310 Semen Analysis

310 Semen Analysis - TECH 105: SEMEN ANALYSIS Accompanied...

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Unformatted text preview: TECH 105: SEMEN ANALYSIS Accompanied Reading: Rick L. Cowell and Ronald D. Tyler Dia nostic C 010 and Hematolo of the Horse, 2"d ed. “Semen Analysis”, pages 187 — 191??? 47 PRINCIPLES OF EQUINE REPRODUCTION Semen analysis is the logistics, quality control, and means of providing a history on every stallion collected for artificial insemination as well as cooled/frozen semen programs. Historical significance of trends can aid in defining reproductive problems early as well as establishing normal values for individual stallions. Analysis of semen can be simply defined as any physical, microsc0pic. and chemical analysis involved in ascertaining the quality of individual ejaculates from the stallion. The most common analyses conducted in normal breeding operations include overall motility, concentration, color, gel- fraction volume, gel free volume, rate, and progressive motility. However, when critical numbers such as concentration and motility drops, this may indicate a deeper physiological or anatomical problem within sperm production or handling processes. Identifying internal spermatogenic problems requires that a technician be familiar with detailed assessments, such as morphology assessment, live-dead staining, longevity, and osmotic factors. Analysis of the ejaculate accompanies a stallion’s breeding soundness report and aids in identifying those stallions that meet the Society for Theriogenology’s standards for satisfactory mating principles. To obtain this rating a stallion must be physically capable of mating, possess good libido, have normal external genitalia with scrotal or testicular dimension consistent with good sperm production, test negative for infections and for venereal, bacterial, and viral pathogens, have good semen quality, and at least 1 X 109 progressively motile, morphologically normal spermatozoa in the second of two ejaculates collected 1 hour apart, preceded by 1 week of sexual rest.6 The following sections will explain the various principles of the individual components of semen analysis. MOTILITY Motility is the broadest of viability defining observations characterizing the overall motile characteristics of an ejaculate. For the most part. we characterize the health of an ejaculate based on this analysis. Sperm cells are simplistic in nature. And, for them to deliver their genetic cargo, they must be motile. However, things are not that simple. In fact. there are three motility requirements that define individual ejaculate health based on a multitude of factors. including: 1. Total Sperm Motility (or Overall Motility) — this establishes the viability of the entire ejaculate regardless of direction of motion. Basically. establishing what percentage of the ejaculate is actually moving. Thus, defining health of the ejaculate based on a perceived percentage (0 — 100%) of how many cells are actually living. 48 NOTES 9 Progressive Motility — defines the number ofindividual cells that are moving in a forward or curvilinear path. Again. we base this observation on perception of the percentage of cells moving in the desired path. This method is more difficult in estimating motility and for a truly accurate count computerized motion characteristics must be employed. However. in the case that PMS (or progressively motile sperm) is a requirement and you are not sure of your assessment, it is plausible to use a standard average deduction of 15 — 20% from the Total Motile Sperm (or TMS) defined by Colorado State University. Keep in mind, this standard deduction is an average and may not accurately represent your sample. It is up to the technician to make the decision of whether the collection represents the average population of sperm generally examined. Extended Motility - represents the number of motile spermatozoa from O —100% after the desired extender is added to the collection. Again, we are assessing Total Sperm Motility with the exception that we are now evaluating it based on the affects of the extender. Generally, the technician will observe a 5 — 15% increase in Total Motile Sperm and a slight increase in rate. Rate will be discussed shortly. Examining extended semen provides technicians and more importantly, the breeder an idea of the effects of specific extenders on individual stallion spermatozoa. It is important to remember that every stallion responds differently to the components of various extenders and it may be necessary to determine which extenders provide the most enhanced sperm motility. Instructions for Preparing a Sample for Microscopic Evaluation 5 J. 0 Raw Semen Obtain a pre-warrned slide and cover slip from a slide warmer or incubator. Remember that sperm are very sensitive to changes in temperature. especially cold. Allowing them to cool will seriously affect the results you obtain and will present inaccurate data. Obtain a disposable pipette in the incubator or on a slide warmer. And. keeping in mind that sperm cells have a tendency to settle. swirl the collection container to resuspend the cells. Now. draw up enough semen to begin filling the wide portion ofthe pipette. This should be done as quickly as possible to reduce exposure ofthe unextended semen to cool air (NEVER set a collection vessel on a cold countertop). Once the sample is drawn into a wami pipette, place as small a drop as is possible on the warmed slide. You may want to place the sample on one end of the slide. so you will be able to use a single slide for all three motility analyses. This reduces waste and clean-up time. TECH 105: SEMEN ANALYSIS NOTES 49 PRINCIPLES OF EQUlNE REPRODUCTION 4. Place a warmed cover slip over the small sample placed on the slide. Simply allow the cover slip to settle over the sample without applying physical pressure to the slide. Any physical pressure applied to the cover slip will suppress sperm movement. Placing the slide under the lens of the microscope, find a sample that is not suppressed by pressures exerted by the cover slip and any inadvertent pressure placed on the cover slip when placing it over the sample. Generally, this is accomplished by scanning multiple segments of the covered sample for the highest concentration and motility. Be careful to stay away from the edges of the cover slip as this suppresses true sperm movement. When you are satisfied that you have a true representative sample of sperm cells, you are now ready to make an educated guess regarding the number of motile sperm. And, it is just that “an educated guess.” Do Not try to physically count the number of moving cells, as it is impossible with the bare eye. Rather, look at the entire view seen through the lens of the microscope and estimate the number of cells you think are moving on the basis of one of the three types of motility. a. For Total Motile Sperm, you are estimating the number of cells that are moving regardless of direction of motion. In Total Motile Sperm motility analysis, we are not concerned with direction, but whether a cell is moving. b. For Progressiver Motile Sperm, you are estimating the number of cells that are moving in a straight to curvilinear direction. In order for a sperm cell to fertilize the ova, they must be moving forward in the uterus. Cells moving in a circular or rearward path of motion are not capable of fertilization and should be discounted in this case. If you are not sure of your estimate, you can compare it to the average deduction of 15 — 20% from the Total Motile Sperm estimation. Progressive Motility is almost always less than overall motility. c. To evaluate Extended Motility, you will need to extend a sample of semen to the desired dilution and evaluate it for overall motility following the same slide preparation and evaluation for overall motility as described above. Keeping in mind that extended motility almost always increases by 5 — 15% from the unextended overall motility. NOTES RATE Rate is characterized by the speed of a sperm cells forward m0tion and is usually estimated in the raw ejaculate: however. it can be characterized in extended aliquots as well. Measured on a l — 4 scale (1 being the slowest — 4 being the fastest), this observation allows a technician to make inferences on viability based on energy consumption and speed. Generally, sperm movement is measured in nanometers per second, but without the aid of computerized flow cytometry an exact Speed cannot be ascertained. Therefore, we utilize the “best guess” evaluation technique here as well. Without sufficient initial forward movement, a sperm cell may not have enough energy to make it up into the oviduct to fertilize the ova. As sperm move forward in the reproductive tract of the mare, sources for extra cellular energy become limited; and, without a healthy rate, the individual cell may not have sufficient character to make it to the oviduct. However, once sperm cells make it to the oviduct, they receive assistance from the oviduct itself. While motion character remains important, the ova duct structure and activity assist sperm cells in theirjourney and reduces the energy usage of the cell. LONGEVITY Longevity characterizes a sperm cells ability to survive extended periods of time and can be analyzed both in regards to the raw fraction as well as an extended fraction. This character provides a breeder some understanding of individual stallions sperm survivability in the reproductive tract of the mare. Longevity allows the technician and breeder to establish survivability from insemination to fertilization for the individual stallion. Does a stallion produce sufficient seminal character or extender compatibility to get sperm through insemination to fertilization? Are the individual sperm cells healthy enough to traverse the reproductive tract of the mare? Does the stallion produce hardy, robust cells capable of surviving extended periods of time in vitro? These are all questions answerable by the longevity test. TECH 105: SEMEN ANALYSIS NOTES l PRINCIPLES OF EQUINE REPRODUCTION Conducting the Longevng Test 1 . Ut Ix.) Remember that longevity can be determined for a multitude of applications. You can utilize longevity for fresh, extended, and cooled/ frozen semen. Each procedure is identical. but utilizes a sample of the desired semen application. Poor off 5 — 10 mL of raw semen into one or more test tube(s) depending on the types of tests you wish to conduct. For raw semen longevity, just place 5 — 10 mL of raw semen in a test tube and keep it in the incubator. To prepare an extended sample, poor off 5 — 10 mL of raw semen and note the volume in the test tube. Then, simply extended the semen to the desired ratio (1 :1, 2:1, etc.) and place in the incubator to keep it warm. Estimate total motile cells for the sample(s) in the test tube(s) immediately after preparation following the procedure described in the motility section of this handout, this will be time zero and the start of the interval you establish based on preference. At the desired interval you established, estimate the motility at each of those times following the Instructions for Preparing a Sample for Microscopic Evaluation. Usually longevity is run on intervals of 30, 60, 90 minutes as well as 12, 24, 36 and 48 hour intervals. Again, it is the technician who should make this decision. For artificial insemination programs, a 30-60-90 minute interval will be sufficient, where as a cooled shipment program would probably want to utilize, in addition to the 30- 60—90 minute interval, a 24, 36, and/or 48 hour interval. . Longevity can also be used on frozen semen. However, cryogenics of sperm damages cells and may alter some function affecting survivability of the cells. Thus, longevity studies are essential to any frozen semen program and should include a 30-60-90 minute longevity study in every report. A 24 plus hour study may not be beneficial, since cell function is altered somewhat during freezing and will not give true metabolic function and survivability. The shorter 30—60-90 minute interval will give indications as to holding time from thawing to insemination. NOTES TECH 105: SEMEN ANALYSIS CONCENTRATION NOTES Often. breeders utilize concentration as a direct means of evaluating a stallion‘s fertility and physical health. It is common to see occasional significant drops in concentration, but any prolonged reduction in Spemt numbers should be seriously explored and corrected as soon as possible. In the past, sperm numbers were counted manually using a hemaCyromeIer. A device that consists of a microscopic grid pattern as reference points for counting sperm in a specific pattern and actual concentration calculated through formulas. Needless to say, more accurate and time saving devices have become commonplace in breeding operations. Most breeders utilize a densimeter. A device that uses laser technology to count spemt in a desired aliquot of semen and is the simplest technology on the market to date. Some operations and universities have upgraded to computerized technologies that are capable of running not only concentration, but an entire semen analysis on the sample itself. Hamilton—Thom produces devices such as the IVOS and CEROS systems based on flow _cytometric analysis. These systems are capable of calculating rate, morphology, and even acrosome integrity, but have yet to become cost effective tools for the average breeding operation. You will be using the SpermaCue to calculate concentration. The procedure for using this device is outlined below. Procedure [or Counting Sperm Cells Using the Minitube SgermaCue l. The SpermaCue requires a very short warm up period, during which time the display will indicate “SP”. Pulling out the black slide mechanism “carrier” (located at the front right side) to the first stop point (you will hear and feel a click at the proper point) resets the unit to zero. After about 5 seconds, the display will read “READY”. And, you can now load the cuvette with semen. 2. Load a sample of the ejaculate directly into a cuvette by first mixing the ejaculate thoroughly. then touch the open end of the cuvette to the ejaculate. The cuvette will load by capillary action. Be sure the entire circle in the center of the cuvette chamber is filled with semen. Wipe any semen off of both sides of the outside of the cuvette with a clean tissue. Be careful not to touch the end of the cuvette. which could wick semen out of it and change the reading. 9) Load the cuvette onto the carrier by placing it onto the carrier with the sample compartment towards the photometer and the raised tab towards the top. This is the only position that will allow the carrier to close properly. Close the carrier and the photometer will automatically take the reading. 4. At this time, the display will indicate, “MEASURING”. When finished measuring the density, the display will indicate the number oi‘Sperm at xlOé cells per 1111.. UI b) PRINCIPLES OF EQUINE REPRODUCTION 5. When you have recorded the concentration, pull out the NOTES carrier and remove the cuvette. And. push the carrier back into place when finished. This will reset the unit to zero. 6. Flush the cuvette chamber with adequate DI water to thoroughly clean the cuvette. Then, using 70% alcohol in a spray bottle, force alcohol into the cuvette by directly spraying into the tip of the cuvette. NOTE: The SpermaCue reads concentrations most accurately in the range from 150 to 450 x 106 cells/mL. If you have a sample that falls outside this range, it will be necessary to dilute a small sample with citric acid. TESTING ALKALINITY AND ACIDITY (PH) Testing a sample for pH is a simple method of determining whether contaminates exist within a collection. Nomial pH of semen ranges from 7.25 — 7.65. Using a calibrated pH meter, this test can indicate acidity or alkalinity without much effort and can give some reasoning to low motility rates. For example, an abnormally high pH value might indicate urine contamination of the ejaculate. This test should be run within 1 hour of collection. Remember that in the raw ejaculate, toxins build quickly and will be a source of contamination affecting pH more and more as time progresses.6 MINOR DATA RECORDED IN A DAILY BREEDING REPORT A V Weight and Temperature —- these values are solely recorded for reference purposes only. This will allow multiple people to construct effective AV’s for individual stallions. An optimal weight and temperature should be provided to the owner in all Soundness Breeding Reports. Gel-Free Volume — while volume of an ejaculate has no real effect on sperm motility, it can give some indication as to developing sexual problems. While extremely low volumes may be an indication that a stallion may need additional sexual rest. a low volume may be the stallion‘s norm and should be verified with historical data before any assumptions are made. Gel Volume — this portion ofthe ejaculate is of no consequence to a breeder as it is discarded after collection. However, it can be used to some extent to help evaluate health of an overall ejaculate. In most cases, experience has shown that a healthy gel fraction directly correlates health of sperm cells. Thus, this number should be recorded to establish normal values for individual stallions as this may provide an indicator of developing problems. Color — directly correlates health of the overall ejaculate and is observed visually by the technician. Nomial semen has a gray- white, opalescent color. A red to brown color usually indicates blood contamination and bright white colors may represent some type of deficiency. TECH 105: SEMEN ANALYSIS NOTES MORPHOLOGY of EQUINE SEMEN Normal Spermalozoa _ . 2 Terminal PrinCIpal Mid - («1; Head Piece I Piece I Piece 1: Head Abnormalities I. Loose head 2. Small 8 narrow 3. Elongated 4. Pyriform 5. Microcephalic 6. Addilional cop Page 2 Neck Abnormalities I. Protoplosmic droplet, proximal 2. Irregular shape 3. Broken VMidpiece Abnormalities l. Protoplasmic droplet, distal 2. Swollen 3. Divided fibers 4. Thread-like 5. Broken Page 3 Principal-piece Abnormalities l. Single loop 2. Double loop 3. Coiled toil 4. Coiled, heod inside ("smile") 5. Thread - like 6. "Two-headed" TSU — Horse Program SEMEN EVALUATION Stallion: Date: Collector: Time: Reaction Time: Jump Mare: W # of Mounts: Phantom: Artificial Vagina Temperature: TVPEZ Weight: VailTiree: Prggiessive Motility Gel‘ m Extended: Color: ____.__~_~_______*‘_fl_~"___ Rate: Concentration %T: ————-———__.______.___._______ Sperm/ml: x 106 Dilution: Hemacytometer: Total Sperm/Ejaculate: m Morphology Primary abnormalities: % Secondary abnormalities: % Tertrary abnormalities: % Live—Dead Live: % Comments: ...
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310 Semen Analysis - TECH 105: SEMEN ANALYSIS Accompanied...

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