Part 3: The ability to recognize that changes are necessary and to accept those changes are essential for any competent breeding manager. Those individuals that have had 25 years experience, one year at a time, cannot compete with those individuals that have had 25 years experience. Managerial techniques that are highly successful on one farm may not be successful on another, particularly with a different population of stallions.
Factors Affecting Bacteria in Semen: 1.) There is extreme variability in microbial content in raw semen among stallions. It is generally assumed that bacterial contamination of semen from normal stallions originates mainly from crypts on the penis and folds of the prepuce. What this means, is that the stallion's penis should be very erect for washing to be effective in removing bacteria. Also, when some stallions lie down and relax their penis comes out of the sheath (prepuce). When the penis is withdrawn, material in the bedding is drawn into the prepuce where the bacteria multiply. This contributes greatly to the variability of the microbial content in raw semen among stallions. This is one of the many reasons that every ejaculate of semen should be placed into an extender containing the appropriate antibiotic, as soon as possible after collection.
2.) Microbial contamination is different from one farm to another. Therefore, on some farms, and with some stallions, an extender containing a single antibiotic may be sufficient to control the bacteria; whereas in other cases a combination of antibiotics in the extender are needed to control the microorganisms.
3.) Such managerial practices as type of bedding, and frequency of changing that bedding greatly influences the bacterial content of the semen; as does the type of flooring under the bedding.
4.) Bacterial content of semen is influenced by the type of material on the floor of the breeding shed, and how often it is changed; as well as how much, if any, dust is raised during the collection process.
5.) Weather conditions, including average daily temperature must be taken into consideration. Bacteria are not nearly as likely to multiply in the bedding or breeding shed in the northern part of the U.S. as in the southern part. Further, areas with high humidity provide a better environment for bacterial growth than dry areas (Florida vs. Arizona), as an example.
6.) Many stallions, with lowered fertility, may ejaculate semen that contains bacteria that as yet have not been identified. Therefore, procedures for control are unknown.
7.) Obviously the mare should also be considered, because it has been shown that cervico/uterine swabs from mares that grew pathogenic bacteria were 2.8 times less likely to conceive than mares with negative cultures.
Fertility has not improved in some breeds of horses in spite of new techniques for treating semen, and vast improvements in equipment. Consequently, it appears that if significant progress is to be made in reproductive efficiency and capacity, control of bacteria in the breeding environment is essential. However, it should also be noted that many breeders cannot or do not utilize the latest, modern equipment such as, high quality phase-contrast microscopy, sperm counter, etc.
Equipment and Facilities: Arrangement of equipment in the laboratory such as appropriate location of the incubator, slide warmer, microscope, etc., are factors determining how quickly semen can be evaluated and processed for insemination. Techniques for handling the semen to prevent heat and/or cold shock can have a profound effect upon fertility. A common mistake is to assume all stallions' semen is the same. The semen from some stallions may tolerate considerable mishandling without affecting fertility, while the slightest error may result in semen from another stallion being subfertile. Consequently, semen from all stallions must be treated as if any error will result in a subfertile ejaculate.
In general, a clean laboratory is indicative of a well-managed operation. All glassware, rubber ware, and equipment should be clean; appropriately stored in dust-free cabinets, when not in use; and all equipment appropriately calibrated and working properly. With respect to equipment, the manufacturer's recommendations on cleaning, care and maintenance should be carefully followed.
Requirements For a Clean Laboratory: 1.) The refrigerator should not be used for medicines, cultures, food and other material not directly associated with the breeding operation. A separate refrigerator should be used to store this material. In addition, refrigerators in a laboratory should be cleaned once weekly, inside and out, and more often if needed.
2.) Unused or rarely used materials should not be stored in the incubator.
3.) For ease of cleaning, there is a tendency to place materials such as paper or aluminum foil on the shelves in the incubator. Most incubators are "gravity-flow" and depend upon free movement of air to maintain the correct temperature throughout. If air flow is restricted, the incubator will heat unevenly, causing some areas within the incubator to be too hot or too cold, this could be damaging to the spermatozoa.
Incubators made from shells of old refrigerators are not uncommon on breeding farms. They are large, thus numerous items can easily be stored. However, they rely entirely upon gravity convection for uniformity of temperature. Unfortunately, the heat source, generally a light bulb, is placed in the bottom with the thermostat. Consequently, the bottom portion is considerably warmer than the top. Also, the door is extremely large in relation to storage space, thus, maintenance of uniformity of temperature is difficult, if not impossible. The incubator (refrigerator) must be appropriately vented at the top for heat to flow properly, which is difficult to accomplish.
The greatest value of these "homemade" units is in the breeding shed for storage of pipettes, etc., objects for which temperature is not quite as critical as for semen and/or extender. Unless it is used in the breeding shed, the refrigerator-incubator is of limited value. In fact, we have heard "horror stories" of semen being severely damaged in these units due to exposure to excessive temperature. In these cases, they became very expensive rather than inexpensive.
Is is not unusual for semen, extender, lubricant, etc., to be spilled in an incubator. The odoriferous effect of such material on freshly-collected equine semen is unknown. However, incubators should be kept clean. Dirty equipment in a breeding laboratory reflects upon the overall operation of the breeding program.
4.) Do not permit people and animals, particularly dogs, to congregate in the laboratory. The laboratory should not be used as a kitchen, lounge or kennel. Having a laboratory in which there is a coffee pot and/or a bathroom, which is even worse than the coffee pot, is an absolute guarantee that the laboratory will not and, in fact, can not be kept clean. Therefore, every effort should be made to reduce the traffic of unneeded personnel in any laboratory.
5.) The laboratory should be constructed so that semen is not processed and evaluated in direct sunlight. Ultraviolet radiation in sunlight is damaging to spermatozoa. Therefore, exposure to sunlight should be avoided.
6.) Reusable material should not be washed in the same laboratory where semen is processed. Ideally, a separate and dedicated area for washing and preparing reusable materials should be available. In general, sinks are too small, without insufficient compartments for washing reusable material. The sink should have three compartments unless part of the washing is done elsewhere. Regardless, two compartments are essential.
7.) The accuracy of thermometers and other temperature measuring devices should be monitored once per week. Generally, this is neglected until equipment is unusable. The accuracy can be checked by using a known standard, such as an accurate mercury or alcohol thermometer. Many times no one on the farm knows how to clean and adjust the laboratory equipment. At least one person in the breeding crew should understand how all of the equipment works and how to clean and adjust that equipment. Often farms will purchase a superior microscope and no one will know how to properly adjust or clean the instrument.
8.) The microscope is one of the most versatile and commonly used instruments in science. Unfortunately, it is also one of the most misused, abused and misunderstood of the precision instruments used on a breeding farm. There are many types of microscopes, with respect to optics and accessories. A phase-contrast microscope is essential in a breeding laboratory for appropriate evaluation of total and/or progressive sperm motility. The phase-contrast microscope, when properly cleaned and adjusted, permits clear observation of individual particles, cells, spermatozoa, etc., of similar optical density without the necessity of staining. Therefore, unseen, transparent cells can then be seen in rich contrast to their background.
As previously indicated, all reasonably modern breeding farms should have a phase-contrast microscope. Unfortunately, most microscopes utilized on farms need adjustment, and most of all need cleaning. Since the microscope is used more than any other instrument on the farm to predict fertility of the semen and indirectly the number of mares that can be bred with that ejaculate, it should have high quality optics, be clean and properly adjusted. Unfortunately, the more complex the microscope, the more difficult is it to keep clean and adjusted. This is the primary reason we encouraged Animal Reproduction Systems to develop their Video Microscope. It is sealed. Therefore, the only area that can get dirty are the stage and the objective, which are easily accessible for cleaning. In the event you have another type of phase- contrast microscope, you should have a professional clean and adjust it just prior to the breeding season, and provide sufficient information on how to keep it clean and adjusted.
9.) Hot water is essential for cleaning, and filling artificial vaginas. However, in many laboratories the hot water tank is too small for a smooth, efficient operation. This is particularly true when hot water is mixed with cold water (which it should be) for washing mares before artificial insemination.
10.) Have plenty of distilled or deionized water, since it must be used to rinse all materials that are washed that will come into contact with semen. Further, if water baths are used in the laboratory they should be washed, rinsed and refilled with distilled water once a week, or more often if something is spilled in the bath. The bath should be kept covered when not in use.
11.) Good breeding programs have a tendency to grow. This is particularly true now that shipped semen has become a large, integral part of getting mares pregnant. Stallion owners are always looking for a farm to stand their stallion that has a reputation for getting mares pregnant with one or two shipments of semen. Thus, there is a tendency for the farm to outgrow its electrical needs in the laboratory. Consequently, the laboratory finds itself with too little power, too few outlets, and too many outlets per line.
12.) Many laboratories are not located or constructed to permit ease of cleaning. Most laboratory floors are covered with vinyl tile. This material is relatively inexpensive, but results in seams in the floor, because the tiles can not be joined closely enough to prevent cracks. Thus, the floor is impossible to clean. Many times rugs or some other fibrous material is placed on the floor in an effort to keep it clean. Although, it may be effective in keeping the floor clean, it results in bacteria, dust and dirt in the atmosphere contributing to an unsanitary environment. Laboratory floors should be covered with seamless vinyl, or if not seamless, thoroughly sealed. The vinyl should extend up under the foot area to the bottom of the cupboards, and up the walls at least 4 inches, and be capped with a metal strip.
In general, the same can be said of the walls in the laboratory. An inexpensive but satisfactory wall covering is sheets of vinyl, in which the seams are filled then covered with strips of vinyl.
13.) Countertops and cupboards should be covered with acid-resistant material that can be easily cleaned. Any countertop that is rough or porous should be avoided. The covering should extend up the wall 6 to 8 inches (back splash) and be sealed. The edge of the countertop should extend beyond the cabinets by 4 inches, with a groove cut into the under side of the overhang, approximately one inch from the outside edge of the overhang. This groove is commonly called a "drip lip." Any liquids that spill over the countertop will either fall directly on the floor or, when it reaches the drip lip, will drip onto the floor rather than onto or into the cabinets.
The front of all cabinets and cupboards should have a bonded layer of vinyl. The vinyl, if properly applied, ensures that these drawers and doors are waterproof, but without the 4 inches overhang water, etc. will run into the drawers. Obviously, this renders the contents unsanitary.
14.) The palpation shed should also be constructed with cleanliness in mind. In an effort to save space and labor, there is a tendency to place sinks, cabinets, stocks, etc. in the breeding shed. This is contraindicated for safety of animals and personnel, if for no other reason. In addition, this adds to the difficulty in keeping the area clean, and can restrict other activities, such as collecting a stallion might prevent mares from being inseminated at the same time. Epoxy paint can be used on walls, which will allow them to be washed with soap and warm water, and steam cleaned periodically. Two of the biggest problems in a palpation shed are; a.) insufficient drains, and b.) insufficient storage apace. Drains should be 2 feet wide and extend behind all stocks. While storage space for waste baskets, brooms, buckets, hoses, cleaning material should not be stored in the palpation area. If so, the area is difficult to clean.
About the Author...
B. W. Pickett, Ph.D. is Professor Emeritus, Colorado State University.