NZ619785B2 - A method for identifying a mammal receptive to embryo transfer - Google Patents
A method for identifying a mammal receptive to embryo transfer Download PDFInfo
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- NZ619785B2 NZ619785B2 NZ619785A NZ61978512A NZ619785B2 NZ 619785 B2 NZ619785 B2 NZ 619785B2 NZ 619785 A NZ619785 A NZ 619785A NZ 61978512 A NZ61978512 A NZ 61978512A NZ 619785 B2 NZ619785 B2 NZ 619785B2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
Abstract
Discloses a method of identifying a cow that is receptive to embryo transfer, the method comprising the steps of: a) synchronising the oestrous cycles of two or more cows in a population; b) identifying a subpopulation of cows which are not in heat or which do not 5 show outward signs of heat, from the population; c) testing at least one cow from the subpopulation identified in step (b) for the presence or absence of a corpus luteum; d) identifying a cow that has a corpus luteum based upon that testing; and e) transferring an embryo to the cow identified as having a corpus luteum, and 10 testing the cow for pregnancy, where a positive pregnancy test indicates the cow’s receptivity to embryo transfer. Further discloses related methods for decreasing the number of cows killed from a herd by selecting a subpopulation receptive to embryo transfer, and a method for maximising the number of cows selected from a herd to be recipients for embryo transfer. rom the population; c) testing at least one cow from the subpopulation identified in step (b) for the presence or absence of a corpus luteum; d) identifying a cow that has a corpus luteum based upon that testing; and e) transferring an embryo to the cow identified as having a corpus luteum, and 10 testing the cow for pregnancy, where a positive pregnancy test indicates the cow’s receptivity to embryo transfer. Further discloses related methods for decreasing the number of cows killed from a herd by selecting a subpopulation receptive to embryo transfer, and a method for maximising the number of cows selected from a herd to be recipients for embryo transfer.
Description
A METHOD FOR IDENTIFYING A MAMMAL RECEPTIVE TO EMBRYO
TRANSFER
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application claims priority to U.S.A. Priority Application 61/496,761,
filed 06/14/2011, and is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present technology relates to the identification of reproductive mammals that,
prior to the inventive technology, would have been classified as unreceptive to embryo
transfer and artificial insemination methods for inducing pregnancy.
BACKGROUND
In recent years, the dairy industry has seen a drastic decrease in the reproductive
efficiency of cows. This is reflected in undesirable decreases in such industry-standard
reproductive performance measurements as (1) days to first service, (2) days to conception,
(3) calving interval, (4) services per conception, (5) non-return rate, (6) oestrus (or “heat”)
detection rate, and (7) pregnancy rate. Oestrus, or heat, detection, in particular, is useful for
identifying cows that are likely to become pregnant if inseminated at the time of oestrus
detection, or shortly thereafter. Wrongly identifying a cow as not being “in heat” (i.e.,
“oestrus-negative”) is a significant contributor to reproductive inefficiency. This has a
corresponding significant impact on the dairy industry because dairy cows that are not
reproductively efficient are culled.
SUMMARY
The present invention therefore generally provides a method of identifying a mammal that is
receptive to embryo transfer, the method comprising the steps of:
a) synchronising the oestrous cycles of two or more mammals in a population;
b) identifying a subpopulation of mammals which are not in heat or which do not
show outward signs of heat, from the population;
c) testing at least one mammal from the subpopulation identified in step (b) for
the presence or absence of a corpus luteum;
d) identifying a mammal that has a corpus luteum based upon that testing; and
e) transferring an embryo to the mammal identified as having a corpus luteum,
and testing the mammal for pregnancy,
where a positive pregnancy test indicates the mammal’s receptivity to embryo
transfer.
In a particularly preferred embodiment, the mammals are cows. In another
embodiment, synchronizing is achieved by administering prostaglandin to the at least two
mammals. In another embodiment, synchronizing is achieved by administering one or more
of prostaglandin, gonadotropin and progestin.
In another embodiment, the mammals are cows and the presence of a corpus luteum
is detected by a method selected from the group consisting of (i) rectal palpation; (ii)
ultrasonography; (iii) measuring the concentration of progesterone in the blood of the at least
one cow; and (iv) measuring the concentration of the progesterone in the milk by the at least
one cow.
In another embodiment, the mammals are cows and the embryo is between 5 days and
10 days old.
In another embodiment, the mammals are “oestrus-negative”, i.e., the mammals are
not “in heat or do not show outward signs of heat.” In a further embodiment, the at least one
oestrus-negative mammal is a cow.
In another embodiment, the synchronizing is achieved by administering prostaglandin
to the at least two oestrus-negative cows. The prostaglandin may be used in conjunction with
other pharmaceuticals in a synchronization program.
In another embodiment, the presence of a corpus luteum in the at least one oestrus-
negative cow is detected by a method selected from the group consisting of (i) rectal
palpation; (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood
of the cow; and (iv) measuring the concentration of the progesterone in the milk produced by
the cow.
In a further embodiment, the embryo to be transferred to the at least one estrus-
negative cow is between 5 days and 8 days old.
In a further embodiment, the embryos to be transferred are fresh embryos, frozen
embryos, vitrified embryos, in vivo embryos, in vitro fertilized embryos, embryos produced
by somatic cell nuclear transfer, embryos produced by embryonic cell nuclear transfer,
embryos produced by embryonic splitting, embryos produced by sperm microinjection,
embryos produced by genetic modification, and/or embryos produced by genetic selection.
In a further embodiment, the mammal to receive the embryo is in the oestrous cycle
between day 6 and day 10.
An additional embodiment comprises detecting whether the mammals are in oestrus
or “in heat”, so as to identify oestrus-positive and oestrus-negative mammals.
An additional embodiment comprises detecting whether an ovarian corpus luteum is
present in the mammals that have been identified as not in oestrus or oestrus-negative.
Another aspect of the invention is a method for selecting mammals receptive to
embryo transfers, comprising: (1) synchronizing the oestrous cycles of at least two mammals;
(2) from the at least two mammals in step (1), selecting a mammal that is oestrus-negative;
(3) from the mammal in step (2), selecting a mammal that has a corpus luteum (CL) present
in its ovary; (4) transferring an embryo to the mammal selected in step (3); and (5) testing
receptivity of the mammal towards pregnancy.
In one embodiment, the method for selecting mammals receptive to embryo transfers
further comprises between step (1) and (2) selecting an estrus-positive mammal from the at
least two mammals, wherein an embryo is transferred to the oestrus-positive mammal and the
CL-bearing mammal selected in step (3) In another embodiment, the at least two mammals
are cows.
In one embodiment, the method for selecting mammals receptive to embryo transfers
further comprises between step (1) and (2) selecting an oestrus-positive mammal from the at
least two mammals, wherein an embryo is transferred to the estrus-positive mammal and the
CL-bearing mammal selected in step (3) and wherein the milk production from the oestrus-
positive mammal and the CL-bearing mammal is at least 1% higher than the milk production
from the oestrus-positive mammal alone. In this regard, the milk production from the oestrus-
positive mammal and the CL-bearing mammal can be 1-5%, 5-10%, 10-15%, 15-20%, 20-
%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50% higher than the milk production from the
estrus-positive mammal alone.
In another embodiment, the oestrus-positive cows are selected by a method selected
from the group consisting of: (i) tail marking; (ii) watching oestrus signs; (iii) using marker
animals; (iv) mounting-activity detectors; (v) measuring methyl heptanol and (vi) measuring
pheromone (vii) measuring motion (viii) measuring temperature.
In another embodiment, the at least two mammals are cows and the synchronizing is
achieved by administering prostaglandin to the at least two mammals. In another
embodiment, the synchronizing is achieved by administering one or more of prostaglandin,
gonadotropin and progestin to the cows.
In another embodiment, the at least two mammals are cows and the presence of a
corpus luteum is detected by a method selected from the group consisting of: (i) rectal
palpation; (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood
of the cow; and (iv) measuring the concentration of the progesterone in the milk produced by
the cow.
In yet another embodiment, the embryo to be transferred to the at least two cows is
between 5 days and 10 days old. In yet another embodiment, the embryos to be transferred
are fresh embryos, frozen embryos, vitrified embryos, in vivo embryos, in vitro fertilized
embryos, embryos produced by somatic cell nuclear transfer, embryos produced by
embryonic cell nuclear transfer, embryos produced by embryonic splitting, embryos produced
by sperm microinjection, embryos produced by genetic modification, and/or embryos
produced by genetic selection.
In yet a further embodiment, the cow to receive the embryo is in the estrous cycle
between day 6 and day 10.
Another aspect of the invention is a kit to increase reproductive efficiency of
mammals, comprising: (1) at least one oestrus-detecting reagent or device,(2) at least one
corpus luteum (CL)-detecting reagent or device, (3) at least one warming or thawing medium
and (4) at least one transfer medium.
In one embodiment, the at least one oestrus-detecting device is a mounting-activity
detector or motion activity detector. In another embodiment, the at least one CL-detecting
reagent comprises an antibody against progesterone and an antibody against the antibody
against progesterone.
Another aspect of the invention is a kit to impregnate one or more oestrus-negative
cows, comprising: (1) at least one corpus luteum detecting reagent or device; and (2) at least
one embryo to be transferred to at least one cow that has been identified to have a corpus
luteum by said corpus luteum detecting reagent or device.
In one embodiment, wherein the corpus luteum detecting reagent comprises an
antibody against progesterone and an antibody against the antibody against progesterone.
In another embodiment, the embryos to be transferred are fresh embryos, frozen
embryos, vitrified embryos, in vivo embryos, in vitro fertilized embryos, embryos produced
by somatic cell nuclear transfer, embryos produced by embryonic cell nuclear transfer,
embryos produced by embryonic splitting, embryos produced by sperm microinjection,
embryos produced by genetic modification, and/or embryos produced by genetic selection.
Another aspect of the invention is a method of increasing milk production,
comprising: (1) synchronizing the estrous cycles of at least two mammals; (2) from the at
least two mammals in step (1), selecting a mammal that is oestrus-negative; (3) from the
mammal in step (2), selecting a mammal that has a corpus luteum (CL) present in its ovary;
(4) transferring an embryo to the mammal selected in step (3); and (5) testing receptivity of
the mammal towards pregnancy.
In one embodiment, the method of increasing milk production further comprising
synchronizing the oestrus cycles of the cows.
In another embodiment, the synchronization is achieved by administering
prostaglandin to the cows. In another embodiment, the synchronizing is achieved by
administering one or more of prostaglandin, gonadotropin and progestin to the cows.
In another embodiment of the method of increasing milk production, the presence of a
corpus luteum is detected by a method selected from the group consisting of: (i) rectal
palpation; (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood
of the cow; and (iv) measuring the concentration of the progesterone in the milk produced by
the cow.
In yet another embodiment of the method of increasing milk production, the embryo
is between 5 days and 10 days old. In yet another embodiment, the embryos to be transferred
are fresh embryos, frozen embryos, vitrified embryos, in vivo embryos, in vitro fertilized
embryos, embryos produced by somatic cell nuclear transfer, embryos produced by
embryonic cell nuclear transfer, embryos produced by embryonic splitting, embryos produced
by sperm microinjection, embryos produced by genetic modification, and/or embryos
produced by genetic selection.
In yet a further embodiment, the cow to receive the embryo is in the oestrous cycle
between day 6 and day 10.
A further aspect of the invention is a method for improving the pregnancy rate of a
cow population, comprising: transferring an embryo into a cow that has been identified as
oestrus-negative, and wherein the oestrus-negative cow has been synchronized by
administering one or more of prostaglandin, gonadotropin and progestin.
It is yet further preferred in this aspect of the invention, that the method is one in
which the cow that is receptive to embryo transfer is not part of an artificial insemination
program.
The present invention further generally provides a method of decreasing the number of cows
culled from a herd of cows, by selecting a subpopulation of cows from the herd that are
receptive to embryo transfer, the subpopulation being cows that would otherwise be culled
from the herd due to not being in heat or not showing outward signs of heat, the method
comprising the steps of:
a) synchronizing the oestrous cycles of two or more cows in the herd;
b) identifying a subpopulation of cows from the herd, being cows which are not in heat
or do not show outward signs of heat;
c) testing one or more cows from the subpopulation identified in step (b) for the
presence or absence of a corpus luteum;
d) identifying a cow that has a corpus luteum based upon that testing; and
e) transferring an embryo to the cow identified as having a corpus luteum,
where a cow that subsequently becomes pregnant after embryo transfer is one that is
receptive to embryo transfer, but is otherwise one that would have been culled from the herd
due to not being in heat or not showing outward signs of heat.
Preferably in this aspect of the invention, the method is one in which the synchronisation in
step (a) is achieved by administering one or more of prostaglandin, gonadotropin and
progestin to the cows. It is particularly preferred in this regard that the synchronisation in step
(a) is achieved by administering prostaglandin to the cows.
Preferably, in some embodiments, the method is one in which the presence of the corpus
luteum in the at least one cow that is not in heat or which does not show outward signs of
heat is detected by:
(i) rectal palpation;
(ii) ultrasonography;
(iii) measuring the concentration of progesterone in the blood of the cow; or
(iv) measuring the concentration of progesterone in milk produced by the cow.
Preferably further, the method is one in which the presence of the corpus luteum in the at
least one cow that is not in heat or does not show outward signs of heat is detected by rectal
palpation.
It is further preferred in some embodiments that the method is one in which the embryo is
between 5 days and 8 days old.
Preferably further, the method is one in which the embryo is selected from the group
consisting of: fresh embryos, frozen embryos, in vivo embryos, in vitro fertilized embryos,
embryos produced by somatic cell nuclear transfer, embryos produced by embryonic cell
nuclear transfer, embryos produced by embryonic splitting, embryos produced by sperm
microinjection, embryos produced by genetic modification, and embryos produced by genetic
selection.
It is further preferred that the method is one in which the embryo is transferred to the
at least one cow that is not in heat or does not show outward signs of heat at a time between
day 6 and day 10 of the cow’s oestrous cycle.
In this aspect of the invention, it is yet further preferred that the method is one in
which the cow that is receptive to embryo transfer is not part of an artificial insemination
program.
The invention yet further generally provides a method of maximizing the number of cows
selected from a herd to be recipients for embryo transfer, by identifying a subpopulation of
cows from the herd that are not in heat or do not show outward signs of heat, but which are
still receptive to embryo transfer, the method comprising the steps of:
a) synchronising the oestrous cycles of two or more cows in the herd;
b) identifying a subpopulation of cows from the herd, being cows which are not
in heat or do not show outward signs of heat;
c) testing one or more cows from the subpopulation identified in (b) for the
presence or absence of a corpus luteum;
d) identifying a cow that has a corpus luteum based upon that testing; and
e) transferring an embryo to the cow identified as having a corpus luteum,
where the embryo recipient cow is one that would not otherwise have been given an embryo.
Preferably in this aspect of the invention, the method is one in which the synchronisation in
step (a) is achieved by administering one or more of prostaglandin, gonadotropin and
progestin to the cows. It is particularly preferred in this regard that the synchronisation in step
(a) is achieved by administering prostaglandin to the cows.
Preferably, in some embodiments of this aspect of the invention, the method is one in
which the presence of the corpus luteum in the at least one cow that is not in heat or which
does not show outward signs of heat is detected by:
(v) rectal palpation;
(vi) ultrasonography;
(vii) measuring the concentration of progesterone in the blood of the cow; or
(viii) measuring the concentration of progesterone in milk produced by the cow.
Preferably further, the method is one in which the presence of the corpus luteum in
the at least one cow that is not in heat or does not show outward signs of heat is detected by
rectal palpation.
It is further preferred in some embodiments of this aspect of the invention that the
method is one in which the embryo is between 5 days and 8 days old.
Preferably further, the method is one in which the embryo is selected from the group
consisting of: fresh embryos, frozen embryos, in vivo embryos, in vitro fertilized embryos,
embryos produced by somatic cell nuclear transfer, embryos produced by embryonic cell
nuclear transfer, embryos produced by embryonic splitting, embryos produced by sperm
microinjection, embryos produced by genetic modification, and embryos produced by genetic
selection.
It is further preferred that the method is one in which the embryo is transferred to the
at least one cow that is not in heat or does not show outward signs of heat at a time between
day 6 and day 10 of the cow’s oestrous cycle.
In this aspect of the invention, it is yet further preferred that the method is one in
which the cow that is receptive to embryo transfer is not part of an artificial insemination
program.
It is to be understood that wherever used in the present specification, the terms
“estrus’ and “oestrus” have the same meaning.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Conception rates for animals in each synchrony group (not different to one
another). (P>0.1).
Figure 2: Conception rates for animals in known and unknown synchrony groups
were found not to be different. (P>0.1)
Figure 3: Pregnancy rates for beef cows, including oestrus-positive cows as identified
by “standing heat”, oestrus-positive cows as identified by “tail chalk”, and oestrus-negative
but CL-positive cows as identified by CL palpation.
Figure 4: Pregnancy rates for repeated breeder cows from Dairy A, including oestrus-
positive cows as identified by “tail chalk”, and oestrus-negative but CL-positive cows as
identified by CL palpation.
Figure 5: Pregnancy rates for oestrus-positive cows as identified by “standing heat”,
oestrus-positive cows as identified by “tail chalk”, oestrus-negative but CL-positive cows as
identified by CL palpation, and cows subjected to timed embryo transfer.
DETAILED DESCRIPTION
Maximizing the number of cows in a herd that can become pregnant – and therefore
begin lactating to produce milk – is a chief goal among dairy farmers. In the dairy industry,
monitoring the pregnancy rate of cows is a standard way to measure the reproductive
performance of individual cows. And the parameter of pregnancy rate is calculated as the
conception rate multiplied by estrus (“in heat”) detection rate. In the estrus phase of the
estrous cycle, females become sexually receptive and are often observed as “in heat.” Each
cow may experience seventeen estrous cycles per year if they do not become pregnant and
they reside in locations where adequate light and nutrition is available year round. The
estrous cycle generally consists of four phases: (1) proestrus; (2) estrus; (3) metestrus and (4)
diestrus. The corpus luteum is a structure that often protrudes from the edge of the ovary and
can be differentiated from the follicle by palpation or ultrasound. The ovarian follicle usually
contains a single oocyte and is found at the cortex, or outer part, of the ovary. The corpus
luteum, which derives from ovarian follicles, is also located at the ovarian cortex
Developmentally, during each estrus phase, ovarian follicles develop into maturity under the
influence of estrogen and gonadotropic hormones. For some mammals such as cows,
spontaneous ovulation may occur in the estrus phase, while in other species, ovulation
requires sexual intercourse.
With respect to cows, and the calculation of pregnancy rates, the heat detection rate is
determined by the fraction of cows that are identified as being in estrus during a 21 day
period, via various methods discussed in methods below. An important point is that
variability in correct heat detection can have a dramatic effect on the pregnancy rate of the
collective herd. For instance, if the conception rate is 40%, and the heat detection rate is
identified as 50%, then the pregnancy rate would equal 20% (PR = 0.4*0.5 = 0.2). However,
if all cows were correctly identified as being in heat, or in estrus, then the pregnancy rate
would double to 40% (PR = 0.4*1.0 = 0.4). Consequently, misidentifying the numbers of
cows that could actually become pregnant by artificial insemination or embryo transfer, but
which were otherwise culled because “no heat” was detected, can significantly reduce the
productivity of the entire herd. In addition, incorrect identification of estrus, and breeding
cows that are not in estrus, costs the dairymen for the wasted semen and lost time while
waiting to determine if the animal is pregnant. Therefore, breeding all animals synchronized
is also costly.
The present invention maximizes the number of cows selected from a herd to be
recipients for embryo transfer by identifying additional animals not seen in estrus “estrus-
negative’ that are still receptive to embryo transfer, and thus pregnancy.. “Receptive”
typically refers to the hormone-induced, thickened condition of the uterine lining that
provides nourishment for the implanted embryo. The present invention allows dairy
producers to impregnate cows that had not shown signs of estrus. Although these cows were
capable of becoming pregnant they would not have been identified using other protocols. In
addition, this new protocol also provides a superior method for impregnating cows over the
standard timed artificial insemination method, because it can permit fewer shots and fewer
drugs to be administered to cows over time.
Artificial insemination refers to the introduction of semen into the vagina or uterus by
mechanical or instrumental means rather than by sexual intercourse. Artificial insemination
is planned to coincide with the expected time of ovulation so that fertilization can occur.
Timed artificial insemination protocols are designed to synchronize ovulation among a group
of cows that are often in various stages of the estrous cycle at the initiation of the program.
In short, the present invention provides an alternative and preferred method to increase
pregnancies and therefore lifetime milk production.
The present invention identified that cows that were classified as estrus-negative, i.e.,
cows that were not identified as in heat, but which had a corpus luteum in the ovary, were in
fact receptive to embryo transfer, and could become pregnant. Thus, the present inventive
method permits the identification of reproductively sound cows from the subpopulation of
cows in the herd that would otherwise have been culled. That is, normally, a dairy farmer
will segregate and cull or first use other strategies such as timed AI to increase the number of
pregnancies and then cull. Typically they would only use those identified as being in heat for
embryo transfer or artificial insemination to become pregnant. Many reproductively sound
cows, particularly on dairy’s, do not show heat because of things like poor footing , negative
energy ballence, excessive heat and inadequate ventilation, inadequate space, inadequate
bedding etc. and therefore have a silent or estrus-negative heat. By using the present
inventive method, however, the dairy farmer can increase the reproductive efficiency of the
cattle herd by recategorizing the to-be-culled subpopulation so as to identify estrus-negative
cows that nonetheless are still able to become pregnant. Thus, any variability in heat
detection can be corrected for by detecting the presence of corpus luteum in the estrus-
negative cows, allowing more cows per herd to be used for reproduction, and thus milk
production.
Accordingly, one aspect of the present inventive method is identifying the presence of
a corpus luteum in estrus-negative cows. The present invention does not require an initial
detection of estrus, or non-detection of estrus, as the case may be. Rather, the present
invention also accommodates simply the detection of corpus luteum in all cows regardless of
whether or not they have been subjected to estrus/heat detection. Furthermore, the present
invention is not limited to use only in cows. Other female mammals can be subjected to the
inventive test to determine whether they are suitably biologically, and receptive for
reproductive activities. Thus, other animals, including horses, dogs, cats, sheep, goats,
camelids, pachyderms, as well as others, can be tested for the presence of a corpus luteum, or
identified as being in heat, or not in heat, and the latter subsequently tested for the presence
of a corpus luteum prior to application of reproductive technology, such as by embryo
transfer. . Thus, citation to “cows” as used elsewhere herein is only an illustrative example,
and not meant to be interpreted as limiting the inventive method to only that species of
animal.
Thus, in one embodiment, a subpopulation of estrus-negative cows is provided that
are nonetheless receptive to embryo transfer. This particular embodiment therefore concerns
identifying those cows from a herd that are not seen in heat, and then testing those estrus-
negative cows to determine which contain a corpus luteum (CL). These estrus-negative, CL-
positive cows are then implanted with embryos, and, of that subpopulation, some will become
pregnant. Accordingly, present invention combined with appropriate synchronization
increases the reproductive efficiency of the herd.
In this regard, the corpus luteum is a temporary endocrine structure developed from
an ovarian follicle during the metestrus and diestrus phases of the estrous cycle in mammals.
The corpus luteum produces high levels of progesterone, which thicken and maintains the
uterine lining in preparation for implantation of an embryo. In the absence of a viable
embryo, the corpus luteum stops secreting progesterone and regresses into a scar tissue, while
the uterine lining consequently degenerates back to normal size. With the presence of a
viable embryo, however, the corpus luteum continues to secrete progesterone, which in turn
maintains the thick uterine lining to provide the necessary nourishment for the development
of the embryo.
There are three standard ways to detect the corpus luteum: (1) rectal palpation;
(2) ultrasonography; and (3) measuring progesterone levels in the blood or milk produced by
the mammal. In the case of rectal palpation in cows, the cervix is first typically detected by
hand, touch and feel in order to identify the uterus, which is characterized by an external
bifurcation between the two uterine horns. The next step is to detect the ovaries, which are
typically located underneath or to the side of the uterus. The corpus luteum is a structure that
often protrudes from the edge of the ovary and can be differentiated from the follicle by
palpation or ultrasound. The ovarian follicle contains a single oocyte and is found at the
cortex, or outer part, of the ovary. The corpus luteum, which derives from ovarian follicles,
is also located at the ovarian cortex.
When performing ultrasonography, a probe is inserted through the rectum and
positioned above the ovary. Pulses of ultrasound are generated by the probes and transmitted
to the tissues adjacent to the probe. From the adjacent tissues, the pulses of ultrasound are
reflected back to the probe. The amount of the reflection depends on the structure of the
adjacent tissues. The corpus luteum is typically seen as a distinct grayish echogenic area
within the ovarian stroma and could contain a cavity. Ultrasonography offers a highly
reliable way of detecting corpus luteum, however, equipment is costly.
Lastly, the corpus luteum can be detected by measuring the concentration of the
hormone progesterone in the cow’s blood or milk. Assay kits are commercially available to
determine progesterone level in blood or milk. For example, a “Target Equine Progesterone
Kit” is available from BioMetallics Incorporated, Princeton, New Jersey, USA. A
QUICKCHECK Canine Progesterone Kit and a QUICKCHECK™ Equine Progesterone
Kit are available from Exodus Breeders Corporation in York, Pennsylvania, USA.
As mentioned above, the present inventive method permits the identification of cows
containing a corpus luteum as suitable candidates for embryo transfer regardless of whether
or not the cows have been classified as in heat or not in heat.
There are various traditional methods for determining whether a cow is in heat.
Mammals in heat, i.e., that are in the estrus phase of the estrous cycle, usually display certain
estrus signs. In the case of cows, these signs include: (1) standing to be mounted by another
cow, which is the most commonly used sign in the industry for estrus detection; (2) greater
physical activity as a result of increased nervousness and restlessness; (3) licking and sniffing
of the genital areas of other cows; (4) bawl or bellow frequently; (5) a thin, clear, watery
mucus discharge from vulva; and (6) reduced feed intake. Because many of these individual
signs require human observation and even then can be subtle in nature, a determination of
whether a cow or other mammal is in heat should be based on the totality of all signs. A
mammal is deemed “estrus-positive” if it is determined that the totality of all signs points to
estrus. Conversely, a mammal is deemed “estrus-negative” if it is determined that the totality
of all signs does not point to estrus.
Specifically, whether a cow is in heat or not can be identified by “standing heat” and
“tail chalk”. Those in “standing heat” showed behavioral estrus by standing to be jumped by
another cow. Further, “tail chalk” can be used to identify cows in estrus – by painting the tail
head of all cows synchronized and observing whether the paint is rubbed off. If the paint is
rubbed off, the cow is recorded as having shown sign of estrus.
The dairy farmer is aware of many of the practical ways in which the aforementioned
signs can be detected. “Marker animals,” for example, are surgically-altered bulls or
androgenized cows or steers, that are sexually active and seek out cows in estrus.
Androgenized cows are cows injected with testosterone and simulate male behavior. These
marker animals are typically equipped with a marking device, such as a halter that works like
a ball-point pen under the marker mammal’s chin. When the marker bull mounts a cow that
is in heat, the chin-ball marker leaves mark on the female mammal’s back. These marks can
then be interpreted to determine whether the female mammal is in heat. Similarly, mounting-
activity detectors attached to a cow can change color upon sexual activity, while electronic
detectors transmit radio signals to a computer that records the identity of the cow, and the
time and duration of the mount. Electronic detectors are more accurate than other types of
detectors because they can record multiple mounts and the duration of the mount.
Alternatively, increased levels of methyl heptanol in a cow’s vaginal secretions indicates it is
in heat. Methyl heptanol can be monitored through gas chromatography as described, for
instance, in U.S. Patent No. 4,467,814, which is hereby incorporated by reference in its
entirety. “Tail marking” is yet another method to detect estrus by placing chalk or paint in a
band along a mammal’s tailhead from hooks to pins. The chalk or paint is rubbed off by the
mammal doing the mounting.
Accordingly, one of ordinary skill in the art would know how to detect cows that are
in heat, or are not in heat, and subsequently identify in the latter (estrus-negative) subgroup,
those cows which have a corpus luteum. The cows that are estrus-negative but CL-positive
then can become candidate recipients of embryos in embryo transfer procedures.
In this regard, embryo transfer refers to various standard methods for establishing a
pregnancy in which one or several embryos are isolated from at least one donor mammal and
placed into the uterus of at least one other mammal. An in vivo embryo is one isolated from
the donor mammal In addition to producing the embryo in a donor mammal, an embryo can
also be produced with the process of in vitro fertilization (IVF). IVF refers to a process by
which an egg is fertilized by a sperm outside the body. Embryos may be transferred fresh or
after preservation by freezing or vitrifying in liquid nitrogen. Frozen/vitrified embryos are
thawed/warmed prior to embryo transfer following protocols dependent on the preservation
process and the tool used to contain the embryo. Frozen embryos can maintain their viability
for long periods of time, likely because the low temperature effectively stops all biological
activities, including biological reactions leading to apoptosis.
In one embodiment, the embryos to be transferred are produced in vitro by somatic
cell nuclear transfer (i.e., cloning), which is exemplified in Kato et al., Science
282(5396):2095-2098 (1998) and Wilmut et al., Nature 385:810-813 (1997), both of which
are hereby incorporated by reference in their entireties.
In another embodiment, the embryos to be transferred are produced in vitro by
embryonic cell nuclear transfer, which is exemplified in Peura et al., Mol. Reprod. Dev.
50:185-191 (1998) and Smith et al., Biology of Reproduction 40(5):1027-1035 (1989), both
of which are hereby incorporated by reference in their entireties.
In a further embodiment, the embryos to be transferred are produced in vitro by
embryo splitting or cleavage, which is exemplified in Gary et al., Therigenology 35(1):37-44
(1991) and Baker et al., Therigenology 23(1):3-12 (1985), both of which are hereby
incorporated by reference in their entireties.
In an additional embodiment, the embryos to be transferred are produced in vitro by
sperm microinjection, which is exemplified in Heuwieser et al., Therigenology 38(1):1-9
(1992), which is hereby incorporated by reference in its entirety.In one embodiment, the
embryos to be transferred are produced in vitro by genetic modification, which is exemplified
in Yang et al., PLoS One 6(3):e17593 (2011); Clark, Am. J. Clin. Nutr. 63:633-638 (1996);
and Lai et al., Reprod. Biol. Endocrinol. 1:82 (2003), all of which are hereby incorporated by
reference in their entireties.
In one embodiment, the embryos to be transferred are produced in vitro by genetic
selection, which is exemplified in Bodo et al., Acta Vet. Hung. 49(1):99-109 (2001) and Chen
et al., Biology of Reproduction. 67(5):1488-1492 (2002), both of which are hereby
incorporated by reference in their entireties.
The embryo transfer procedure with respect to fresh or in vivo donor-generated
embryos, generally comprises seven steps: (1) selection of donor mammal; (2)
synchronization of donor mammal and recipient mammals; (3) superovulation of donor
mammal (a process of manipulating the donor mammal to produce maximum number of
eggs); (4) insemination of the donor (fertilization of donor eggs by sperm from selected
males); (5) flushing the embryo (separating the embryos from the donor uterus); 65)
evaluation of the embryo (classifying the embryos into different grades, such as excellent,
good, fair, poor and dead); (7) selection of the recipient mammal (selecting female mammals
that are receptive to embryo transfer); (8) transferring the embryos, which is a process of
“loading” the embryo into a transfer gun, inserting the transfer gun through the cervix and
then expelling the embryo in the forward tip of a uterine horn ipsilateral to the corpus luteum.
In the case of in vitro produced embryo, only steps (5), (6) and (7) typically would apply.
Sometimes it is desirable to select a donor mammal that is regarded to be “genetically
superior” than other members of its species. Thus, in one embodiment of the present
invention, embryo transfer can be used to increase the number of offsprings from genetically
superior mammals. For instance, artificial insemination is a low cost alternative compared
with maintaining bulls. Artificial insemination also provides the ability to select from a large
number of bulls those that have particularly desirable traits. Embryo transfer has been used
to maximize the reproductive potential of such elite cows that can be bred with those bulls.
With the help of hormone stimulation, for example, elite cows can be subject to
superovulation to produce 7-12 embryos per ovulation cycle to be transferred to other
surrogate cows. At about 50% successful transfer rate, the number of calves from an elite
cow therefore increases from one to more than six per ovulation cycle. Cows can be subject
to superovulation multiple times in a year.
Various media used in embryo transfer are known in the art. For example, embryo
flush can be used when removing in vivo embryos from a donor’s uterus. Commercially
available embryo flush include, for example, EmCare™ Complete Flush Media and Vigro™
Complete Flush Media from Reproduction Resources, as well as BoviPro™ Recovery Media
from Minitube of America. Further, holding media can be used when manipulating embryos
on a benchtop. Commercially available holding media include, for example, EmCare™
Holding Media, Vigro™ Holding Plus Media and Syngro Holding Media from Reproduction
Resources, as well as BoviPro™ Holding Media from Minitube of America. Additionally,
thawing media can be used for thawing frozen embryos. Commercially available thawing
media include, for example, EmCare™ CSU Thawing Kit from Reproduction Resources, as
well as BoviPro™ Rehydration Media from Minitube of America.
Before estrus detection and/or corpus luteum detection are performed, it can
sometimes be desirable to “synchronize” a herd of cows to ensure substantially all members
of the dairy herd are staged at the same or similar reproductive period of their respective
estrous cycles. Synchronizing the estrous cycles generally means manipulating the
reproductive process of a group of female mammals so that ovulation occurs at
approximately the same time. Synchronized estrous cycles in cows, for instance, allow a
breeder to schedule breeding activities for a group of cows during a predetermined period,
thereby increasing the efficiency of artificial insemination, embryo transfer or other breeding
techniques, useful for maximizing the reproductive efficiency of the entire herd.
Synchronization can be achieved through the application three primary groups of
hormones: prostaglandins, progestins, and gonadotropins. There are commercial products
available for each group of hormones. Prostaglandin products include Lutalyse, Estrumate,
and IN-SYNCH. Progestin products include the vaginal implant CIDR and the feed
additive Melengestrol Acetate (MGA). Gonadotropins products include Cystrorelin, Factrel,
and Fertagyl. Antibodies can be used to detect these hormones. As is well known, an
antibody is a protein employed by the immune system to recognize and neutralize foreign
objects such as bacteria and virus. The target of an antibody is termed an antigen. The
antibody typically recognize a unique part (“epitope”) of an antigen. In addition to bacteria
and viruses, modern molecular biology techniques now allow a skilled artisan to raise
antibodies against almost any protein and then use the antibody as an assay tool as well as
diagnostic or therapeutic tool.
“Prostaglandin” is a group of 20-carbon fatty acid hormones, secreted by the uterine
endometrium, which causes the regression of corpus luteum and thereby induces a sharp drop
of progesterone produced by the corpus luteum. With the removal of progesterone,
gonadotropin-releasing hormone (GnRH) starts to exert greater influence. The higher level
of GnRH leads to higher level of follicle-stimulating hormone (FSH) and luteinizing hormone
(LH), which supports follicular development as the mammal enters the proestrus phase of the
estrous cycle. “Progesterone” is a 21-carbon hormone that works through the intracellular
progesterone receptor to exert a number of physiological effects, including (1) converting the
endometrium, or uterine lining, to its secretory stage to prepare for the implantation of
fertilized egg; (2) decreasing maternal immunity during implantation for the female
mammal’s body to accept pregnancy without an immune reaction; (3) decreasing the
contractibility of the uterine smooth muscle; and (4) inhibiting lactation during pregnancy.
During the metestrus phase of the estrous cycle, the newly formed corpus luteum starts to
secret low level of progesterone. The corpus luteum then secretes high levels of progesterone
during the diestrus phase.
A number of protocols can be employed to achieve synchronization, such as
administering one hormone or a combination of hormones. For instance, prostaglandin may
be injected individually in one of the four ways:
(1) A single injection of prostaglandin is administered and estrus is detected in the
following 2-5 days.
(2) Estrus is detected in the first five days, and all female mammals found to be in
heat were bred. Then a single injection of prostaglandin is administered on the fifth day to
the female mammals that were not in heat.
(3) A first injection of prostaglandin is administered and estrus is detected in the next
days. On the 11 day, a second injection of prostaglandin is administered to the female
mammals that were not in heat.
(4) A first injection of prostaglandin is administered and estrus is detected in the next
days. On the 14 day, a second injection of prostaglandin is administered to the female
mammals that were not in heat.
The administration of multiple shots of prostaglandins is typically referred to by a
person skilled in the art as the two-shot method. See DuPonte MW and Lee KK, Three
Simplified Estrus Synchronization Programs for Hawaii’s Beef Breeding Season, Livestock
Management (Cooperative Extension Service, University of Hawaii at Manoa), April 2007,
LM-14, 1-2, which is hereby incorporated by reference in its entirety.
Prostaglandin can also be administered together with a progesterone-like feed additive
Melengestrol Acetate (MGA). MGA can be fed for 14 days and then withdrawn. A single
injection of prostaglandin then is administered 17 to 19 days after the MGA withdrawal.
In one embodiment, after synchronizing the cows with a single injection of
prostaglandin, no other synchronizing agent is administered to the cows before the detection
of corpus luteum. In another embodiment, after synchronizing the cows with a single
injection of prostaglandin, no other synchronizing agent is administered to the cows before
embryo transfer.
Synchronization can also be achieved through the combination of prostaglandin and
vaginal implant CIDR . Typically the CIDR implant is inserted on Day 0, one injection of
prostaglandin is given on Day 6, and the CIDR implant is removed on Day 7. Estrus can be
detected from Day 8 to Day 11. Further examples of synchronization methods include the
combination of gonadotropins and prostaglandin. In the so-called “Select Synch protocol”
(See Lamb GC, et al, Effectiveness of Select Synch (Gonadotropin-Releasing Hormone and
Prostaglandin F2 ) for Synchronizing Estrus in Replacement Beef Heifers, The Professional
Animal Scientist, 20:27-33 (2004), incorporated by reference in its entirety), an injection of
gonadotropin is administered on Day 0 with an injection of prostaglandin administered on
Day 7. Estrus typically occurs 36 hour before and up to 6 days after the prostaglandin
injection. The “Select Synch protocol” can be further modified to include a further
gonadotropin injection 2 days after the prostaglandin injection.
Accordingly, one embodiment of the present methodology entails synchronizing a
number of cows, detecting estrus in the synchronized cow population, and then detecting the
presence of a corpus luteum in cows that are estrus-negative.
By increasing the numbers of cows identified as receptive to embryo transfer and
subsequent pregnancy, the present invention also increases the number of lactating cows that
correspondingly increases the quantity of milk that can be produced per unit time. Dairy
cattle such as Holstein have been repeatedly and selectively bred for increased milk
production, and such cows can be used in the context of the present inventive technology.
While milk production level varies from breed to breed, one cow can typically produce
around 15,000 to 25,000 lbs of milk per lactation period. Ten or more lactations are possible
during a cow’s lifetime. But most cows are culled after two lactations, mainly for four
reasons: (1) infertility; (2) persistent and potentially fatal mammary gland infection; (3)
persistent foot infection or leg problems causing infertility and loss of production; and (4)
failure to produce economic levels of milk to justify their feed costs. The present invention
therefore addresses the number one reason for culling dairy cows and maximizes the
efficiency of the rate of pregnancy by identifying additional cows receptive to embryo
transfer, and also maximizes the efficiency of milk production.
It was shown as disclosed elsewhere herein, that estrus was synchronized using a
single intramuscular injection of Lutalyse injectable prostaglandin into cows, and estrus
detected by observing tail chalk once daily. Fresh in vitro produced (IVP) embryos at Day
6.5 and 7.5, were then transferred to cows observed to be in estrus 11 days after Lutalyse
injection on cow estrous cycle Days 6 -10. Those cows that did not show signs of estrus were
then subsequently palpated for a corpus luteum (CL). If a CL was detected an IVP embryo
was transferred. Pregnancy was detected by palpation at approximately 40 days.
Significantly, the cows which did not show estrus but did have a corpus luteum, had a
conception rate of 38.7% (Figure 1 and Table 1).
Similarly, in the same study disclosed herein, thirty-one additional cows were
identified through CL palpation for embryo transfer which increased the total number of
embryos transferred by 26%. This study shows that these cows, which would have remained
undetected, were capable of becoming pregnant with an IVP embryo.
Furthermore, embryos have the unique ability to speed up or delay development in
order to fit the stage of the uterine environment. Therefore the embryo and the uterus do not
need to be in exact synchrony for a pregnancy to occur. This cannot be said for artificial
insemination (AI). When AI occurs, it must be done within a very short time period (12 hour
window). There are some protocols for timing AI so heat detection is unnecessary.
However, these protocols require giving numerous injections and can take several weeks
from beginning of the protocol to AI. If the service is unsuccessful repeating the protocol
again takes many additional weeks.
Because the corpus luteum structure does not exist on the ovary until day 4-5 of the
estrous cycle, 4-5 days after the necessary time for artificial insemination, the process of
palpating a corpus luteum structure after synchronization is not applicable for artificial
insemination programs. However, corpus luteum palpation is effective in embryo transfer, as
the embryo is transferred on days 6-10 of the estrous cycle. The results from this study, for
instance, as explained in detail in the Examples, showed that the conception rates of cows
which did not show signs of estrus but had a corpus luteum, and in which an embryo was
transferred, were not different than cows that were observed in estrus and known to be
synchronized.
Thus, this new protocol will save dairy producers money in synchronization costs, as
well as increasing the efficiency of obtaining pregnant animals, such as cows, that had not
shown signs of estrus. Although these animals were capable of becoming pregnant they
would not have been identified using current, existing protocols.
* * *
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. Generally, the nomenclature used herein, and the laboratory procedures described
herein, are those well known and commonly employed in the art. Standard techniques are
used for mammal breeding and maintenance, analytical chemistry, molecular biology,
enzymatic assays and pharmaceutical formulation and delivery. Generally, enzymatic
reactions and purification and/or isolation steps are performed according to the
manufacturers' specifications.
The following examples serve to illustrate various embodiments of the present
invention and should not be construed, in any way, to limit the scope of the invention.
All references cited herein, including patents, patent application and publications, are
hereby incorporated by reference in their entireties, where previously specifically
incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in
the art that the same can be performed within a wide range of equivalent parameters,
concentrations and conditions, without undue experimentation. This application is intended to
cover any variations, uses, or adaptations of the invention, following in general the principles
of the invention, that include such departures from the present disclosure as come within
known or customary practice within the art to which the invention pertains and as may be
applied to the essential features hereinbefore set forth.
EXAMPLES
Example 1
Between July 2010 and January 2011 one hundred forty-eight Holstein, Holstein
crossbred and Jersey cows were identified as repeat breeders by being over 120 days in milk
or having had over 4 unsuccessful artificial insemination services. Estrus was synchronized
using a single intramuscular injection of Lutalyse injectable prostaglandin (25mg, Pfizer,
New York, NY). Estrus was detected by observing tail chalk once daily. Fresh in vitro
produced (IVP) embryos, Day 6.5 and 7.5, were transferred to cows observed in estrus 11
days after Lutalyse injection on cow estrous cycle Days 6 -10. Those cows that did not show
signs of estrus were palpated for a corpus luteum (CL). If a CL was detected an IVP embryo
was transferred. Pregnancy was detected by palpation at approximately 40 days.
Synchrony of cows and embryos were calculated as cow day minus embryo day and
ranged from -0.5 to 2.5 Days. The highest proportions of animals were 0.5 and 1.5 Days. (n
= 85). Thirty-one animals that received embryos but did not show signs of estrus had an
unknown synchrony. The overall Day 40 conception rate was 31.3% for animals in this
study. Those cows with unknown synchrony, which did not show estrus but did have a CL,
had a conception rate of 38.7% (Figure 1 and Table 1).
In this study, dairy cattle were synchronized with one shot of Lutalyse. Lutalyse
(prostaglandin) is able to cause regression of the corpus luteum (CL) on days 5 through 17 of
the estrous cycle when a fully functional CL is present on the ovary. Luteolysis is the
regression of the CL which allows onset of estrus. Those animals past day 17 of the cycle
would have natural CL regression if a pregnancy had not been established. Only those
animals between days 0 and 5 would not be synchronized. Therefore, on average the
percentage of animals that would be synchronized for estrus within the next 5 days would be
76.2%. Many of these animals, however, would not show or may not be seen in heat. In this
study tail chalk was used to detect estrus by once daily observation. The animals that were
not detected were palpated for a CL. If a CL was present an embryo was transferred.
Thirty-one additional animals were identified for embryo transfer which increased the total
number of embryos transferred by 26%. This study shows that those undetected animals
were capable of becoming pregnant with an IVP embryo.
Embryos have the unique ability to speed up or delay development in order to fit the
stage of the uterine environment. Therefore the embryo and the uterus do not need to be in
exact synchrony for a pregnancy to occur. This cannot be said for artificial insemination
(AI). When AI occurs it must be done within a very short time period (12 hour window).
There are some protocols for timing AI so heat detection is unnecessary. However, these
protocols require giving numerous injections and can take several weeks from beginning of
the protocol to AI. If the service is unsuccessful repeating the protocol again takes many
weeks.
Because the CL structure does not exist on the ovary until day 4-5 of the estrous
cycle, 4-5 days after the necessary time for artificial insemination, the process of palpating a
CL structure on the ovary post synchronization is not applicable to AI programs. However,
CL palpation is effective in embryo transfer, as the embryo is transferred on days 6-10 of the
estrous cycle. Results from this study showed that the conception rates of animals which did
not show signs of estrus but had a CL, and in which an embryo was transferred, were not
different than animals that were observed in estrus and known to be synchronized. This new
protocol saves dairy producers money in synchronization costs, as well as impregnating
animals that had not shown signs of estrus. Although these animals were capable of
becoming pregnant they would not have been identified using other protocols. . In addition,
this new protocol also provides a superior method for impregnating animals over the standard
timed artificial insemination method, because it permits fewer shots and fewer drugs to be
administered to animals over time. This protocol provides an alternative and preferred
method to increase pregnancies and therefore lifetime milk production.
Synchrony of cows and embryos were calculated as cow day minus embryo day and
ranged from -0.5 to 2.5 Days. The highest proportions of animals were 0.5 and 1.5 Days. (n
= 85). Thirty-one animals that received embryos but did not show estrus had an unknown
synchrony. The overall Day 40 conception rate was 31.3%. Those cows with unknown
synchrony, which did not show estrus but did have a CL, had a conception rate of 38.7%
(Figure 1) and Table 1 (no. of animals in each group and the % of those that conceived,
P>0.1).
Table 1
Conception
Synchrony Pregnant Open Total Rate
-0.5 4 8 12 33.3%
0.5 9 27 36 25.0%
1.5 15 33 48 31.3%
2.5 1 3 4 25.0%
Unknown 12 19 31 38.7%
Total 41 90 131 31.3%
Example 2
Figure 3 and Table 2 show the results from 490 IVF embryo transfers to older beef
recipients from January 2011 to present. Animals were given one shot of Lutalyse and
observed for signs of estrus. Those in standing heat showed behavioral estrus by standing to
be jumped by another animal. Another method used to identify animals in estrus is by
painting the tail head of all cows synchronized. If the paint is rubbed off the animal is
recorded as having showed sign of estrus. By palpating for the presence or absence of a
corpus luteum (CL) we were also able to infer that estrus had occurred when the CL is
present. In vitro produced (IVP) embryos, Days 6.5, 7, 7.5 and 8, were transferred to both
estrus-positive cows (cows observed in heat or showing outward signs of heat), and estrus-
negative but CL-positive cows (cows not observed in heat or does not show outward signs of
heat, but has CL present) 10 to 12 days after Lutalyse injection on cow estrous cycle Days 6
-10. Due to our synchronization efforts, the estrus is assumed to have occurred in the time
frame necessary for embryo transfer. Using CL palpation we were able to identify 129
additional animals for embryo transfer. Of those, 34 were confirmed pregnant at 30 days of
gestation (26.4%). However, none of these animals would have had the opportunity to
become pregnant. The drop in pregnancy rate is expected as the synchrony of the animals
uterus and the embryo are unknown, but the fact that pregnancies do occur is significant.
Table 2
Estrus PREG OPEN
Detection PREG OPEN TOTAL RATE RATE
Standing Heat 106 107 213 49.8% 50.2%
Tail Chalk 73 75 148 49.3% 50.7%
CL Palpation 34 95 129 26.4% 73.6%
TOTAL 213 277 490 43.5% 56.5%
Example 3
Figure 4 and Table 3 shows the results from 138 IVF embryo transferred at a working
dairy (Dairy A) to repeat breeder animals from July to December 2010. Animals were given
one shot of Lutalyse and observed for signs of estrus by looking for the absence of tail chalk.
Twenty-one additional animals were identified for embryo transfer by using CL palpation.
Of those, nine were confirmed pregnant at 45 days of gestation (42.9%). The 21 animals in
the CL palpation group had not shown signs of estrus as the tail chalk had not been rubbed
off the tail head. These animals would not have had the opportunity to become pregnant
without the use of CL palpation and embryo transfer. Fresh in vitro produced (IVP) embryos,
Day 6.5 and 7.5, were transferred to both estrus-positive cows (cows observed in heat or
showing outward signs of heat), and estrus-negative but CL-positive cows (cows not
observed in heat or does not show outward signs of heat, but has CL present) 11 days after
Lutalyse injection on cow estrous cycle Days 6 -10
Table 3
Estrus PREG OPEN
Detection PREG OPEN TOTAL RATE RATE
Tail Chalk 32 85 117 27.4% 72.6%
CL Palpation 9 12 21 42.9% 57.1%
TOTAL 41 97 138 29.7% 70.3%
Example 4
Figure 5 shows results of 861 IVF embryo transfers to beef and dairy cattle from
January 2010 to present. Using CL palpation we were able to identify a significant number
of estrus-negative cows for embryo transfer. Of those, 28.7% were confirmed pregnant at 30-
45 days of gestation. None of these animals would have had the opportunity to become
pregnant if the embryos are only transferred to cows that were identified as “in heat”, or
estrus-positive.
INTERPRETATION OF THIS SPECIFICATION
It will therefore be understood that the invention could take many forms and be put to
many different uses. All such forms and uses are embodied within the spirit and scope of the
invention, which is to be understood as not being limited to the particular details of the
embodiments or the examples discussed previously, but which extends to each novel feature
and combination of features disclosed in or evident from this specification (including in the
accompanying claims and drawings). All of these different combinations constitute various
alternative aspects of the invention.
Throughout this specification, unless the context requires otherwise, the word
"comprise" is (and variants, variations or other forms of that word, such as "comprises" or
"comprising" are) to be understood as implying the inclusion of a stated element, feature or
integer or group of elements, features or integers, but not the exclusion of any other element,
feature or integer or group of elements, features or integers. Further, wherever used in this
specification, the term “includes” is not a term of limitation, and is not to be taken as
excluding the presence of other any element, feature or integer or group of elements, features
or integers.
It is further to be understood that any discussion in this specification of
background or prior art documents, devices, acts, information, knowledge or use
(‘Background Information’) is included solely to explain the context of the invention. Any
discussions of such Background Information is not to be taken as an admission in any
jurisdiction that any such Background Information constitutes prior art, part of the prior art
base or the common general knowledge in the field of the invention on or before the priority
date of the appended claims or any amended claims later introduced into this specification.
Claims (30)
1. A method of identifying a cow that is receptive to embryo transfer, the method comprising the steps of: a) synchronising the oestrous cycles of two or more cows in a population; 5 b) identifying a subpopulation of cows which are not in heat or which do not show outward signs of heat, from the population; c) testing at least one cow from the subpopulation identified in step (b) for the presence or absence of a corpus luteum; d) identifying a cow that has a corpus luteum based upon that testing; and 10 e) transferring an embryo to the cow identified as having a corpus luteum, and testing the cow for pregnancy, where a positive pregnancy test indicates the cow’s receptivity to embryo transfer.
2. A method as claimed in claim 1 in which the synchronisation in step (a) is achieved 15 by administering one or more of prostaglandin, gonadotropin and progestin to the cows.
3. A method as claimed in claim 2, in which the synchronisation in step (a) is achieved by administering prostaglandin to the cows.
4. A method as claimed in any one of claims 1 to 3, in which the presence of the corpus luteum in the at least one cow that is not in heat or which does not show outward signs of heat is detected by: 25 (i) rectal palpation; (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood of the cow; or (iv) measuring the concentration of progesterone in milk produced by the cow. 30
5. A method as claimed in claim 4, in which the presence of the corpus luteum in the at least one cow that is not in heat or does not show outward signs of heat is detected by rectal palpation.
6. A method as claimed in any one of claims 1 to 5, in which the embryo is between 5 days and 8 days old.
7. A method as claimed in any one of claims 1 to 6, in which the embryo is selected 5 from the group consisting of: fresh embryos, frozen embryos, in vivo embryos, in vitro fertilized embryos, embryos produced by somatic cell nuclear transfer, embryos produced by embryonic cell nuclear transfer, embryos produced by embryonic splitting, embryos produced by sperm microinjection, embryos produced by genetic modification, and embryos produced by genetic selection.
8. A method as claimed in any one of claims 1 to 7, in which the embryo is transferred to the at least one cow that is not in heat or does not show outward signs of heat at a time between day 6 and day 10 of the cow’s oestrous cycle. 15
9. A method as claimed in any one of claims 1 to 8, in which the cow that is receptive to embryo transfer is not part of an artificial insemination program.
10. A method of decreasing the number of cows culled from a herd of cows, by selecting a subpopulation of cows from the herd that are receptive to embryo transfer, the 20 subpopulation being cows that would otherwise be culled from the herd due to not being in heat or not showing outward signs of heat, the method comprising the steps a) synchronizing the oestrous cycles of two or more cows in the herd; b) identifying a subpopulation of cows from the herd, being cows which are not 25 in heat or do not show outward signs of heat; c) testing one or more cows from the subpopulation identified in step (b) for the presence or absence of a corpus luteum; d) identifying a cow that has a corpus luteum based upon that testing; and e) transferring an embryo to the cow identified as having a corpus luteum, 30 where a cow that subsequently becomes pregnant after embryo transfer is one that is receptive to embryo transfer, but is otherwise one that would have been culled from the herd due to not being in heat or not showing outward signs of heat.
11. A method as claimed in claim 10, in which the synchronisation in step (a) is achieved by administering one or more of prostaglandin, gonadotropin and progestin to the cows. 5
12. A method as claimed in claim 11, in which the synchronisation in step (a) is achieved by administering prostaglandin to the cows.
13. A method as claimed in any one of claims 10 to 12, in which the presence of the corpus luteum in the at least one cow that is not in heat or which does not show 10 outward signs of heat is detected by: (i) rectal palpation; (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood of the cow; or 15 (iv) measuring the concentration of progesterone in milk produced by the cow.
14. A method as claimed in claim 13, in which the presence of the corpus luteum in the at least one cow that is not in heat or does not show outward signs of heat is detected by rectal palpation.
15. A method as claimed in any one of claims 10 to 14, in which the embryo is between 5 days and 8 days old.
16. A method as claimed in any one of claims 10 to 15, in which the embryo is selected 25 from the group consisting of: fresh embryos, frozen embryos, in vivo embryos, in vitro fertilized embryos, embryos produced by somatic cell nuclear transfer, embryos produced by embryonic cell nuclear transfer, embryos produced by embryonic splitting, embryos produced by sperm microinjection, embryos produced by genetic modification, and embryos produced by genetic selection.
17. A method as claimed in any one of claims 10 to 16, in which the embryo is transferred to the at least one cow that is not in heat or does not show outward signs of heat at a time between day 6 and day 10 of the cow’s oestrous cycle.
18. A method as claimed in any one of claims 10 to 17, in which the cow that is receptive to embryo transfer is not part of an artificial insemination program.
19. A method of maximizing the number of cows selected from a herd to be recipients for 5 embryo transfer, by identifying a subpopulation of cows from the herd that are not in heat or do not show outward signs of heat, but which are still receptive to embryo transfer, the method comprising the steps of: a) synchronising the oestrous cycles of two or more cows in the herd; b) identifying a subpopulation of cows from the herd, being cows which are not 10 in heat or do not show outward signs of heat; c) testing one or more cows from the subpopulation identified in (b) for the presence or absence of a corpus luteum; d) identifying a cow that has a corpus luteum based upon that testing; and e) transferring an embryo to the cow identified as having a corpus luteum, 15 where the embryo recipient cow is one that would not otherwise have been given an embryo.
20. A method as claimed in claim 19, in which the synchronisation in step (a) is achieved by administering one or more of prostaglandin, gonadotropin and progestin to the 20 cows.
21. A method as claimed in claim 20, in which the synchronisation in step (a) is achieved by administering prostaglandin to the cows. 25
22. A method as claimed in any one of claims 19 to 21, in which the presence of the corpus luteum in the at least one cow that is not in heat or which does not show outward signs of heat is detected by: (i) rectal palpation; 30 (ii) ultrasonography; (iii) measuring the concentration of progesterone in the blood of the cow; or (iv) measuring the concentration of progesterone in milk produced by the cow.
23. A method as claimed in claim 22, in which the presence of the corpus luteum in a cow that is not in heat or does not show outward signs of heat is detected by rectal palpation. 5
24. A method as claimed in any one of claims 19 to 23, in which the embryo is between 5 days and 8 days old.
25. A method as claimed in any one of claims 19 to 24, in which the embryo is selected from the group consisting of: fresh embryos, frozen embryos, in vivo embryos, in 10 vitro fertilized embryos, embryos produced by somatic cell nuclear transfer, embryos produced by embryonic cell nuclear transfer, embryos produced by embryonic splitting, embryos produced by sperm microinjection, embryos produced by genetic modification, and embryos produced by genetic selection. 15
26. A method as claimed in any one of claims 19 to 25, in which the embryo is transferred to the at least one cow that is not in heat or does not show outward signs of heat at a time between day 6 and day 10 of the cow’s oestrous cycle.
27. A method as claimed in any one of claims 19 to 26, in which the cow that is receptive 20 to embryo transfer is not part of an artificial insemination program.
28. A method as claimed in any one of claims 1 to 9 of identifying a cow that is receptive to embryo transfer, the method being substantially as disclosed in this specification, and with reference to the examples given in the description, claims and accompanying 25 drawings.
29. A method as claimed in any one of claims 10 to 18 of decreasing the number of cows culled from a herd of cows, the method being substantially as disclosed in this specification, and with reference to the examples given in the description, claims and 30 accompanying drawings.
30. A method as claimed in any one of claims 19 to 27 of maximizing the number of cows selected from a herd to be recipients for embryo transfer, the method being substantially as disclosed in this specification, and with reference to the examples 5 given in the description, claims and accompanying drawings. DATED 27 April 2016 J.R. Simplot Company By its Patent Attorneys KNIGHTSBRIDGE PATENT ATTORNEYS
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161496761P | 2011-06-14 | 2011-06-14 | |
US61/496,761 | 2011-06-14 | ||
PCT/US2012/042212 WO2012174086A1 (en) | 2011-06-14 | 2012-06-13 | A method for identifying a mammal receptive to embryo transfer |
Publications (2)
Publication Number | Publication Date |
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NZ619785A NZ619785A (en) | 2016-05-27 |
NZ619785B2 true NZ619785B2 (en) | 2016-08-30 |
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