Breeding without boundaries; it sounds
like science fiction. Yet today we can breed two animals from
different time zones, even different continents, through
advances in artificial insemination. In this article, we will
delve into the present and future of this advancing technology,
discussing how it can be used for the convenience of owners and
the betterment of breeds.
But first, a little
history...
Artificial insemination (AI)
is the collection of semen from a male, usually of superior
genetic merit, and its transfer into an ovulating female to
achieve fertilization. It is practiced in numerous mammals
including humans, livestock, and exotic zoological species. It
has a long history, with the first reputed use being in the 14th
Century. However, significant development of the technique did
not occur until the end of the 19th Century, when it was first
used commercially in Russian horses.
Before horse AI could become
widely established, the advent of the combustion engine and the
subsequent decline in horse population drove AI research toward
use in other livestock. Although some countries continued their
interest in equine AI on a small scale, many concentrated on
bovine, ovine, and porcine AI with their greater earning
potential. The upsurge in interest in equine AI during the last
20 years has been a reflection of the increase in horse numbers
along with the developing leisure interest in equestrian
activities and the realization of the economic advantages of AI.
Despite this recent increase
in interest, equine AI is still a developing technology that has
yet to reach the sophistication of cattle AI.
There are many reasons why AI
is now used in horses.
Advantages of AI:
-
Removal of geographical
restrictions.
-
Storage of semen for
posterity.
-
Increasing the number of
mares covered per stallion.
-
Facilitation and
acceleration of genetic improvement of stock.
-
Ensuring routine semen
evaluation and monitoring.
-
Improving the reproductive
potential of sub-fertile stallions.
-
Allowing breeding of
problem mares that are precluded from natural service.
-
Allowing mares with a
heightened post-coital immunological response (severe
post-coital endometritis) to be bred.
-
Allowing stallions to run
concurrent performance and breeding careers.
-
Helping preserve rare
breeds.
-
Potentially reducing labor
costs through the use of fixed-time AI.
-
Allowing stock to be bred
that are isolated for health reasons.
-
Helping control disease.
-
Reducing the risk of
injury.
-
Permitting the use of
injured stallions.
-
Reinforcing natural
service.
-
Encouraging routine
examination of the mare's reproductive tract.
-
Extending the breeding
season.
Despite these advantages,
there are several potential disadvantages as well.
Disadvantages of AI:
-
Reducing the genetic pool.
-
Reducing the potential
income from mare boarding fees.
-
Significant variation in
the quality of semen available.
-
Ethical dilemmas, such as
breeding from deceased stallions.
-
Problems over semen
ownership--for example, upon the sale or death of the
stallion.
-
Increasing the opportunity
for fraud.
-
Increasing the risk of
disease transfer.
-
Increasing the cost of
covering mares.
-
Responsibility for
conception lies with mare owner/manager.
-
Requirement for an
increased degree of knowledge from both the veterinarian and
mare manager.
-
Risks to handlers at semen
collection.
It is largely due to these
perceived disadvantages and the continued reluctance of some
breed registries--most notably Thoroughbreds--to accept AI that
its use in horses was slow to take off and its development has
not been as rapid as might have been expected. However, despite
this reluctance, many equine breed registries, such as the
United States Trotting Association, American Quarter Horse
Association, and the United States Polo Association, have
embraced AI and the new reproductive technologies available and
encouraged research and development. Because of that, some
exciting advances have been made.
Semen Collection
Little has changed in the ways
of collecting semen; the Missouri, Colorado, or Cambridge
artificial vagina remain the routine collection method. The
open-ended AV is increasingly used to collect separate semen
fractions in problem stallions, such as those with urospermia
(urine is ejaculated in semen) or significant bacterial
contamination, or to obtain high concentrate fractions for
freezing. Experimentally, sperm can now be collected from the
epididymis (a long, narrow, convoluted tube that lies on the
posterior aspect of each testicle, connecting to the vas
deferens). This has potential uses in stallions who have to have
their testes removed or who have died unexpectedly. If sperm are
collected immediately, they can be frozen and used at a later
date.
Semen Evaluation
All semen should be evaluated
prior to use. The traditional parameters assessed in a full
evaluation are still the most popular today: Volume, gross
appearance, motility, morphology, concentration, longevity,
percentage dead, osmolarity, pH, cytology, bacteriology, and
virology (see "What Does That Mean?" on page 37). In commercial
practice, however, a quick assessment of appearance, volume,
concentration, and motility remains the most common evaluation.
Ideally, examination of a
semen sample should allow the fertilizing capacity of that
stallion to be predicted. It would seem probable that parameters
such as sperm progressive motility (forward movement) and sperm
morphology (structure) would be correlated strongly with
fertilizing capacity. Based upon this assumption, these
parameters have traditionally been the ones measured as
indicators of semen quality.
Until relatively recently,
these parameters have been assessed by means of a microscope--an
inexpensive but rather laborious job that often gave variable
results. Today, sophisticated, computerized sperm analysis
systems can automatically record the percentage motility,
patterns of movement, and sperm concentration. Morphometric
analysis systems (to assess sperm morphology) have taken longer
to develop, and their use is not widespread, largely due to
cost. Despite the sophistication of the equipment, it is
increasingly obvious that there is only a poor correlation
between sperm motility and/or gross morphology (structure and
form) and fertilizing capacity of a semen sample and, therefore,
these two parameters are only of limited use in predicting the
potential success of AI.
Thus, the search is on for
other indicators.
The common opinion is that
even if gross morphology is not a good indicator of fertilizing,
the key might lie in functional or microscopic morphology. To
this end, there has been recent research interest in sperm
function tests. Although many of these are only at the
experimental stage and are costly and labor-intensive, they
could hold the key to the future of semen evaluation.
Let's take a look at some of
the most promising.
Biochemical Analysis
Biochemical analysis of semen
might give indirect information on the quality of the sample.
The total number of sperm present within a sample is reflected
in the concentration of certain enzymes found within sperm--for
example, hyaluronidase and acrosin (enzymes present in the sperm
head and essential for fertilization) or adenosine triphosphate
(ATP, an energy source). Their concentration can be used to
indicate sperm integrity, as high levels free within the seminal
plasma or surrounding extender indicate sperm damage.
Similarly lactate, pyruvate,
carnitine, and acetylcarnitine (by-products of sperm activity)
can be used to indicate sperm movement and, therefore,
viability. Additionally, as many of these chemicals are located
in specific parts of the sperm--for example, acrosin and
hyaluronidase in the sperm head--they can indicate the site of
damage.
Based on the same principle,
other biochemical components have also been suggested for use,
but further research is required to determine the normal
concentrations of these components.
Membrane Integrity Tests
An intact sperm membrane is
essential for successful fertilization; a defective membrane
indicates poor sperm viability. Sperm integrity has been
investigated using antibodies to test various components of the
sperm membrane along with fluorescent probes or stains combined
with flow cytometry (more on this in a moment).
In other words, antibodies can
be labeled with fluorescent dyes, and as they attach to specific
areas of the sperm membrane, the dyes outline the integrity of
the membrane. Using this principle, a monoclonal antibody test
(in which antibodies are used to determine the presence of an
organism or substance) for the outer acrosomal membrane has been
developed.
Alternatively, the integrity
of the membrane can be assessed by its permeability to stains
that are normally fluorescent for identification. One example is
carboxyfluorescein diacetate/propidium iodide stain, where
carboxyfluorescein passes through the sperm membrane and is
transformed into the fluorescent green form. Propidium iodide is
not membrane permeable and thus can only enter membranes of
damaged sperm (in which it binds to the DNA and fluoresces red).
Based on these dyes, viable
sperm fluoresce green and dead sperm or those with defective
membranes appear red (see images above left).
Other probes have been
developed, including those that cause specific areas such as the
acrosome (compartment at the tip of a sperm's head) to fluoresce
more brightly than others. Some are specific to certain
structures, such as mitochondria (organelles that are
responsible for a cell's energy production and cellular
respiration), and thus allow the integrity of specific areas to
be evaluated. These techniques are available commercially.
Flow Cytometry
The assessment of stained
sperm in biochemical analysis and sperm integrity tests was
originally carried out by light or electron microscopy, which is
an accurate, but laborious, slow process. However, the advent of
flow cytometry has revolutionized the measurement of stained
sperm and hence sperm evaluation.
Flow cytometry allows
measurements to be made as a series of cells (in this case,
sperm) pass in a fluid stream through a measuring point with an
array of detectors. This allows information on specific aspects
of sperm morphology or concentration to be measured as they pass
through the flow cytometer.
"Gates" can be added that
allow sperm to be sorted (see "Sexing Semen" section on page
37). Recent work has shown flow cytometry to be a rapid and
objective means to assess the functional characteristics of
sperm stained with various structure-specific stains.
Filtration Assay
Various filters can be used to
filter out less viable sperm. This filtration gives a
correlation between the number of sperm passing through to the
filtrate and the fertilizing potential of the original sample.
This method yields a filtrate of highly viable sperm.
Hypo-Osmotic Stress Test
The hypo-osmotic test, now
commercially available for stallion semen, relies upon the fact
that the sperm membrane is semi-permeable, allowing passage of
water through it along an osmotic pressure gradient. If sperm
are placed in a hypotonic solution (high water content), water
passes into the sperm, causing the head to balloon and the tail
to deform by bending and coiling. This only occurs in intact
sperm, hence the extent of sperm deformation indicates
viability.
Function Testing
A number of other tests have
been proposed for evaluating the functional abilities of sperm.
Some of these include the oviductal epithelial cell explant
test, zona-free (having no outer layer, or envelope, of the
ovum) hamster ova penetration assay, and hemizona (the result of
an oocyte being manually sectioned, resulting in two identical
hemizonae) assay. All of these tests measure the ability of
sperm to bind to and activate oviductal epithelial cells (cells
that line the fallopian tube in the mare's reproductive tract),
zona-free hamster oocytes, horse oocytes, and hemizona from
single horse oocytes. By measuring sperm binding, an indication
of sperm viability can be obtained. However, these techniques
are currently only experimental.
Semen Storage
Another key to successful AI
is the perfection of long-term sperm storage. Currently, success
rates with fresh and chilled semen are reasonably comparable
with those for natural service. Recent research has
re-emphasized the vulnerability of sperm to cold shock,
particularly in the range from 20ºC to 5ºC, where the rate of
drop is critical. Several new containers for transporting cooled
semen have been developed over the last 10 years, but studies
have shown containers that maintain precise cooling at a
controlled rate remain the most effective.
It has been appreciated for
some time that seminal plasma has an adverse affect on sperm
motility during cooling, and that its removal and replacement
with an extender enhances sperm motility and fertility rates.
Recent research supports this.
Protocols for the removal of
seminal plasma and replacement with an extender are still being
investigated. However, it is not common practice to replace
seminal plasma with an extender immediately on collection. This
might become future standard practice, especially in stallions
whose semen does not fare well during cooling.
The extenders used are likely
to affect cooling success, so recent research has investigated a
host of extenders. Traditionally, commercially available
extenders for chilled semen are based upon non-fat dried skim
milk, and these are still in common use. Other extenders have
been suggested, such as purified milk fraction (native
phosphocaseinate) plus micellar caseins and beta lactoglobulin
(milk proteins, INRA96), or cream-gel and egg yolk-based
extenders (such as INRA82-Y).
Work has also been done on
storing semen at 15ºC (59ºF) using INRA96 as the extender. The
results looked promising and might open the door to
room-temperature sperm storage in the future.
Although chilling semen is now
relatively successful, poor and variable results are still
obtained with frozen (cryopreserved) semen. This lack of
reliability discourages many from using AI and realizing its
advantages.
Cryopreservation of stallion
semen has been certainly less successful than cryopreservation
of bull semen. However, it must be remembered that cattle are
selected for their reproductive ability; one of these criteria
is the ability of a bull's semen to survive freezing. Hence,
bulls whose semen does not freeze well are selected out of the
population.
Stallions, on the other hand,
are bred for attributes other than semen freezability; thus,
significant variation in the success of cryopreservation of
individual stallion semen remains.
The key to freezing semen is
thought to lie with the cryoprotectant and the freezing
technique, so considerable experimental work has been devoted to
these areas.
Cryoprotectants
Cryoprotectants are required
to prevent damage to sperm during freezing. This damage is
caused by internal ice formation, which alters sperm structure,
causes physical damage, and increases solute (substances other
than water) concentrations as water is withdrawn to form ice in
both the intra- (within) and extra-cellular (outside the cell)
fluid. Any changes in solute concentration as water is withdrawn
to form ice also might result in changes to osmotic pressure,
especially if differences occur in the rate of ice formation in
the extra- and intracellular fluid. This will then cause
dehydration or hydration of the sperm as water moves across the
sperm membrane.
Cryoprotectants can be divided
into two groups depending upon their actions. Penetrating
cryoprotectants are able to penetrate the plasma membrane of the
sperm and act intracellularly as well as extracellularly.
Non-penetrating cryoprotectants cannot penetrate the plasma
membrane and only act extracellularly.
The first cryoprotectant
identified was glycerol, and it remains one of the most favored
cryoprotectants. Glycerol is a penetrating cryoprotectant. Its
presence, both intracellularly and extracellularly, acts to
lower the freezing point of the medium to a temperature much
lower than that of water. This reduces the proportion of the
medium that is frozen at any one time, and hence spreads out the
formation of ice crystals over a temperature range and reduces
sudden changes to solute concentrations and osmotic pressure
differences.
Other penetrating
cryoprotectants include dimethyl sulfoxide, propylene glycol,
dimethyl formamide, and ethylene glycol dimethyl formamide. The
last three show particular promise with stallions whose semen
does not freeze well using conventional extenders.
Non-penetrating
cryoprotectants include many sugars such as lactose, mannose,
raffinose, trehalose, and polyvinylpyrrolidone; some proteins,
such as egg yolk lipoprotein and hysteresis (the lagging of an
effect behind its cause) proteins; and specific amino acids.
These cryoprotectants are thought to act by increasing the
osmotic pressure of the extracellular fluid, which draws water
out from the sperm, thus decreasing the risk of ice crystals
forming and physical damage. However, they do not alleviate, and
might even exacerbate, sperm dehydration.
Further alternative
cryoprotectants have been used, including Orvus ES paste (a mix
of anionic--negatively charged ion--detergents), the synthetic
detergent O.E.P., amino-sodium lauryl sulphate, and liposomes
(phosphatidylserine and cholesterol).
However, regardless of the
cryoprotectant used, freezing success remains variable, and it
is evident that cryoprotectants damage sperm via changes in
osmotic pressure gradients or biochemical disruption.
Additionally, the mitochondria
appear to be affected by freezing more than the acrosome region
(responsible for fertilization). Hence, post-thaw motility rates
are low, resulting in even poorer viability than fresh and
chilled samples.
Recent work indicates that
this detrimental effect of cryoprotectants such as glycerol is
also evident at thawing, where again changes in osmotic pressure
gradients cause water influx into sperm.
However, in the absence of a
successful alternative and despite reports that suggest
glycerol has a greater detrimental effect on stallion sperm than
in any other livestock, glycerol remains the cryoprotectant of
choice in most commercial equine semen freezing. Some reduction
in the detrimental effects of glycerol can be achieved by
altering the inclusion rates and the timing of glycerol
addition.
Ultimately, the protocol for
using cryoprotectants is a compromise between their advantageous
and disadvantageous effects, and might ideally need to vary with
individual stallions. However, such individual tailoring is not
practical in a commercial situation, and further com promise in
the form of reduced fertility rates is accepted.
Freezing Technique
Semen is traditionally frozen
in liquid nitrogen, but some success has been
reported using a unique freezing technique (UFT) adapted from
the high-speed freezing technique used for human feedstuffs. The
UFT has a relatively high freezing rate that appears to vitrify
(to change or make into glass or a glassy substance) water.
Results of studies done with the UFT and stallion spermatozoa
vary, as some studies suggest liquid nitrogen and UFT produce
very similar results.
Filters have also been used to
improve a semen sample pre-thaw. The filtrate obtained has high
fertilizing potential that improves AI fertilization rates
post-thaw.
Sexing Semen
Sexing semen is an exciting
area of growing interest. This technique allows the breeder to
select the desired sex of the offspring. Sperm are stained with
a fluorescent DNA stain, and since X- and Y-chromosome-bearing
sperm (female and male) contain different amounts of DNA, they
can be sorted into separate collecting channels by high-speed
flow cytometry. The success of separation is reported to be more
than 90%, and fertilization rates using sexed semen are
comparable to those of unsorted semen.
Although the process is now
commercially available for horses, only fresh semen is sorted,
meaning that stallions have to be transported to one of the very
few sorting centers. Sorting rates are very slow, with only
5,000 sperm of each sex sorted per second (stallions normally
ejaculate 100 million to 1 billion sperm on average, which means
it would take about 2.5 to 23 days to sort one ejaculate).
Recent research, however, looks promising and suggests that sex
sorting of semen stored at 5ºC or 15ºC for short periods is
possible, and new low-dose insemination techniques now allow the
successful insemination of small doses of sexed semen.
Insemination Techniques
Not only have there been
advances in the evaluation and storage of semen, but also in
insemination techniques. For good semen samples--whether they
are fresh, chilled, or frozen--the standard insemination
technique calls for depositing 500 x 106 (500
million) motile sperm into the uterus using an insemination
pipette. However, semen that does not store well is inherently
poor, and sometimes only a small volume/ number of sperm are
available (such as with sex-sorted semen). New insemination
techniques now allow such samples to be used quite successfully.
During natural service, 100
million to 1 billion sperm are normally deposited into the
mare's uterus, but of these, relatively few (200,000-300,000)
reach the fallopian tube (site of fertilization), and of these
only one is required for fertilization.
If the mare's uterus could be
by-passed, it should be possible to deposit a much smaller
number of sperm at the utero-tubular junction (the junction
between the uterus and the fallopian tube) and achieve good
fertility rates. Indeed, it has been demonstrated that
acceptable pregnancy rates can be achieved by depositing 20-50
million sperm at the utero-tubular junction. This technique is
now used commercially.
The major challenge to
depositing semen at the utero-tubular junction is access without
trauma to the mare's reproductive tract. There are two
techniques: Hysteroscopic AI and deep intrauterine AI. For both
techniques, the mare is prepared as for standard AI, but also
sedated and a similar number of sperm/volume of semen is
deposited at the utero-tubular junction, which is ipsilateral to
(on the same side as) the ovary bearing the large pre-ovulatory
follicle.
Hysteroscopic AI involves
inserting an endoscope (usually a video endoscope) through the
mare's vagina, cervix, and uterus and up to the top of the
appropriate uterine horn. Once in place, the long insemination
catheter is introduced into the endoscopic channel so that it
reaches the utero-tubular junction, where the semen is then
deposited.
Deep intrauterine insemination
is very similar to conventional insemination except it uses a
much longer, flexible-ended insemination catheter that is passed
up through the vagina, cervix, and uterus as per normal AI, but
then pushed higher up to the appropriate utero-tubular junction,
where the catheter is guided per rectum as in rectal palpation.
Once the catheter is in place, the semen is deposited onto the
utero-tubular junction.
Both methods are reported to
have similar success rates, although it might be argued that
greater skill is required with deep intrauterine AI to ensure
the catheter has reached the utero-tubular junction than in
hysteroscopic AI, in which the junction can be visualized via
the endoscope. Deep intrauterine insemination is also less
invasive and might not always require sedation.
The Future
Many developments have already
been mentioned, but researchers are refining other techniques
that might hold the key to future success and use of equine AI.
Freeze-dried sperm--Freeze
drying sperm is now possible; freeze-dried sperm have been
successfully used in intracytoplasmic sperm injection (ICSI;
more on this shortly). In the future, it might prove possible to
freeze dry equine sperm rather than conventional freezing in
bulky containers of liquid nitrogen.
Spermatogonal
transplantation--It is now possible to transfer stem cells
(which can produce sperm) from the testes of some male animals
(goats, mice, and rats) into the testes of others, where they
then produce sperm with the donor's DNA. Although not done yet
in horses, this might have a potential use in transferring
testicular tissue into the testis of sterile stallions.
In vitro
spermatogenesis--Instead of transferring testicular tissue to
another animal, it might prove possible to transfer the tissue
into a culture medium for development and sperm production in
vitro. Mature sperm have not yet been produced by this method,
but development through some of the stages of spermatogenesis
has been achieved.
In vitro fertilization--There
have only been three foals born as a result of in vitro
fertilization, the first one in 1990. Although this would seem
to be a potential area of development, little progress has been
made to date largely due to problems with achieving penetration
of the ovum by viable sperm in vitro.
Intracytoplasmic sperm
injection (ICSI)--The problems encountered in in vitro
fertilization in the horse have led to the development of ICSI
as an alternative technique. ICSI involves the injection of a
single sperm directly into the cytoplasm of the ovum; as such it
avoids the problems of natural penetration. This technique might
prove of particular use in stallions with very low sperm counts.
Gamete intrafallopian tube
transfer (GIFT)--GIFT might be another alternative to in vitro
fertilization. GIFT involves the deposition of 50,000-200,000
sperm plus the ovum into the oviduct of a recipient mare. This
allows fertilization to occur in vivo, which is most successful,
but still requires only a very small number of sperm.
Don't Forget the Mare...
When considering AI,
significant attention is placed upon the stallion and his semen.
However, it must remembered that any AI program is only as good
as the mares inseminated. Although not considered here, the
mare's reproductive competence and stage of cycle are of
paramount importance if successful fertilization is to be
achieved, whether it be by natural service or AI.
Take-Home Message
Although equine breeding stock
is selected for performance and conformation reasons rather than
fertility, several methods exist to gain offspring from those
star performers. Many researchers are focused on achieving
maximum fertility in horses with AI and other means, and their
efforts will give us the ability to gain more foals from less
fertile horses.
Bibliography
Ball, B.A.; and Mohammed, H.O.
(1995) Morphometry of stallion spermatozoa by computer-assisted
image analysis. Theriogenology 44, 3, 367-377.
Batellier, F.; Vidament, M.;
Fauquant, J.; Duchamp, G.; Arnaud, G.; Yvon, J.M.; and
Magistrini, M. (2001) Advances in cooled semen technology.
Animal Reproduction Science 68, 181-190.
Batellier, F.; Gerard, N.;
Courtens, J.L.; Palmer, E.; and Magistrini, M. (2000)
Preservation of stallion sperm quality by phosphocaseinate: A
direct or indirect effect. Journal of Reproduction and
Fertility Supplement. 56, 69-77.
Bhowmick, S.; Zhu, L.;
McGinnis, L.; Lawitts, J.; Nath, B.D.; Toner, M.; and Biggers,
J. (2003) Desiccation tolerance of spermatozoa dried at ambient
temperature: production of fetal mice. Biology of
Reproduction 68, 1779-1786.
Brinsko, S.P.; Rowan, K.R.;
Varner, D.D.; and Blanchard, T.L. (2000a) Effects of transport
container and ambient storage temperature on motion
characteristics of equine spermatozoa. Theriogenology
53, 1641-1655.
Brinsko, S.P.; Crockett, E.C.;
and Squires, E.L. (2000b) Effect of centrifugation and partial
removal of seminal plasma on equine spermatozoal motility after
cooling and storage. Theriogenology 54, 129-136.
Bruemmer, J.E.; Reger, H.P.;
Zinbinski, G.; and Squires, E.L. (2002) Effect of storage at 5
deg C on the motility and cryopreservation of stallion
epididymal spermatozoa. Theriogenology, 58, 405-407.
Carnevale, E.M.; Maclellan,
L.J.; Countinho da Silva, M.A.; Scott, T.J.; and Squires, E.L.
(2000) Comparison of culture and insemination techniques for
equine oocyte transfer. Theriogenology, 54, 981-987.
Casey, P.J.; Hillman, R.B.;
Robertson, K.R.; Yudin, A.I.; Lui, I.K.; and Drobonis, E. (1993)
Validation of an acrosomal stain for equine sperm that
differentiates between living and dead sperm. Journal of
Andrology 14, 282-297.
Castro, T.A.M.; Gastal, E.L.;
Castro, F.G., Jr.; and Augusto, C. (1991). Physical, chemical
and biochemical traits of stallion semen. In: Anais, IX
Congresso Brasileiro de Reproducao Animal, Belo Horizonte,
Brazil, 22 a 26 de Junho de 1991. Vol II. Belo Horizonte,
Brazil; Colegio Brasileiro de Reproducao Animal, pp. 443.
Christanelli, M.J.; Squires,
E.L.; Amann, R.P.; and Pickett, B.W. (1984) Fertility of
stallion semen processed, frozen and thawed by a new procedure.
Theriogenology 22, 1, 39-45.
Colenbrander, B.; Gadella,
B.M.; and Stout, T.A. (2003) The predictive value of semen
analysis in the evaluation of stallion fertility.
Reproduction in Domestic Animals 38, 305-11.
Coutinho da Silva, M.A.;
Carnevale, E.M.; Maclellan, L.J.; Preis, K.A.; Leao, K.M.; and
Squires, E.L. (2002a) Use of fresh, cooled and frozen semen
during gamete intrafallopian transfer in mares.
Theriogenology, 58, 763-766.
Coutinho da Silva, M.A.;
Carnevale, E.M.; Maclellan, L.J.; Seidel, G.E. Jr;. and Squires,
E.L. (2002b) Effect of time of oocyte collection and site of
insemination on oocyte transfer in mares. Animal Science,
80, 1275-1279
Coutinho da Silva, M.A.;
Carnevale, E.M.; Maclellan, L.J.; Preis, K.A.; Seidel, G.E.; and
Squires, E.L (2004) Oocyte transfer in mares with intrauterine
or intraoviductal imsemination using fresh, cooled and forxen
semen. Theriogenology 61, 705-713.
Denniston, D.J.; Graham, J.K.;
Squires, E.L;. and Brinsko, S.P. (1997) The effect of liposomes
composed of phosphatidylserine and cholesterol on fertility
rates using thawed equine spermatozoa. Journal of Equine
Veterinary Science 17, 12, 675-676.
Devireddy, R.V.; Swanlund,
D.J.; Olin, T.; Vincente, W.; Troedsson, M.H.T.; Bischof, J.C.;
and Roberts, K.P. (2002) Cryopreservation of equine sperm:
optinmal cooling rates in the presence and absence of
cryoprotective agents determined using differential scanning
calorimetry. Biology of Reproduction 66, 222-231.
Evenson, D.P.; Sailer, B.L.;
and Josh, L.K. (1995) Relationship between stallion sperm
deoxyribonucleic acid (DNA), susceptibility to denaturation in
situ and presence of DNA strand breaks: implications for
fertility and embryo viability. Biology of Reproduction,
Monograph Equine Reproduction VI, 1, 655-659.
Fiser, P.S.; Hansen, C.;
Underhill, K.L.; and Shrestha, J.N.B. (1991) The effect of
induced ice nucleation (seeding) on the [post thaw motility and
acrospmal integrity of boar spermatozoa. Animal Reproduction
Science 24, 293-304.
Goolsby, H.A., Elanton, Jr.,
J.R. and Prien, S.D. (2004) Preliminary comparisons of a unique
freezing technology to traditional cryopreservation methodology
of equine spermatozoa. Journal of Equine Veterinary Science
24, 314-318.
Graham, J.K. (1996)
Cryopreservation of stallion spermatozoa. Veterinary Clinca
of North America Equine Practioners 12, 131-147.
Gravance, C.G.; Garner, D.L.;
Baumber, J.; and Ball, B.A. (2000) Assessment of equine sperm
mitochondrial function using JC-1. Theriogenology, 53,
1691-703.
Hochi, S.; Korosue, K.; Choi,
Y.H.; and Oguri, N. (1996) In vitro capacitation of stallion
spermatozoa assessed by the lysophosphatidylcholine-induced
acrosome reaction and the penetration rate into in-vitro
matured, zona free mare oocytes. Journal of Equine
Veterinary Science 16, 6, 244-248.
Katila, T. (2001) In vitro
evaluation of frozen-thawed stallion semen: A review. Acta
Vet Scand. 42, 199-217.
Kavak, A.; Johannisson, A.;
Lundeheim, N.; Rodriguez-Martinez, H.; Aidnik, M.; and
Einarsson, S. (2003) Evaluation of cryopreserved stallion semen
from Tori and Estonian breeds using CASA and flow cytometry.
Animal Reproduction Science 76, 205-16.
Kenney, R.M.; Evenson, D.P.;
Garcia, M.C.; and Love, C.C. (1995) Relationships between sperm
chromatin structure, motility and morphology of ejaculated sperm
and seasonal pregnancy rate. Biology of Reproduction
Monograph Equine Reproduction VI, 1, 647-653.
Keskintepe, L.; Pacholczyk,
G.; Machnicka, A.; Norris, K.; Curuk, M.A.; Khan, I.; and
Brackett, B.G. (2002) Bovine blastocyst development from oocytes
injected with freeze-dried spermatozoa. Biology of
Reproduction 67, 409-415.
Klinc, P.; Kosec, M.; and
Majdic, G. (2005) Freezability of equine semen after glass beads
column separation. Equine Veterinary Journal 37, 43-47.
Lindsey, A.C.; Bruemmer, J.E.;
and Squires, E.L. (2001) Low dose insemination of mares using
non-sorted and sex-sorted sperm. Animal
Reproduction Science, 68, 279-89.
Lindsey, A.C.; Schenk, J.L.;
Graham, J.K.; Bruemmer, J.E.; and Squires, E.L. (2002)
Hysteroscopic insemination of low numbers of flow sorted fresh
and frozen/thawed stallion spermatozoa. Equine Veterinary
Journal 34, 121-7.
Lindsey, A.C.; Varner, D.D.;
Seidel, G.E. Jr.; Bruemmer, J.E.; and Squires, E.L. (2005)
Hysteroscopic or rectally guided, deep-uterine insemination of
mares with spermatozoa stored 18 h at either 5 degrees C or 15
degrees C prior to flow-cytometric sorting. Animal
Reproduction Science, 85,125-30.
Loomis, P.R. (2001) The equine
frozen semen industry. Animal Reproduction Science, 68,
191-200.
Magistrini, M.; Guitton, E.;
Levern,Y.; Nicolle, J.C.; Vidament, M.; Kerboeuf, D.; and
Palmer, E. (1997) New staining methods for sperm evaluation
estimated by microscopy and flow cytometry. Theriogenology
48, 1229-1235.
McKinnon, A.O.; Lackey, B.R.;
and Trounson, A.O. (2000) Pregnancies produced from fertile and
infertile stallions by intracytoplasmic sperm injection (ICSA)
of single frozen/thawed spermatozoa into in-ivtro matured mare
oocytes. Journal of Reproduction and Fertility, Supplement,
56, 513-517.
Merkies, K.; Chenier, T.;
Plante, C.; and Buhr, M.M. (2000) Assessment of stallion
spermatozoa viability by flow cytometry and light microscope
analysis. Theriogenology. 54, 1215-24.
Meyers, S.A.; Liu, I.K.M.;
Overstreet, J.W.; Vadas, S.; and Drobnis, E.Z. (1996) Zona
pellucida binding and zona induced acrosome reactions in horse
spermatozoa: Comparisons between fertile and sub fertile
stallions. Theriogenology 46, 7, 1277-1288.
Montovani, R.; Rota, A.;
Falmo, M.E.; Baloni, L.; and Vincenti, L. (2002) Comparison
between glycerol and ethylene glycol for the cryopreservation of
equine spermatozoa: semen quality assessment with standard
analyses and with hypoosmotic swelling test. Reproduction
Nutrition and Development 42, 217-226.
Moffett, P.D.; Bruemmer, J.E.;
Card, C.E.; and Squires, E.L. (2003) Comparison of Dimethyl
formaamide and glycerol for cryopreservation of equine
spermatozoa. Proceedings of the Annual Meeting of the
Society for Theriogenology 42.
Morris, L.H.; Tiplady, C.; and
Allen, W.R. (2003) Pregnancy rates in mares after a single fixed
time hysteroscopic insemination of low numbers of frozen-thawed
spermatozoa onto the uterotubal junction. Equine Veterinary
Journal, 35, 197-201.
Morris, L.H. (2004) Low dose
insemination in the mare: An update. Animal Reproduction
Science, 82-83, 625-32.
Neild, D.M.; Chaves, M.G.; Flores, M.; Miragaya, M.H.; Gonzalez,
E.; and Aguero, A. (2000) The HOS test and its relationship to
fertility in the stallion. Andrologia 32, 351-5.
Nie, G.J.l and Wenzel, J.G.
(2001) Adaptation of the hypoosmotic swelling test to assess
functional integrity of stallion spermatozoal plasma membranes.
Theriogenology. 55, 1005-18.
Nie, G.J.; Wenzel, J.G.; and
Johnson, K.E. (2002) Comparison of pregnancy outcome in mares
among methods used to evaluate and select spermatozoa for
insemination. Animal Reproduction Science 69, 211-22.
Nie, G.J.; Johnson, K.E.; and
Wenzel J.G. (2003) Pregnancy outcome in mares following
insemination deep in the uterine horn with low numbers of sperm
selected by glass wool/Sephadex filtration, Percoll separation
or absolute number. Animal Reproduction Science, 79,
103-9.
Padilla, A.W.; and Foote, R.H.
(1991) Extender and centrifugation effects on the motility
patterns of slow cooled spermatozoa. Journal of Animal
Science 60, 3308-3313.
Palmer, E.; Bezard, J.;
Magistrini, M.; and Duchamp, G. (1991) In vitro fertilisation in
the horse: A retrospective study. Journal of Reproduction
and Fertility, Supplement, 44, 375-384.
Pantke, P.; Hyland, J.H.;
Galloway, D.B.; Liu, D.Y.; and Baker, H.W.G. (1995) Development
of a zona pellucida sperm binding assay for the assessment of
stallion fertility. Biology of Reproduction Monograph Equine
Reproduction VI 1, 681-687
Papaioannou, K.Z.; Murphy,
R.P.; Monks, R.S.; Hynes, N.; Ryan, M.P.; Boland,M.P.; and
Roche, J.F. (1997) Assessment of viability and mitochondrial
function of equine spermatozoa using double staining and flow
cytometry. Theriogenology 48, 299-312.
Parks, J.E.; Leee, D.R.;
Huang, S.; and Kaproth, M.T. (2003) Prospects for
spermatogenesis in vitro. Theriogenology 59, 73-86.
Polge, C.; Smith, A.U.; and
Parkes, A.S. (1949) Revival of spermatozoa after vitrification
and dehydration at low temperature. Nature 164, 666.
Rigby, S.L.; Brinsko, S.P.;
Cochran, M.; Blanchard, T.L.; Love, C.C.; and Varner, D.D.
(2001). Advances in cooled semen technologies: seminal plasma
and semen extender. Animal Reproduction Science 68,
171-80.
Rota, A.; Furzi, C.; Panzani,
D.; and Camillo, F. (2004) Studies on motility and fertility of
cooled stallion spermatozoa. Reproduction in Domestic
Animals 39, 103-9.
Samper, J.C. (1995) Stallion
semen cryopreservation: Male factors affecting pregnancy rates.
Proceedings of the Society for Theriogenology, San
Antonio, Texas, pp. 160-165.
Samper, J.C. (2001) Management
and fertility of mares bred with frozen semen. Animal
Reproduction Science 68, 219-28.
Samper, J.C.; Vidament, M.;
Katila, T.; Newcombe, J.; Estrada, A.; and Sargeant, J. (2002)
Analysis of some factors associated with pregnancy rates of
frozen semen: A multi-centre study. Theriogenology, 58,
647-650.
Schulman, M.L.; Gerber, D.;
Nurton, J.; Guthrie, A.J.; Jourbert, K.; and Vilkmann, D.H.
(2003) Effects of halothane anaesthesia on the cryopreservation
of epididymal spermatozoa in pony stallions. Equine
Veterinary Journal, 35, 93-95.
Seidel, G.E. Jr.; and Garner,
D.L. (2002) Current status of sexing mammalian spermatozoa.
Reproduction, 124, 733-43.
Sieme, H.; Martinsson, G.;
Rauterberg, H.; Walter, K.; Aurich, C.; Petzoldt, R.; and Klug,
E. (2003) Application of techniques for sperm selection in fresh
and frozen-thawed stallion semen. Reproduction in Domestic
Animals 38, 134-40.
Sieme, H.; Troedsson, M.H.;
Weinrich, S.; and Klug, E. (2004) Influence of exogenous GnRH on
sexual behavior and frozen/thawed semen viability in stallions
during the non-breeding season. Theriogenology, 61,
159-71
Sieme, H.; Katila, T.; and
Klug, E. (2004).Effect of semen collection practices on sperm
characteristics before and after storage and on fertility of
stallions. Theriogenology, 61, 769-84.
Sieme, H.; Bonk, A.; Hamann,
H.; Klug, E.; and Katila, T. (2004) Effects of different
artificial insemination techniques and sperm doses on fertility
of normal mares and mares with abnormal reproductive history.
Theriogenology 62, 915-28.
Squires, E.L.; Keith, S.L.;
and Graham, J.K. (2004) Evaluation of alternative
cryoprotectants for preserving stallion spermatozoa.
Theriogenology 62, 1056-65.
Stradaioli, G.; Sylla, L.;
Zelli, R.; Verini Supplizi, A.; Chiodi, P.; Arduini, A.; and
Monaci, M. (2000) Seminal carnitine and acetylcarnitine content
and carnitine acetyltransferase activity in young Maremmano
stallions. Animal Reproduction Science 64, 233-45.
Thomas, P.G.A.; and Ball, B.A.
(1996) Cytofluorescent assay to quantify adhesion of equine
spermatozoa to oviduct epithelial cells in vitro. Molecular
Reproduction and Development 43, 1, 55-61.
Trimeche, A.; Yvon, J.M.;
Vidament, M.; Palmer, E.; and Magistrini, M. (1999) Effect of
glutamine, praline, histidine, and betaine on post thaw motility
of stallion spermatozoa. Theriogenology 52, 181-191.
Vidament, M.; Daire, C.; Yvon,
J.M.; Doligez, P.; Bruneau, B.; Magistrini, N.; Ecot, P. (2002)
Motility and fertilisation of stallion semen with frozen
glycerol and/or dimethyl formamide. Theriogenology 58,
249-251.
Watson, P.F.; and Duncan, A.G.
(1988) Effect of salt concentration and unfrozen water fraction
on the viability of slowly frozen ram spermatozoa.
Cryobiology 25, 131-142.
Wilhelm, K.M.; Graham, J.K.;
and Squires, E.L. (1996) Comparison of the fertility of
cryopreserved stallion spermatozoa with sperm motion analyses,
flow cytometry evaluation and zona-free hamster oocyte
penetration. Theriogenology 46, 4, 559-578.
"What Does This Mean?"
-
Motility--Moving or having the power to move spontaneously.
-
Morphology--Form and structure of an organism.
-
Osmolarity--Of or pertaining to osmosis (diffusion of fluid through a
semi-permeable membrane from a solution with a low solute
concentration to a solution with a higher solute
concentration until there is an equal concentration of fluid
on both sides) and the concentration of a solution expressed
as osmoles of solute per liter of solution.
-
Cytology--The branch of biology that deals with the formation, structure,
and function of cells.
-
Bacteriology--The study of bacteria, especially in relation to medicine and
agriculture.
-
Virology--The study of viruses and viral diseases.
