General Considerations For Testing Ability Of Spermatozoa To Survive Cooled Storage

By | 2012-10-24

Preservation of semen begins with the collection process. Accurate assessment of semen quality relies heavily on proper semen collection techniques. Ejaculated semen is susceptible to environmental influences. Therefore mishandling semen samples before evaluation can lead to erroneous interpretation of results, thereby negating their value for representing the ability of a stallion’s spermatozoa to survive the cooling process.

Semen should be collected using a properly prepared artificial vagina. The interior of the artificial vagina should be clean and free of potentially toxic substances such as soap or tapwater residues. Between uses, artificial vaginas should be rinsed thoroughly with deionized water to remove impurities, rinsed with 70% isopropyl or ethyl alcohol to eliminate growth of microorganisms, and allowed to air dry. Before collection, artificial vaginas should be lubricated with a nonspermicidal product. Additionally, the semen collection receptacle should be nonspermicidal and fitted with a filter to allow separation of gel from the gel-free portion of the ejaculate.

After collection, semen should be processed in a careful and efficient manner. The semen should be placed immediately in a light-shielded incubator adjusted to 37° C to 38° C. All items that come in contact with raw semen should be prewarmed to 37° C to 38° C to prevent cold shock to the spermatozoa. The filtered gel-free semen should be poured into a graduated cylinder to measure volume accurately. Some types of specimen cups have inaccurate graduated markings for volume.

Sperm concentration of the gel-free semen is determined using either a hemacytometer or properly calibrated photometric instrument. The total spermatozoal number in the ejaculate is calculated by multiplying spermatozoal concentration by volume of gel-free semen. This calculation is necessary (when the percentage of progressively motile spermatozoa is taken into account) to aid in determination of the number of inseminations possible from an ejaculate and determination of the amount of semen extender that should be added to the raw semen to maximize longevity of motility following cooled storage. A portion of the gel-free semen should be diluted in a suitable prewarmed extender, then incubated at 37° C to 38° C for 5 to 10 minutes before estimation of percentage of progressively motile spermatozoa in the sample.

Motility assessment using raw (unextended) semen can yield erroneous measurements. Warmed nonfat dry skim milk-glucose (NFDSM-G) extender serves this purpose well because it sustains spermatozoal motility and does not interfere with microscopic visualization of the spermatozoa. To standardize the spermatozoal motility testing protocol, all semen samples should be diluted to a specific concentration (i.e., 25 x 106 spermatozoa/ml) with extender before analysis. Ideally, spermatozoal motility should be estimated at a magnification of 200 to 400 times, using a microscope equipped with phase-contrast optics and a warming stage.

Screening against ejaculates of poor quality is necessary to maximize success with preserved semen. If fresh stallion semen is poor quality, successful results most likely cannot be obtained by breeding with preserved semen. Extended semen from fertile stallions often can be stored in a cooled state for hours to days before insemination without a significant reduction in pregnancy rate. Longevity of spermatozoal viability in vitro may be maximized by properly diluting semen with a high quality extender, cooling the extended semen at the proper rate, and holding the cooled semen at the proper temperature until it is used.

Semen extenders contain protective ingredients that permit spermatozoal survival outside the reproductive tract. Lipoproteins, such as those contained in milk, protect spermatozoa against cold shock by stabilizing cellular membranes. Metabolizable substrates, such as glucose, provide a plentiful source of energy for spermatozoa. Antibiotics are added to extenders to retard or eliminate growth of bacterial organisms.

Osmotic pressure and pH of extenders also are adjusted to maximize spermatozoal survival. Extenders may be homemade formulations or commercially available preparations. Potassium penicillin G (1000 units per ml of extender), amikacin sulfate (100-1000 M-g/ml of extender), amikacin sulfate plus potassium penicillin G in combination, or ticarcillin (100-1000 μg/ml of extender) have been found to be acceptable antibiotics for inclusion in NFDSM-G extender formulation. These antibiotics do not impair motility of stored spermatozoa and inhibit the growth of most bacteria present in equine semen. The combination of potassium penicillin G and amikacin provides better control of bacterial growth than ticarcillin or either antibiotic used singularly.

Ideally, semen should be mixed with a prewarmed (37° C to 38° C) extender within minutes after ejaculation. A minimum of a 1:1 ratio of semen to extender is recommended if semen is to be inseminated immediately. If semen is to be stored for a period longer than 2 to 4 hours before insemination, greater dilution (i.e., more extender to semen) is required. A final concentration of 25 to 50 million spermatozoa per ml in extended semen generally maximizes spermatozoal survivability in vitro. Alternatively, extender can be added to semen at a 1:4 to 1:19 (semen: extender) ratio to reduce seminal plasma in the ejaculate to 5% to 20% of the extended volume. Seminal plasma can be detrimental to longevity of spermatozoal viability during storage of the semen if it occupies more than 20% of the total volume of extended semen; however, retention of some seminal plasma generally improves longevity of spermatozoal motility. The concentration of spermatozoa in extended semen should not be below 25 million spermatozoa per ml. When a stallion ejaculates relatively dilute semen (e.g., £100 million spermatozoa per ml), dilution in extender to arrive at a final concentration of 25 million spermatozoa per ml may fail to provide protection against environmental influences for spermatozoa, thereby resulting in a low rate of spermatozoal survival following cooled storage. In such instances, it may be beneficial to mix the raw semen with extender, then centrifuge the extended semen at 500 x g for 10 minutes, aspirate the supernatant, and resuspend the spermatozoal pellet in additional fresh extender. The majority of seminal plasma is removed after centrifugation and aspiration of the supernatant, so the remaining spermatozoal pellet can be resuspended in extender to arrive at a final concentration of 25 to 100 million spermatozoa per ml. For some stallions, centrifugation of extended semen, followed by resuspension in fresh extender, has been shown to improve spermatozoal motility characteristics after 24 hours of cooled storage at 5° C.