Why is semen cooled?
Fresh semen stored at 370C deteriorates rapidly. Most raw ejaculates will have no motile sperm after 3-4 hours at 370C and 5-6 hours at room temperature (20-240C ). Cooling stallion sperm to 50C induces a transition in sperm membranes from the liquid crystalline to the gel state. Since damage can arise from these changes why would we want to cool semen ? At body temperature spermatozoal metabolism is maximal and at room temperature it is much lower. Waste products (lactic acid and or CO2) can increase the acidity of semen causing permanent cellular damage; peroxidation of membrane lipids occurs resulting in membrane damage and cells in general wear out. When the temperature of cells is reduced, for each 100C reduction, cellular metabolism is reduced by 50%. Therefore if spermatozoa are stored at 50C their metabolic needs are only about 10% of what they would have been at 370C (body temperature). Consequently, spermatozoa stored at 50C do not produce as many waste products, lipid peroxidation occurs more slowly and the cells do not wear out as quickly. All of these tend to lengthen the life of fertile spermatozoa compared to those stored at body temperature or room temperature. Cooling however stresses spermatozoa and often causes cellular damage. Cellular injury can be caused by directly affecting cellular structure (such as rupturing membranes) or indirectly by altering cellular functions (such as slowing down metabolic processes). Rapid cooling of stallion spermatozoa from room temperature to 50C induces partially irreversible damage characterised by an abnormal pattern of swimming (circular or backwards), rapid loss of motility, acrosome damage, plasma membrane damage, reduced metabolism and loss of intracellular components. Collectively this damage is referred to as "cold shock". Indirect cooling damage is more difficult to discern and may not be evident for some time (sometime hours) after the spermatozoa have reached 50C or after they are re-warmed to 370C. Some of the damage occurring to spermatozoa during cooling and cold shock is due to changes that occur to the plasma membrane in the transition from the liquid-crystalline to the gel state. This damage can be minimised by including additives to the extender and also by careful slow cooling through the temperature zone where the transition from the fluid phase to the solid phase occurs. Egg yolk is a common lipid additive to stallions spermatozoal extenders. The protective action is provided by the low-density lipoproteins in egg yolk, in particular phospholipid (stabilise spermatozoal membranes). Extenders containing skim milk have also been used to protect spermatozoa from cold shock. Little research has been conducted in investigating the mechanism by which milk protects spermatozoa but it is likely that lipoproteins in milk act similarly to those in egg yolk. It appears that the critical temperature range for induction of cold shock is rapid cooling between 200C and 50C. Semen should be cooled slowly in this range.
Currently several cooling devises are available which use either a passive cooling system (semen is cooled by passive heat transfer to a coolant) or an active cooling system (electricity is used to regulate cooling similar to a small refrigerator) to cool semen from about 200c to 50c in a controlled manner. Passive cooling systems (ie., the Equitainer) have limitations as larger volumes will cool more slowly than smaller volumes and cooling rate is affected by the initial temperature of the spermatozoal suspension. In addition these systems cool the semen in a curvilinear fashion with the semen cooling at the greatest rate initially and progressing to slower rates as the spermatozoa near the final temperature (Figure 1). Despite this, these systems work very well, are relatively inexpensive and are commercially available. An average cooling rate of -0.080C between 200C and 120C with this system was reported (Douglas-Hamilton et al., 1984) as being appropriate for stallion spermatozoa.
Figure 1. The interaction of temperature versus time with a passive cooling system.
Active cooling systems (548 MOD-X Semen Cooler. Animal Reproduction Systems Chino California) have permitted investigations on the effects of cooling rate utilising programmed linear cooling rates over defined temperature ranges. Such experiments revealed that stallion spermatozoa, initially diluted in a skim milk extender, can be cooled rapidly (-0.70C/min) from 370C to 190C without any deleterious effects on % of motile spermatozoa. Between 190C and 80C, however the cooling rate had to be reduced to 0.050C/min to prevent loss of motile sperm. After spermatozoa had reached 80C rapid cooling could be resumed (Figure 2).
Figure 2. The interaction of temperature versus time with an active cooling system.
Type of extender
When semen is collected for utilisation on the farm with AI, in many cases it is not essential, although it is often desirable, to place the semen in an extender. To allow spermatozoa to withstand the rigours of cooling below room temperature and to survive any length of time at all, semen must be mixed with a seminal extender. An extender should provide;
1) an osmotic pressure compatible with spermatozoa,
2) a proper balance of ions and mineral elements,
3) a proper balance of electrolytes and non-electrolytes,
4) a proper combination of nutrients,
5) chemicals for neutralising the toxic by-products of spermatozoa,
6) ingredients to minimise the damage to spermatozoa for cooling beneath 200C,
7) ingredients to stabilise enzyme systems and integrity of spermatozoal membranes and 8) a carrier free of infectious organisms.
An extender must maintain the quality of spermatozoa for longer when compared with survival in the raw state (suspended in seminal plasma). The following advantages are associated with extending stallion semen;
1) to permit effective antibiotic or antibacterial treatment of semen contain either pathogenic or potentially pathogenic organisms,
2) to prolong the life of spermatozoa,
3) enhance the viability from some low fertility stallions,
4) to protect the spermatozoa from unfavourable environmental conditions,
5) increase the volume of the inseminate, and
6) to aid in proper evaluation of spermatozoal motility.
Numerous seminal extenders (Table 1) have been used with semen intended for storage at either 200C or 50C and most have been based on milk by-products plus glucose, egg yolk plus a buffer or cows milk. Results reported in most studies should be viewed with caution as many studies have used progressive motility (PMS) as a predictor of fertility rather than pregnancy rate per cycle. Even studies that include fertility data should be examined with caution because frequently there are no controls or inadequate experimental animals. The costs of breeding a sufficiently large number of mares to truly determine an individual stallions fertility are prohibitive. The confidence interval for any fertility is extremely variable, particularly if less than 50 services are involved (Table 2, Figure 3 and 4). Despite these limitations it is evident that extenders based on a combination of non-fat dried milk solids (powdered milk) and glucose and either identical to or derived from the formula provided by Kenny in 1975 (Table 1)(Kenney et al., 1975) are useful in prolonging fertile life of stallion spermatozoa after cooling slowly to 50C. Currently it is apparent that some stallions tolerate cooling quite well, while others, although having normal fertility with fresh semen, have quite poor fertility with cooled semen.
1) Non Fat Dry Skim Milk (NFDSM)- Glucose Extender (Adapted from:(Kenney et al., 1975))
Sterile De-ionised Water qs 100 ml
Antibiotics per 100 ml (select one or two)
a) Penicillin, crystalline 150,000 IU
b) Streptomycin, crystalline 150,000 m g
c) Gentamicin Sulphate 100,000 m g
8.4% NaHCO3 2 ml
(Mix all liquids before adding NFDSM)
d) Polymixin B Sulphate 100,000 IU
2) Cream-Gel Extender (Adapted from (Voss and Pickett 1976))
1.3 g Knox gelatin
10 ml deionised water
90 ml half-and-half cream
a) Add gelatin to deionised water
b) Autoclave gelatin and deionised water for 20 min.
c) Heat half-and-half in double boiler for 10 min at 920C (not > 950C)
d) Remove scum from surface
e) Add half-and-half to gelatin (total 100 ml)
3) Skim Milk Extender Adapted from (Voss and Pickett 1976)
100 ml of Skim Milk (non-fortified)
Heat Skim Milk in a double boiler for 10 min at 920C (not > 950C)
4) Skim Milk Gel Extender Adapted from (Voss and Pickett 1976)
1.3 g Knox gelatin
100 ml of Skim Milk (non-fortified)
a) Add Skim Milk and agitate for 1 min.
b) Heat Skim Milk in a double boiler for 10 min at 920C (not > 950C). Swirl mixture periodically during the process.
TABLE 2. SELECTED 95% CONFIDENCE INTERVALS FOR A THEORETICAL BINOMIAL SUCH AS PREGNANCY
TRUE FERTILITY (Pregnancy/cycle %)
* Implies that if 10 mares are bred for one cycle the fertility may be represented by results of anywhere from 0 to 4 mares pregnant with a true fertility of 15% per cycle. Adapted from (Pickett et al., 1987)
Figure 3. Demonstrating the large possible variation in outcomes (95% confidence limits) when breeding small numbers of mares when the true fertility is 15% per cycle.
Figure 4. Demonstrating the large possible variation in outcomes (95% confidence limits) when breeding small numbers of mares when the true fertility is 50% per cycle.
Volume of extender
The amount of extender necessary in relation to the volume of semen to be cooled will vary with the composition of the extender and the concentration of spermatozoa. Although there are many extenders available, only one(Kenney et al., 1975) has been thoroughly investigated for its ability to maintain spermatozoal motility at different dilution rates after cooling and prolonged storage. Varner et al (Varner et al., 1988) reported on the effect of different dilution rates on spermatozoal motility after cooling and storage (Figure 5).
Figure 5. Effect of spermatozoal dilution with extender on % PMS after storage.
These workers concluded that the greater the dilution rate the better the maintenance of spermatozoal motility and that an ideal dilution rate was one that resulted in 25X106 spermatozoa per ml. In order to study the effects of seminal plasma on spermatozoal motility for stored samples, workers at Colorado State University (CSU) (Jasko et al., 1992b) diluted semen at various levels (Figure 6) whilst maintaining the same concentration of spermatozoa per ml (initial semen concentrations were varied). Their results suggested higher dilution rates (>1:3) promote the maintenance of PMS during cooling and storage (Jasko et al., 1992b). From this experiment it appeared that seminal plasma must be diluted. However a small amount of seminal plasma may be essential for the maintenance of spermatozoal motility during storage of semen that has been cooled. An additional experiment (Jasko et al., 1992a) was performed wherein spermatozoa were resuspended in extender to which either 0, 5, 10 or 20% of seminal plasma had been added. Samples were cooled and maintained at 50C for 72 hours. The retention of 5-20% of seminal plasma in extended semen was better when compared to the extended semen with no seminal plasma. Practically speaking, this can be achieved without centrifugation by the dilution of semen in the range or 1:4 to 1:20, however the higher dilution rates (1:20) are probably detrimental to fertility of long term stored spermatozoa as they result in excessive dilution of seminal plasma and may effect spermatozoal concentration.
Figure 6. The effect of ratio of extender dilution to % PMS maintaining the same concentration per ml.
The next question that needed to be answered was what effect a large volume of extender would have with cooled transported semen. An initial study at CSU (Squires et al., 1989) demonstrated that using 250 million PMS plus extender to make a volume of 10 ml resulted in an embryo recovery rate (pregnancy rate per cycle) of 70.6% which was significantly different (p<0.001) than the same number of spermatozoa diluted in extender to a volume of 100 ml (13.6%). Clearly, although the numbers were adequate, the dilution due to large volume has effected the ability of spermatozoa to effect fertilisation (perhaps mares are unable to achieve adequate numbers in the oviduct). A further study diluting 250 million PMS to a total volume of 50 ml (5 million per ml) compared to 250 million PMS in 10 ml (25 million per ml) still resulted in a significant difference in pregnancy rate of 34.3% compared to 65.7% respectively (P<0.05). The conclusion for this experiment can be that a) when breeding with 250 million PMS total volume should not exceed 10 ml (sperm concentration 25 X 106 PMS) (Figure 7). An additional experiment (Jasko et al., 1992b) demonstrated that breeding with 1250 X 106 in 50 ml resulted in identical embryo recovery as breeding with 250 X 106 in 10 ml.
Figure 7. Effect of extender volume while maintaining the same sperm numbers (250 million PMS) on embryo recovery.
From the above information it is apparent that we should be aiming to breed with an extended semen concentration of approximately of 25 million sperm per ml and a raw sperm dilution of at least 1 in 4 (raw semen to extender). If a stallion ejaculates a large volume of dilute semen (for example 200 mls with a concentration of 15-25 X 106) it may be difficult to ensure transport of sufficient numbers of spermatozoa (properly extended) without providing an excessively large volume. One method to circumvent this problem is to centrifuge and resuspend the semen. However caution is advised as centrifugation has been demonstrated to effect the fertility of stallion semen under some conditions (Pickett and Amann 1993). Processed semen resulted in an average of 67% embryo recovery of fresh. With the information above in mind, it is important not to excessively tease stallions that are to be used for semen cooling and storage, as teasing will increase the volume of the ejaculate dramatically in most stallions(Pickett et al., 1987). Most clients will be surprised to learn that volume does not equate with the quality of the sample and that sperm numbers ejaculated are relatively consistent not volume.
Timing and frequency of insemination
Even the best techniques for depositing the correct number of live spermatozoa in the right place are useless if the time of insemination is wrong. Ideally mares should be inseminated as close as possible (prior) to ovulation. Pregnancy rates drop after ovulation and fertilisation that occurred from semen deposited after ovulation resulted in an increase in early embryonic death (Woods et al., 1990). Although to is tempting to speculate on the life of the ovum, current recommendations are to breed with fresh semen at a maximum of 6 hours after ovulation (Woods et al., 1990; Pickett and Amann 1993).
Figure 8. Effect of teasing on sperm parameters.
No data are available for use of cooled semen relative to ovulation. Similarly no data are available as to seminal longevity in-vivo after semen has been cooled and transported, however the optimum time for insemination of cooled spermatozoa may include a shorter interval (closer to ovulation) than if fresh semen or natural service is used. In summary insemination with the use of cooled transported semen under ideal conditions would include the insemination of mares zero to 24 h prior to ovulation such as the schedule for natural breeding programs.
Number of spermatozoa
Presented in figure 9 are pregnancy rates from non-cooled spermatozoa in raw and extended semen based on pooled data over many experiments conducted at CSU. First cycle pregnancy rates were 22.2%, 40.6% and 54.5% for mares bred with 50, 100 or 500 million PMS per insemination, respectively. As a result of these experiments it is now recommended that for most stallions it is best to breed mares with 500 X 106 PMS. Occasionally a particularly fertile stallion will still have good pregnancy rates when sperm numbers per insemination are allowed to drop down to 100 X 106 PMS. It has been estimated that approximately 50% of spermatozoa are lost in the process of cooling and transporting semen (will be non-viable at the time of insemination). Accordingly our aim is to start with one billion PMS per insemination dose in a cooled transported semen program.
Figure 9. Effect of insemination of various numbers of progressively motile spermatozoa on pregnancy rate per cycle.
Clearly this halves the numbers of mares that can be bred compared to normal AI with fresh semen. The following formula is used to calculate the volume of raw semen to be placed in an extender for shipment:
1 X 109 PMS
(conc X106/ml) X % motility
If we assume that a stallion ejaculates 160 X 106/ml of gel free semen and that the progressive motility is 60% then the formula is:
1 X 109
160 X 106 X 0.6
=10.4 mls of raw semen.
A 1:3 dilution in extender will give » 25X 106/ml in a 40 ml volume.
Concern has been expressed by some breeders that approval of transported semen will result in too many mares being bred to too few stallions. This is not likely to occur due to the high number of progressively motile spermatozoa required with cooled transported semen. Presented in Figure 10 are the expected numbers of mares that could be bred from varying total sperm counts (5 to 14 billion) and varying PMS (40-80%).
Fertility of cooled semen
From a series of experiments conducted at CSU it was concluded that there was an interaction between storage temperatures and extenders (Pickett et al., 1987; Francl et al., 1987; Jasko et al., 1992c). The results of these studies demonstrated clearly that as storage time increased, there was a trend towards reduction in fertility, particularly when semen was stored at 200C for longer than 12 hours (Figure 11). Therefore current recommendations are that if semen is to be transported, it should be cooled to 50C and mixed in a non-fat dry skim milk extender (NFDSM) extender (Table 1). Short term storage (6-12 h) is possible by cooing to room temperature.
Figure 10. Number of mares calculated for breeding (1 X 109) varying PMS and total sperm in the ejaculate.
Figure 11. Fertility of cooled semen stored at varying temperatures for varying lengths of time.
Semen can conveniently be handled in 'Whirl-pak' bags (6 oz.) and the appropriate cooling rate to 40C can be obtained by the use of an 'Equitainer' for most stallions. The Equitainer utilises frozen canisters (-300C), insulation, thermal ballast bags and an isothermalizer. The procedures to follow are to add the frozen canisters to control the cooling rate, extend the semen in the Whirl-pak bags and surround the semen with thermal ballast bags inside the isothermalizer container. The ballast bags should be placed against the semen at 370C, similar to the temperature of the sample. It is best for the stallion owner or veterinarian to save a small sample that will be cooled similarly for future analysis in case of problems with motility at the time of mare insemination. The Equitainer is relatively rugged and designed to handle the stresses of transport.
Another technique to cool equine spermatozoa is to place the Whirl-pak bags or other semen/extender containing canister into a beaker/container of water at 370C which is placed inside a refrigerator. However, it is important to do trial runs with a thermocouple to measure the temperature decline over time. Once the semen has reached 40C it can be packaged with cooled ballast bags and sent to a remote location.
Note: Semen handled this way and placed in Styrofoam containers can easily be damaged by heat stresses such as being placed in the cargo areas on buses that occasionally are extremely warm.
Transport of Semen.
The time between collection and insemination of the mare has an important implication on pregnancy rates. Even for semen stored at 40C, mares inseminated < 24 hours have a significantly higher pregnancy rate than those inseminated > 48 hours after the collection of semen. Close co-ordination of stallion owners and mare owners should identify the most expedient route for semen transport.
Normal artificial insemination procedures are employed. Each Whirl-pak bag containing one insemination dose for each mare is inseminated immediately it arrives whilst it is still cool. Evaluation of semen may then be performed later (from the small sample left over) using a pre-warmed 370C slide and microscope stage warmer. Chilled semen may require 5 minutes of warming to 370C before demonstrating its real motility. No attempt should be made to warm the semen prior to insemination of the mare.
Reasons for Failure.
Management is the most important key for successful insemination of mares with cooled, transported semen. There are many reasons for failure of this technique. Some of these are:
1. Early in the breeding season, it is not uncommon for mares to either not ovulate or ovulate later than expected.
2. Some mares have genital tract infections that may not be identified.
3. Inadequate teasing may have failed to identify mares that are in heat or may result in mares being bred at the wrong stage of the reproductive cycle.
4. Inadequate fertility data from testing of the stallion prior to beginning the program.
Recommendations for Mare Owners.
1. A pre-breeding reproductive examination to identify mares that are of normal fertility. Avoid using old barren mares.
2. Careful monitoring of the mare's oestrus cycle using a teaser stallion and then veterinary examination to determine the appropriate time of breeding.
3. Early contact of the stallion owner to co-ordinate breeding plans.
4. Examination of the mare frequently during oestrus to give the stallion owner at least 24 hours (preferably 48 hours) advanced warning prior to the desired time of breeding.
5. Aim at breeding once per cycle by careful use of veterinary management.
6. Veterinary use of reproductive hormones to ensure ovulation occurs at the expected time.
7. Examination of mares for pregnancy as early as possible using ultrasonography 14-16 days after breeding.
8. Inform the stallion owner of the results of the breeding and keep them well informed of all transported semen quality.
Recommendations for Stallion Owners.
1. Have a veterinarian perform a thorough evaluation prior to the start of the breeding season which includes the response of that stallion's spermatozoa to cooling to 4-5oC for 24 and 48 hours after the semen is extended. Test for the appropriate dilution ratio and any interaction with extenders.
2. Organise to have bacterial cultures from the stallion's semen, urethra and prepuce submitted to a laboratory to identify if any potential pathogens are present. Once identified, antibiotic sensitivity tests can be used to determine if special antibiotics need to be added to the extender.
3. Keep equipment clean and sterile. Use only proven techniques for packaging semen.
4. Keep the stallion on a regular collection schedule. Most stallions should be collected every other day. Failure to collect a stallion for 4-5 days may result in stored semen being harvested that is of inferior quality.
5. Keep accurate records of semen volume, concentration and motility and any changes that occur in procedures, extenders and antibiotics that may effect sperm quality.
6. Keep a control sample for each batch of transported semen and notify mare owners if the sample appears to be poor. Transport only excellent quality semen.
7. Try to avoid teasing with the stallion to be collected unless it is necessary.
8. Include with each shipment instructions for owners (motility, concentration, dilution rate, type of extender, antibiotics used, etc.). Always transport 1 X 109 PMS.
9. Use the fastest express service to have the semen arrive to the mare.
10. Take an active role in educating mare owners by providing instructions, newsletters, reference manuals, etc.
11. Keep records of all mares bred to that stallion.
12. Insist on early pregnancy detection in order to quickly and accurately determine the success of each stallion after transport of his semen.
13. Be prepared for frustrating client communications and remain flexible.
14. Transport semen only to those clients that have proper management skills, personnel and equipment necessary to ensure ultimate success.
Recommendations for Veterinarians.
1. Before embarking on breeding with cooled semen, carefully explain to clients the difficulties associated with collection and transport, and accurately impart the expected results (ie. 60-70% of mares pregnant per cycle). Because clients are emotionally and physically involved with the palpation, scheduling and breeding with transported semen, they often expect the success to be higher. It is important for all parties to recognise that results with this technique will not improve fertility.
2. Make sure the client understands the costs and the procedures. Although clients request breeding with transported semen because of poor stallion availability, many of them expect costs to be decreased because of no mare transport and expect less injuries to the mare and or foal and are frustrated if the mare is not pregnant and there is a large veterinary bill. Bills from the stallion owner and veterinarian involved with collection and transporting of the semen are often higher than expected ($100-300/collection).
3. Semen collection and processing: Immediately after collection, measure the progressive motility (extended) and concentration of the sample and calculate the amount of fresh semen required to breed with 1 billion progressively motile spermatozoa. Add this to an appropriate extender (at 37oC) and organise to have a breeding dose of between 30-60 ml, while not having a ratio of semen to extender less than 1:3 and » 25 X 106 PMS/ml. On occasion it may be necessary to increase the volume of the inseminate rather than trying to centrifuge low concentration spermatozoa. It is recommended that semen transported long distances for >6 hours should be cooled in an Equitainer. There are many seminal extenders available and some veterinarians mix their own. We recommend the use of commercially prepared extenders to provide consistent quality. A convenient equine extender has been prepared by Animal Reproduction Systems (Chino, California, Fax - 909 597 3043) Three formulas are available:
a) Original Formula (which contains Polymixin B Sulphate)
b) CST (cooled and stored semen extender containing Amikacin))
c) Basic (has no antibiotics)
These extenders are purchased in two components. The nutrient component is in a sachet which is added to purified, distilled, de-ionised water (the other component).
4. Examination of the mare: In order to avoid disappointment, barren mares should be subjected to a full reproductive evaluation including uterine culture, cytology and biopsy. Mares should be examined ultrasonographically as soon as possible after they have been identified to be in oestrus. We recommend the use of ovulation induction with "Ovuplant" (McKinnon et al., 1993), or hCG (3000 IU i.v.)(Voss 1993) once a 30-35 mm follicle has been detected in a mare displaying oestrus, but not until it is confirmed that the semen can be collected and transported to arrive at a pre-determined time prior to ovulation. Ovulation will be induced approximately 36 hours after mares are administered hCG. Mares should always be examined 24-48h post-breeding to determine if they need intra-uterine or systemic antibiotics and to perform post- breeding reproductive procedures such as a Caslick. Mares should examined for pregnancy detection, 14-16 days after the expected ovulation time. Good management and communication are the key to success of cooled, transported semen.
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