Breeding Globally--AI Advances
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.
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.
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 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.
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.
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.
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.
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 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.
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 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.
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.
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.
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.
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"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.
About the Author
Mina Davies Morel, PhD, is head of the equine group at the Institute of Biological, Environmental and Rural Sciences at Aberystwyth University in the United Kingdom. She has particular interest in equine reproductive physiology and its application to stud management, and she is the author of a number of scientific papers and text books on the subject. She is a leisure rider and owner of Welsh Cob Section Ds.