EASTERN KENTUCKY UNIVERSITY

McNair Summer Research Program

Summer 2011

Relationship between Embryo Quality, Stage of Embryo, and Conception Rates of

Recipients Following Implantation of Embryos from Brown Swiss Cows
By Stacy Thacker
Pre-Veterinary Medicine

Abstract
This study was conducted at Eastern Kentucky Universitys Stateland Dairy. The objective of this
study was to evaluate if a relationship existed between conception rates, embryo stage and
embryo grade following the transfer of Brown Swiss embryos in reproductively mature Holstein
heifers and cows. Another objective was to establish a small Brown Swiss herd at EKU. This
herd would allow for future research collaboration and financial support from the Brown Swiss
Association.
Five heifers and six cows were subject to the same estrus synchronization program:
implantation of a CIDR (progesterone), removal of the CIDR one week later, and an injection of
a luteinizing hormone. Signs of estrus were closely monitored and recorded. One week post
estrus date, the recipients were palpated by a veterinarian. Four heifers had a corpus luteum
(CL) on their ovaries and were therefore considered to be able to maintain pregnancy and
receive an embryo. Four frozen Brown Swiss embryos were implanted, one in each eligible
heifer. Two months later, three of the four recipient heifers were observed in estrus and

concluded to have experienced early embryonic death. Possible reasons for this include the
failure of the blastula (early embryo) to implant in the uterine wall, rejection of the blastula by
the recipient female, improperly met nutritional demands, or expression of a lethal gene in the
developing embryo terminating further development (Lamb 2011). One heifer remains pregnant.
This study is a second trial of a previous study. In the first study, none of the embryos that
were transferred produced a viable pregnancy. Due to the lack of pregnancies, the relationships
between embryo quality, stage, grade, and their effects on conception rates could not be
measured.
Introduction
The use of Embryo Transfer (ET) in cattle production and dairy operations is an important
advancement in the genetic enrichment of offspring in that it increases the reproductive capacity
of a superior female. Embryo transfer is a way to speed up the rate of genetic change by
increasing selection intensity of animals with highly desirable traits. This procedure, however
beneficial, is expensive and has limitations. Those limitations are being improved by
agriculturalists and farmers all over the world in order to make this valuable practice more
commonplace in livestock operations of every size.
Embryo transfer in dairy operations is a major advantage in increasing the heritance of
desirable traits such as high milk production, high butterfat content, and desirable type traits.
Several factors, however, make this procedure expensive, such as the fact that it must be
performed by a licensed embryologist veterinarian and that not every embryo implanted will
produce a viable offspring. This means that the procedure may have to be repeated several times
in order to establish a pregnancy. So, in order to drive costs down and increase the use of this
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valuable procedure in American livestock operations, sound methods must be established to

improve conception rates and ensure a viable pregnancy in each cow for every transfer.
Materials and Methods

Herd
The 11 subjects used in this experiment were registered Holstein Dairy Cattle, belonging to the
dairy herd of Eastern Kentucky Universitys Stateland Dairy. Of the 11 subjects, there were 5
heifers and 6 cows. The cows and heifers were fed daily a nutritional feed specific to their state
(lactating feed, heifer feed). All subjects were kept current on all vaccinations, were housed in
the same facility, provided with free-choice minerals, and exposed to the same amount of
roughage (grass/hay). The subjects were allowed free choice of remaining in the open housing
facility, which provides shade from the sun and cool bedding, or going out to pasture at all times.
Each cow and heifer was subject to the same estrus synchronizing program. On Monday,
May 8th eleven cows received a CIDR (CIDR; 1.9g progesterone; Pfizer). CIDRs are an intravaginal, progesterone releasing device used in synchronizing heat periods in cattle. On Monday,
May 15th the CIDRs were removed and each cow received 2ccs of luteinizing hormone
(estroPLAN; AgriLabs). A luteinizing hormone will regress the corpus luteum (CL) and induce
estrus. This progesterone treatment before an injection of the luteinizing hormone ensures
regression of the CL in response to the progesterone because most cattle will have a CL that has
developed over the seven day period. Progesterone will also delay estrus in cattle that undergo
natural regression of the CL during the seven day period before the injection of the luteinizing
hormone. The subjects were then watched closely thereafter for signs of estrus. When estrus was

detected in a subject, the date and time of the heat was recorded in a chart next to her
identification number.
A week after the cows were observed in heat, veterinarian and embryologist Dr. LW
Beckley palpated each female to determine whether the structures on the ovaries were capable of
maintaining a pregnancy. A corpus luteum, Latin for yellow body, is the desired ovarian structure
when working with embryo transfer recipients because this is the progesterone producing
structure. Progesterone is the hormone which maintains pregnancy. Also during palpation, Dr.
Beckley would make note as to which ovary (right or left) that the CL was found on. This is
important in order to know which uterine horn to implant the embryo. Implanting in the wrong
horn (the horn of the ovary that didnt have a CL) would kill the embryo for lack of hormone
support from the CL. The recipients that were deemed capable of becoming pregnant were then
implanted with an embryo of a particular grade and stage, determined by Stateland Dairy
manager Chad Powers. These decisions were made based on the genetic history of the sire and
dam of the particular embryo. In this case, calving ease, or low birth weight, was selected for in
the sires of the embryos.

Embryos

The embryos used in this experiment were donated by the Brown Swiss Association of America.
Ninety-five of these embryos, of varying grades and stages, were donated to Stateland Dairy. The
stage and grade each recipient female received was decided by Dr. LW Beckley, the veterinarian
and embryologist performing the transfer.
Stages of Embryo Development
Stage
Description
1
Unfertilized
2
2- to 12-cell
3
Early Morula
4
Morula
5
Early Blastocyst
6
Blastocyst
7
Expanded Blastocyst
8
Hatched Blastocyst
9
Expanded Hatched
Blastocyst

Grades of Embryos
Grade
1
2
3

Description
Excellent or good
Fair
Poor
Dead or degenerating

Table 2. Grades of Embryos, described

(Selk 2002)

Table 1. The Stages of Embryo

Development, described (Selk, 2002).

Post ET Maintenance

After the transfer of the embryos, the recipient females were fed an appropriate diet formulated
by a nutritionist to meet the nutritional demands of the heifers. The cows were also clipped of
winter hair, to reduce heat stress, and were provided fans and misters depending on the weather.
Fresh, clean water was provided at all times. The cattle were carefully observed daily for any
signs of heat. There are several signs a female is coming into heat: swollen vulva, a thick
discharge from the vulva, mounting other females, and/or bawling. Signs of heat would
determine that the female did not accept the embryo or that an early embryonic death ocurred.

Results

Heat Synchronization
The estrus synchronization method used in this project was successful in inducing heat in all
eleven head.
Cow

Date Seen in Heat

Time Seen in Heat

919

May 18th

AM

1001

May 18th

AM

1005

May 18th

AM

1007

May 18th

AM

922

May 18th

NOON

924

May 18th

NOON

1008

May 18th

NOON

1009

May 18th

PM

921

May 19th

AM

1003

May 19th

AM

1004

May 19th

AM

Table 3. The date and time each recipient was observed in estrus after being subject to the heat
synchronization procedure.

Palpating and Transfer Recipients

Of the 11 subjects that were heat synchronized, only four were determined capable of producing
a viable pregnancy through ET after palpation by Dr. LW Beckley. In other words, only four
heifers had a viable CL on one of their ovaries. The other cows were found to have had luteal and
follicular cysts on their ovaries, which prevented a normal estrus cycle.
Recipient

Date of Heat
th

Stage of

Grade of

Horn

Cane

Embryo
4

Embryo
2

11328B

921

May 19

922

May 18th

11328B

1008

May 18th

10689

1009

May 18th

10689

Table 4. The four recipients that were determined capable of producing a viable
pregnancy

Pregnancies
The recipient females were observed closely after the transfer of the embryos. Two months later,
three of the four recipients were observed in estrus. This was proof those three heifers did not
become pregnant with the embryo that was implanted in them.

Conclusions
There are many reasons why a recipient female loses the embryo she is implanted with.
However, each possible reason is only a speculation as to why the abortion or embryonic death
occurred. Without costly equipment and lengthy examinations, it is impossible to pinpoint the
exact reason an embryo failed to produce a pregnancy.
There are several possible explanations as to why these heifers did not become pregnant.
Increased body heat, as a result of hot, humid weather conditions has been proven to have a
negative effect on the conception rates of embryo recipient females (Demetrio et al 2007). Even
though fans, misters, and fresh, cool water were provided at all times, the cows were given free
opportunity to stay outside. However, in the group of recipients in the earlier trial were
inseminated in cooler weather conditions where heat stress was not an issue and low conception
rates were still experienced. Another possible explanation is that in this experiment we used
frozen embryos. The use of frozen embryos as opposed to fresh embryos has been shown to
result in a decline in pregnancy rates (Ambrose et al. 1999). In order to transport and store
embryos, however, they must be frozen. With the advantage in the ability to store and transport
embryos for later use comes the disadvantage in a decline in conception rates.
Improper nutrition can also negatively affect the conception rates of ET in cattle (Lamb
2011). In the first trial (mentioned above), no viable pregnancies were produced because of
nutritional problems. CPC Commodities, where Stateland Dairy has hired a nutritionist to
formulate the ration and mix the base feed for the herd mistakenly increased the energy and
protein concentrations of the base feed mix for Stateland. As a result of this mistake, the
increased concentration caused the cows to develop severe rumen acidosis. This condition

changes the pH of the rumen of the cow and in turn has a negative effect on reproduction, causes
ulcers and abscess wounds which bring about lameness, affects the gastro-intestinal system and
decreases milk production. The mistake was found and corrected, but the effects may still have
lingered and caused the low conception rates in this trial as well. No correlation between the
stage and grade of the embryos could be found because only one female remains pregnant.
Improvements may be made to projects in the future that may increase conception rates.
Keeping the recipients in doors at all times under fans, or breeding in a cooler season may help
the conception rates. It is not an option to use fresh embryos in this case because the embryos
that were given to the University are all frozen. Nutrition should also be monitored for several
months prior to the transfer date to ensure no lingering nutritional problems will affect the
pregnancies.
Projections
Embryo transfer was first performed and recorded by Walter Heape in 1890 with Angora
rabbits. Embryo transfer in food animals began in the 1930s using sheep and goats, but it was
not until the 1950s that successful embryo transfers were reported in cattle and pigs by Jim
Rowson at Cambridge, England. After AI and estrus synchronization, embryo transfer (ET) is the
third-most commonly used biotechnology. In 1987, 3.6 percent (5,105) of all calves registered in
the Angus breed were a result of embryo transfer. In 2002, 25,093 calves resulting from ET were
registered. This was 8.9 percent of all calves registered. While Angus is one of the largest breeds
in the United States, similar numbers (percentages) have been documented in other breeds as
well.

Using growth percentages from previous years, I have calculated the approximate
increase in use of ET per year at 0.6%. However, I project the this percentage will increase every
year (and grow exponentially) as new technologies arise, more successful methods are
established, and cost is driven down. It is my prediction that by the year 2040, at least 30% of
food animals will be products of embryo transfer.

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Works Cited
Ambrose, J. D., M. Drost, R. L. Monson, J. J. Rutledge, M. L. Leibfried-Rutledge, M-J Thatcher,
T. Kassa, M. Benelli, P. J. Hanson, P. J. Chenoweth, and W. W. Thatcher. "Efficacy of
Timed Embryo Transfer with Fresh and Frozen In Vitro Produced Embryos to Increase
Pregnancy Rates in Heat Stressed Dairy Cattle." Journal of Dairy Science 82 (1999):
2369-376. Print.
Demetrio, D. G. B., R. M. Santos, C. G. B. Demetrio, and J. L. M. Vasconcelos. "Factors
Affecting Conception Rates Following Artificial Insemination or Embryo Transfer in
Lactating Holstein Cows." Journal of Dairy Science 90.11 (2007): 5073-082. Print.
Lamb, Cliff. "Embryonic Mortality in Cattle." Beef Cattle Handbook. Extension Beef Cattle
Resource Committee. 1-3. Beef Cattle Handbook. Extension Beef Cattle Resource
Committee. Web. 19 July 2011.
Selk, G. (2002). Embryo Transfer in Cattle. Oklahoma, Oklahoma State University.

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