823.
103 King, K. K., G. E. Seidel, Jr., and R. P. Elsden.
1985. Bovine embryo transfer pregnancies. 1. Abor-
tion rates and characteristics of calves. J. Anim. Sci.
61:758.
104 Kinis, A., E. Vergos, P. Lonergan, H. Sharif, M.
Gallagher, and 1. Gordon. 1991. The incidence of
polyspermy and the developmental potential of bo-
vine ova fertilized in vitro after zona drilling. Theri-
ogenology 35:226.
105 Kippax, 1. S., W. B. Christie, and T. G. Rowan.
1991. Effects of methods of splitting, stage of de-
velopment and presence or absence of zona pellucida
on foetal survival in commercial bovine embryo
transfer of bisected embryos. Theriogenology 35:25.
106 Ko. J.C.H., J. P. Kastelic. M. R. Del Campo, and O.
J. Ginther. 1991. Effects of a dominant follicle on
ovarian follicular dynamics during the oestrous cycle
in heifers. J. Reprod. Fertil. 91:511.
2875
107 Krimpenfort, P., A. Rademakers, W. Eyestone. A.
van der Schans, S. van der Brock. P. Kooiman, E.
Kootevijk. G. Platenburg, F. Pieper, R. Strijker, and
H. de Boer. 1991. Generation of transgenic dairy
canle using 'in vitro' embryo production. BioI
Technology 9:844.
108 Kuehl, T. J., S. P. Leibo. and W. F. Rall. 1987. The
role of a single injection of HCG in superovulation
of the cow. Theriogenology 27:244.
109 Lambert, R. D., M. A. Sirard, C. Bernard, R. Beland,
1. B. Rioux, P. Leclerc, D. P. Menard, and M.
Bedoya. 1986. In vitro fertilization of bovine oocytes
matured in vivo and collected at laparoscopy. Theri-
ogenology 25:117.
110 Lawlor, T. 1991. How similar are identical twins?
Holstein World (August):60.
III Lawson, R.A.S., L.B.A. Rowson, and C. B. Adams.
1972. The development of cow eggs in the rabbit
oviduct and their viability after re-transfer to heifers.
1. Reprod. Fertil. 28:313.
112 Lehn-Jensen. H., T. Greve, and A. P. Navas. 1981.
Two-step freezing of cow embryos in 1.4 M
glycerol. Theriogenology 15:427.
113 Lehn-Jensen, H., and W. F. Rall. 1983.
Cryomicroscopic observations of cattle embryos dur-
ing freezing and thawing. Theriogenology 19:263.
114 Leibfied, M. L., B. S. Critser. 1. 1. Parrish, and N. L.
First. 1989. In vitro maturation and fertilization of
bovine oocytes. Theriogenology 31:61.
115 Leibo. S. P. 1983. Embryo transfer method and
apparatus. Rio Vista Int., Inc., assignee. US Pat. No.
4,380,997.
116 Leibo, S. P. 1984. A one-step method for direct
nonsurgical transfer of frozen-thawed bovine em-
bryos. Theriogenology 21:767.
117 Leibo, S. P. 1986. Commercial production of preg-
nancies from one-step diluted frozen-thawed bovine
embryos. Theriogenology 25:166.
118 Leibo, S.P. 1989. Equilibrium and nonequilibrium
cryopreservation of embryos. Theriogenology 31 :85.
119 Leibo, S. P., and W. F. Rall. 1987. Increase of
production of pregnancies by bisection of bovine
embryos. Theriogenology 27:245.
120 Leibo, S. P., A. W. West, III, and B. Perry. 1982. A
one-step method for direct nonsurgical transfer of
frozen-thawed bovine embryos. 1. Basic studies.
Cryobiology 19:673.
121 Leibo, S. P., and D. Winninger. 1986. Production of
bovine pregnancies from embryos transported at O'C
by air. Theriogenology 25:165.
122 Lenz, R. W., G. D. Ball, M. L. Leibfried, R. L. Ax,
and N. L. F'U'St. 1983. In vitro maturation and fertili-
zation of bovine oocytes are temperature-dependent
processes. BioI. Reprod. 29:173.
123 Leonard, M., M. Kirszenbaum, O. Cotinot. P.
Chesne, Y. Heyman, M. G. Stinnakre, C. Bishop, C.
Oelouis, M. Vaiman, and M. Fellous. 1987. Sexing
bovine embryos using Y chromosome specific DNA
probe. Theriogenology 27:248.
124 Lerner, S. P.• W. V. Thayne, R. D. Baker, T. Hen-
schen, S. Meredith. B. K. Inskeep, R. A. Dailey, P.
E. Lewis, and R. L. Butcher. 1986. Age, dose of FSH
and other factors affecting superovulation in Holstein
cows. J. Anim. Sci. 63:76.
Journal of Dairy Science Vol. 75. No. 10, 1992
2876 HASLER
125 Lindsell, C. E., V. Pawlyshyn, A. Bielanski, and R. J.
Mapletoft. 1985. Superovulation of heifers with
FSH-P beginning on four different days of the cycle.
. Theriogenology 23:203.
126 Lindsell, C. E., K. Rajkumar, A. W. Manning, S. K.
Emery, R. J. Mapleloft, and B. D. Mwphy. 1986.
Variability in FSH:LH ratios among batchs of com-
mercially available gonadotrophins. Theriogenology
25:167.
127 Looney, C. R. 1986. Superovulation in beef females.
Page 16 in Proc. Am. Embryo Transf. Assoc. Annu.
Mtg., Hastings, NE.
128 Looney, C. R., A. J. Oden, 1. M. Massey, C. A.
Johnson, and R. A. Godke. 1984. Pregnancy rates
following HCG administration at the time of transfer
in embryo-recipient cattle. Theriogeno10gy 21:246.
129 L6pez-Gatius, F., and J. Cam6n-Urge!. 1988. In-
crease of pregnancy rate in dairy cattle after
preovulatory follicle palpation and deep cornual in-
semination. Theriogenology 29:1099.
130 L6pez-Gatius, F., J. Cam6n-Urgel, and E. Angula
Asensio. 1988. Effects of single deep insemination
on transferrable embryo recovery rates in superovu-
lated dairy cows. Theriogenology 30:877.
131 Loskutoff, N. M., I. W. Pollard, I. M. Scodras, R. De
la Fuente, W. A. King, and K. I. Betteridge. 1991.
Production of blastocysts from single blastomeres
cultured after isolation from 4-cell bovine embryos
generated in vitro. Theriogenology 35:233.
132 Mapletoft, R. 1., C. E. Lindsell, and V. Pawlyshyn.
1986. Effects of clenbuterol, body condition and
non-surgical embryo transfer equipment on preg-
nancy rates in bovine recipients. Theriogenology 25:
172.
133 Massey, I. M. 1990. Animal production in the year
2000 AD. I. Reprod. Fertil. 41(Supp!.):199.
134 Massip, A., P. Van Der ZWalrnen, and F. Betors.
1987. Recent progress in czyopreservation of cattle
embryos. Theriogenology 27:69.
135 McGrath, I., and D. Solter. 1983. Nuclear transplan-
tation in the mouse embryo by microsurgery and cell
fusion. Science 220:1300.
136 McGrath, 1., and D. Solter. 1984. Inability of mouse
blastomere nuclei transferred 10 enucleated zygotes
to support development in vitro. Science 226:1317.
137 Medrano, I. F., and E. Aquilor-Cordova. 1990.
Genotyping of bovine x-casein loci following DNA
sequence amplification. BiolTechnology 8:144.
138 Mintz, B. 1962. Formation of genotypically mosaic
mouse embryos. Am. Zool. 2:432.
139 Momont, H. W., B. E. Seguin, G. Singh, and E.
Stasiukynas. 1989. Does intrauterine site of insemi-
nation in cattle really matter? Theriogenology 32: 19.
140 Moore, K., and K. Bondioli. 1991. Amino acid sup-
plementation of culture medium enhances develop-
ment of bovine IVMIIVF embryos. Page 39 in Proc.
Serono Symp. Preimplantation Embryo Dev., New-
ton, MA, Springer-Verlag, New York, NY.
141 Moore, N. W., C. E. Adams, and L.E.A. Rowson.
1968. Developmental potential of single blastomeres
of the rabbit egg. 1. Reprod. Ferti!. 17:527.
142 Moore, N. W., C. Polge, and L.E.A. Rowson. 1969.
The survival of single blastomeres of pig eggs trans-
ferred to recipient gilts. Aust. I. BioI. Sci. 22:979.
Journal of Dairy Science Vol. 75, No. 10, 1992
143 Munar, C. I., and I. F. Hasler. 1989. Results of the
flISt frozen bovine embryos exported from the USA
to Argentina. Theriogenology 3I :230.
144 Nicolas, F. W., and C. Smith. 1983. Increased rates
of genetic change in dairy cattle by embryo transfer
and splitting. Anim. Prod. 36:341.
145 Niemann, H. 1985. Freezing of bovine embryos:
effects of a one-step addition of 1.4M glycerol.
Theriogenology 23:369.
146 Niemann, H., W. W. Lampeter, B. Sacher, and B.
Kruff. 1982. Comparison of survival rates of day 7
and day 8 bovine embryos after fast freezing and
thawing. Theriogenology 18:445.
147 Niemann, H., B. Sacher, and F. Elsaesser. 1985.
Pregnancy rates relative to recipieDl plasma
progesterone levels on the day of non-surgical trans-
fer of frozen-thawed bovine embryos. Theriogenol-
ogy 23:631.
148 Northey, D. L., F. L. Barnes, W. H. Eyestone, and N.
L. First. 1985. Relationship of serum progesterone,
luteinizing hormone and the incidence of pregnancy
in bovine embryo transfer recipients. Theriogenology
23:214.
1490zil, J. P. 1982. Production of identical twins by
bisection of blastocysts in the cow. I. Reprod. Fertil.
69:463.
150 Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E.
Trumbauer, M. G. Rosenfeld, N. C. Birnberg, and R.
M. Evans. 1982. Dramatic growth of mice that de-
velop from eggs microinjected with metallothionein-
growth hormone fusion genes. Nature (Lond.) 300:
611.
151 Palmiter, R. D., G. Norstedt, R. E. Galinas, R. E.
Hammer, and R. L. Brinster. 1983. Metallothionein-
human GH fusion genes stimulate growth of mice.
Science 222:809.
152 Parrish, I. J., 1. Susko-Parrish, M. A. Winner, and N.
L. First. 1988. Capacitation of bovine sperm by
heparin. BioI. Reprod. 38: 1171.
153 Pawlyshyn, V., C. E. Lindsell, M. Brouthwaite, and
R. J. Mapletoft. 1986. Superovulation of beef heifers
with FSH-P; a dose response trial. Theriogenology
25:179.
154 Payas, A. I., P. I. Broadbent, D. F. Dolman, and W.
B. Christie. 1989. Factors affecting pregnancy rate in
embryo transfer recipients with reference to plasma
progesterone. Theriogenology 31:238.
ISS Petr, J., 1. Mika, and F. Jilek. 1990. The effect of
PMSG-priming on subsequent superovulatory
response in dairy cows. Theriogenology 33:1151.
156 Petters, R. M., and C. L. Markert. 1980. Production
and reproductive performance of hexaparental and
octaparental mice. 1. Reprod. Fertil. 71 :70.
157 Pettit, W. H., Jr. 1985. Commercial freezing of bo-
vine embryos in glass ampules. Theriogenology 23:
13.
158 Peura, T. I.-M. Hyttinen, I. Janne, and I. Aalto.
1991. Transfer of in vitro matured, fertilized and
cultured bovine embryos of predetermined sex.
Theriogenology 35:254.
159 Peura, T., I.-M. Hyttinen, M. Turunen, and 1. Ifume.
1991. A reliable sex determination assay for bovine
preimplantation embryos using the polymerase chain
reaction. Theriogenology 35:547.
 SYMPOSIUM: REPRODUcrlVE TECHNOLOGY
160 Pieterse, M. C., P.LAM. Vos, TAM. Kroip, Y. A.
Woo, T. H. van Beneden, A. H. Willemse, and
M.A.M. Taverne. 1991. Transvaginal ultrasound
guided follicular aspiration of bovine oocytes. Theri-
ogenology 35:857.
161 Pinkel, D., D. L. Garner, B. L. Gledhill, S. Lake, D.
Stephenson, and L. A. Johnson. 1985. Row cytomet-
ric detennination of the proportions of X- and Y-
chromosome-bearing sperm in samples of purport-
edly separated bull sperm. J. Anim. Sci. 60:1303.
162 Pinkel, D., B. L. Gledhill, S. Lake, D. Stephenson,
and M. A. Van Dilla. 1982. Sex selection in mam-
mals? Separation of sperm bearing Y and "0" chro-
mosomes in the vole Microtus oregoni. Science 218:
904.
163 Posadas, E., 1. Valencia, L. Zarco, and J. Avila.
1991. Evaluation of the incorporation of GnRH into
a superovulatory regimen for Zebu cattle. Therioge-
nology 35:761.
164 Prado Delgado. A. R., R. P. Elsden. and G. E. Seidel,
Jr. 1989. Effects of GnRH on superovulated cattle.
Theriogenology 31 :317.
165 Prather, R. S.• F. L. Barnes, M. M. Sims, J. M. Robl,
W. E. Eyestone, and N. L. First. 1987. Nuclear
transplantation in the bovine embryo: assessment of
donor nuclei and recipient oocyte. BioI. Reprod. 37:
859.
166 Pursel, V., C. A. Pinkert, K. F. Miller, D. J. Bolt. R.
G. Campbell, R. D. Palmiter, R. L. Brinster, and R.
E. Hammer. 1989. Genetic engineering of livestock.
Science 244: 1281.
167 Pursel, V. G., D. J. Bolt, K. F. Miller, C. A. Pinkert,
R. E. Hammer, R. D. Palmiter, and R. L. Brinster.
1990. Expression and performance in transgenic
pigs. 1. Reprod. Fertil. 4O(SuppI.):235.
168 Putney, D. J., W. W. Thatcher. M. Drost, 1. M.
Wright, and M. A. DeLorenzo. 1988. Influence of
environmental temperature on reproductive perfor-
mance of bovine embryo donors and recipients in the
southwest region of the United States. Theriogenol-
ogy 30:905.
169 Qvist, R., L. F. Blackwell, H. Bourne, and 1. B.
Brown. 1990. Development of mouse ovarian folli-
cles from primary preovulatory stages in vitro. J.
Reprod. Fertii. 89:169.
170 Raines, L. J. 1990. Public policy aspects of patenting
transgenic animals. Theriogenology 33:129.
171 Rajamahendran, R., R. S. Canseco, and C. 1. Den-
bow. 1987. Effect of low dose of FSH given at the
beginning of the estrous cycle and subsequent su-
perovulatory response in Holstein cows. Therioge-
nology 28:59.
172 Rail, W. F., and S. P. Leibo. 1987. Production of
sexed bovine pregnancies by cytogenetic analysis of
cultured demi-embryos. Theriogenology 27:269.
173 Rail, W. F., M. J. Wood, C. Kirby, and D. G.
Whittingham. 1987. Development of mouse embryos
cryopreserved by vitrification. J. Reprod. Fertii. 80:
499.
174 Remsen, L. G., 1. D. Roussel, and A. K. Karihaloo.
Pregnancy rates relating to plasma progesterone lev-
els in recipient heifers at day of transfer. Therioge-
nology 18:365.
175 Renard, 1.-P., Y. Heyman. P. Leymonie. and J.-C.
Plat. 1983. Sucrose dilution: a technique for field
2877
transfer of bovine embryos frozen in the straw.
Theriogenology 19:145.
176 Rieger, D., D. Desaulnier. and A. K. Goff. 1988.
Ovulatory response and embryo yield in superovu-
lated Holstein heifers given a priming dose of FSH-P
at day 2 of the estrous cycle. Theriogenology 30:695.
177 Robertson, E. 1987. Embryo-derived stem cell lines.
Page 71 in Teratocarcinomas and Embryonic Stem
Cells-a Practical Approach. IRC Press, Oxford,
Engl.
178 Robertson, E. J. 1991. Using embryonic stem cells to
introduce mutations into the mouse germ line. BioI.
Reprod. 44:238.
179 Robl. 1. M.• B. Gilligan, E. S. Crister, and N. L.
First. 1986. Nuclear transplantation in mouse em-
bryos: assessment of recipient cell stage. BioI.
Reprod. 34:733.
180 Roche, J. F., M. P. Boland. T. A. McGeady, and 1. J.
Ireland. 1981. Reproductive wastage following artifi-
cal insemination of heifers. Vet. Rec. 109:401.
181 Romero, A.. J. Albert, Z. Brink, and G. E. Seidel, Jr.
1991. Numbers of small follicles in ovaries affect
superovulation response in cattle. Theriogenology
35:265.
182 Rorie, R. W., C. W. McFarland, T. L. Overskei, S.
A. Voelkel, and R. A. Godke. 1985. A new method
of splitting embryos without the use of a commercial
micromanipulator unit. Theriogenology 23:224.
183 Rowe. R. F.• M. R. Del Campo, C. L. Eilts, L. R.
French, R. P. Winch, and O. J. Ginther. 1986. A
single cannula technique for nonsurgical collection
of ova from cattle. Theriogenology 6:471.
184 Rowson, L.EA, and D. F. Dowling. 1949. An ap-
paratus for the extraction of fertilized eggs from the
living cow. Vet. Rec. 61:191.
185 Rowson, L.EA, R.A.S. Lawson, R. M. Moor, and
A. A. Baker. 1972. Egg transfer in the cow: syn-
chronization requirements. 1. Reprod. Fertil. 28:427.
186 Rowson, L.EA, R. M. Moor, and R.A.S. Lawson.
1969. Fertility following egg transfer in the cow:
effect of method, medium and synchronization of
oestrus. J. Reprod. Fertil. 18:517.
187 Saacke, R. G., J. H. Bame, D. S. Karabinus, K. J.
Mullins. and S. Whitman. 1988. Transport of abnor-
mal spermatozoa in the artificially inseminated cow
based upon accessory spermatozoa in the zona pel-
lucida. Proc. Int. Congr. Anim. Reprod. Artif. Inse-
min. 3:292.
188 Salgado, R.. and L. E. Donaldson. 1984. The effect
of intravaginal progesterone on pregnancy rates in
cows receiving embryo transfers. Theriogenology 21:
258.
189 Savio, 1. D.• H. Bongers, M. Drost, M. C. Lucy. and
W. W. Thatcher. 1991. Follicular dynamics and su-
perovulatory response in Holstein cows treated with
FSH in different endocrine states. Theriogenology
35:915.
190 Schiewe, M. C.• C. R. Looney, C. A. Johnson, K. G.
Hill, and R. A. Godke. 1987. Transferrable embryo
recovery rates following different insemination sche-
dules in superovulated beef cattle. Theriogenology
28:395.
191 Schneider. H. J., Jr., R. S. Castleberry, and 1. L.
Griffin. 1980. Commercial aspects of bovine embryo
transfer. Theriogenology 13:73.
Journal of Dairy Science Vol. 75, No. 10, 1992
2878 HASLER
192 Seidel, G. E., Jr. 1977. Short-tenn maintenance and
culture of embryos. Page 20 in Embryo Transfer in
Fann Animals: A Review of Techniques and Appli-
cations. Monogr. No. 16. Agric. Canada, Ottawa,
ON.
193 Seidel, G. E., Jr. 1983. Production of genetically
identical sets of mammals: cloning? J. Exp. BioI.
228:347.
194 Seidel, G. E., Jr. 1985. Economic aspects of sexing
embryos. Page 48 in Proc. Am. Embryo Transf.
Assoc. Annu. Mtg., Hastings, NE.
195 Seidel, G. E., Jr. 1991. Embryo transfer: the next 100
years. Theriogenology 35:171.
196 Seidel, G. E., Jr., R. P. Elsden, L. D. Nelson, and J.
F. Hasler. 1978. Methods of ovum recovery and
factors affecting fertilization of superovulated bovine
ova. Page 268 in Control of Reproduction in the
Cow. Martinus Nijhoff, The Hague, Neth.
197 Seike, N., K. Saeki, K. Utaka, M. Sakai, R.
Takakura, Y. Nagao, and H. Kanagawa. 1989.
Production of bovine identical twins via transfer of
demi embryos without zonae pellucidae. Therioge-
nology 32:211.
198 Senger, P. L.. W. C. Becker, S. T. Davidge, J. K.
Hillers. and J. J. Reeves. 1988. Influence of cornual
insemination on conception in dairy cattle. J. Anim.
Sci. 66:3010.
199 Shea, B. F., D. J. Hines, D. E. Lightfoot, G. W. Ollis,
and S. M. Olson. 1976. The transfer of bovine
embryos. Page 145 in Egg Transfer in Cattle. Eur.
Econ. Comm. Publ. EUR5491, Luxembourg.
200 Shea, B. F., R. E. Janzen, R. J. McAlister, and D. P.
McDennand. 1983. Freezing of bovine embryos: ef-
fects of embryo quality. time from thawing to trans-
fer and number frozen per vial. Theriogenology 20:
205.
201 Shi, D. S., K. H. Lu, and I. Gordon. 1990. Effects of
bulls on fertilization of bovine oocytes and their
subsequent development in vitro. Theriogenology 33:
324.
202 Singh, E. L. 1987. The disease control potential of
embryos. Theriogenology 27:9.
203 Skrzyszovska, M., and Z. Smorag. 1989. Cellloss in
bisected mouse, sheep and cow embryos. Therioge-
nology 32:115.
204 Smith, L. C., and I. Wilmut. 1990. Factors affecting
the viability of nuclear transplanted embryos. Theri-
ogenology 33:153.
205 Sreenan, J. M., and J. P. Gosling. 1977. The effect of
cycle stage and plasma progesterone level on the
induction of multiple ovulations in heifers. J. Reprod.
Fertil. 50:367.
206 Strelchenko, N., S. Saito, and H. Niemann. 1991.
Towards the establishment of bovine embryonic stem
cells. Theriogenology 35:274.
207 Stubbings, R. B., and J. S. Walton. 1986. Relation-
ship between plasma progesterone concentrations
and pregnancy rates in cattle receiving either fresh or
previously frozen embryos. Theriogenology 26:145.
208 Sugie, T., T. Soma, S. Fukurnitsu, and K. Otsuki.
I 972. Studies on the ovum transfer in cattle, with
special reference to collection of ova by means of
non-surgical techniques. Bull. Nat!. Inst. Anim. Ind.
(lbaraki) 25:27.
Journal of Dairy Science Vol. 75, No. 10, 1992
209 Summers, P. M., J. M. Shelton, and K. Bell. 1983.
Synthesis of primary Bos taurus-Bos indicus chi-
meric calves. Anim. Reprod. Sci. 6:91.
210 Takeda, T., S. V. Hallowell, A. D. McCauley, and J.
F. Hasler. 1986. Pregnancy rates with intact and split
bovine embryos transferred surgically and nonsurgi-
cally. Theriogenology 25:204.
211 Tarkowski, A K. 1959. Experiments on the develop-
ment of isolated blastomeres of mouse eggs. Nature
(Lond.) 184:1286.
212 Tarkowski, A K. 1961. Mouse chimaeras developed
from fused eggs. Nature (Lond.) 190:857.
213 Tarkowski, A. K., and J. Wroblewska. 1967. De-
velopment of blastomeres of mouse eggs isolated at
the 4- and 8-cell stage. J. Embryol. Exp. Morphol.
18:155.
214 Thibier, M., and M. Nibart. 1987. Disease control
and embryo importations. Theriogenology 27:37.
215 Touati, K., P. van Der Zwalmen, F. J. Ectors, J. F.
Beckers. and F. Eetors. 1989. Low dose ofFSH early
in estrous cycle enhances superovulatory response in
heifers. Theriogenology 31 :269.
216 Tsunoda, Y., T. Yasiu, Y. Shioda, K. Nakamura, T.
Uchida, and T. Sugie. 1987. Full-tenn development
of mouse blastomere nuclei transplanted into
enucleated two-cell embryos. J. Exp. Zool. 242:147.
217 van der Schans, A, L.AJ. Van der Westerlaken,
A.A.C. de Wit, W. H. Eyestone, and H. A. de Boer.
1991. Ultrasound-guided transvaginal collection of
oocytes in the cow. Theriogenology 35:288.
218 van Vliet, R. A, A. M. Verrinder Gibbins, and J. L.
Walton. 1989. Livestock embryo sexing: a review of
current methods, with emphasis on V-specific DNA
probes. Theriogenology 32:421.
219 Voss, H. J., D. Landmark, G. Wilke, J. F. Hasler, R.
O. Whitaker, and R. D. Heilman. 1986. Pregnancy
rate after surgical transfer of imported frozen bovine
embryos. Theriogenology 25:209.
220 Wall, R. J., D. J. Bolt, W. 1. Frels, H. W. Hawk, D.
King, V. G. Pursel, C. E. Rexroad, Jr., and R. M.
Rohan. 1990. Transgenic fann animals: current state
of the art. Agric. Biotecthnol. News 2:391.
221 Wall, R. J., and H. W. Hawk. 1988. Development of
centrifuged cow zygotes cultured in rabbit oviducts.
J. Reprod. Fertil. 82:673.
222 Wall, R. J., V. G. Pursel, R. E. Hammer, and R. L.
Brinster. 1985. Development of porcine ova that
were centrifuged to permit visualization of pronuclei
and nuclei. BioI. Reprod. 32:645.
223 Walton, J. S., N. A. Martineau, and R. B. Stubbings.
1986. Pregnancy rates in Holstein embryo transfer
recipients: effect of treatment with progesterone or
clenbuterol and of natural versus induced cycles.
Theriogenology 26:837.
224 Ware, C. B., D. C. Northy, and N. L. First. 1987.
Effect of administration of FSH at the beginning of
the cycle on the subsequent response to superovula-
tion treatment in heifers. Theriogenology 27:292.
225 West, G., C. West, D. Risley, and L. Donaldson.
1984. Effect of breeding regime on percent ova
fertilized in superovulated cows.Theriogenology 21:
273.
226 White, K. L., G. B. Anderson, and R. H. Bondurant.
1987. Expression of a male-specific factor on various
 SYMPOSIUM: REPRODUCTIVE TECHNOLOGY
stages of preimplantation bovine embryos. BioI.
Reprod. 37:867.
227 Whittingham, D. G., S. P. Leibo, and P. Mazur.
1972. Survival of mouse embryos frozen to -196'C
and -269'C. Science 178:411.
228 Willadsen, S. M. 1986. Nuclear transplantation in
sheep embryos. Nature (Lond.) 320:63.
229 Willadsen, S. M., R. E. Janzen, R. J. McAlister, B. F.
Shea, G. Hamilton, and D. McDennand. 1991. The
viability of late morulae and blastocysts produced by
nuclear transplantation in cattle. Theriogenology 35:
161.
230Willadsen, S. M., and C. Polge. 1981. Attempts to
produce monozygotic quadruplets in cattle by blasto-
mere separation. Vet. Rec. 108:211.
231 Willadsen, S., C. Polge, and L.E.A. Rowson. 1978.
The viability of deep-frozen cow embryos. 1.
Reprod. Fertil. 52:391.
232 Williams, T. J., R. P. Elsden, and G. E. Seidel, Jr.
1984. Pregnancy rates with bisected bovine embryos.
Theriogenology 22:521.
233 Williams, T. J., and L. Moore. 1988. Quick-splitting
of bovine embryos. Theriogenology 29:477.
234 Willmott, N., J. Saunders, G. A. Bo, A. Palasy, R. A.
Pierson, and R. J. Mapletoft. 1990. The effect of
FSH-LH ratio in pituitary extracts on superovulatory
response in the cow. Theriogenology 33:347.
235 Wilmut, I., A. L. Archibald, S. Harris, M. McClena-
ghan, J. P. Simons, C.BA Whitelaw, and A. J.
Clark. 1990. Modification of milk composition. J.
Reprod. Fertil. 41 (Suppl.): 135.
2879
236 Wilmut, I., M. L. Hooper, and 1. P. Simons. 1991.
Genetic manipulation of mammals and its application
in reproductive biology. 1. Reprod. Fertil. 92:245.
237 Wilmut, I., and L.EA Rowson. 1973. Experiments
on the low-temperature preservation of cow em-
bryos. Vet. Rec. 92:686.
238 Wilson, 1. M., A. L. Jones, and D. R. Miller. 1990.
Influence of a dominant follicle on the superovula-
tion response. Theriogenology 33:349.
239 Wilson, J. M., K. Moore, A. L. Jones, and C. R.
Looney. 1989. Recombinant bovine follicle-
stimulating honnone: dose and duration regimens for
superovulation of embryo donors. Theriogenology
31:273.
240 Wright, G., A. Carver, D. Cottom, D. Reeves, A.
Scott, P. Simons, 1. Wilmut, I. Garner, and A. Col-
man. 1991. High level expression of active human
alpha-I-antitrypsin in the milk of transgenic sheep.
Biotrechnology 9:830.
241 Wright, J. M. 1985. Commercial freezing of bovine
embryos in straws. Theriogenology 23:17.
242 Xu, K., M. Wu, H. Wang, and R. 1. Mapletoft. 1988.
FSH-P Lot-Number and superovulation response in
beef heifers. Theriogenology 29:335.
243 Younis, A. I., and B. G. Brackett. 1991. Importance
of cumulus cells and insemination intervals for de-
velopment of bovine oocytes into morulae and
blastocysts in vitro. Theriogenology 36: 11.
244 Younis, A. I., C. L. Keefer, and B. G. Brackett.
1989. Fertilization of bovine oocytes by spenn injec-
tion. Theriogenology 31 :276.
Journal of Dairy Science Vol. 75, No. 10, 1992