310 Semen Collection

310 Semen Collection - TECH 104: SEMEN COLLECTION AND...

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Unformatted text preview: TECH 104: SEMEN COLLECTION AND ARTIFICIAL INSEMINATION PROCEDURES Accompanied Reading: Juan C. Samper fluine Breeding Management and Artificial Insemination CHAPTER 7: Semen Collection CHAPTER 9: Artificial Insemination PRINCIPLES OF EQUINE REPRODUCTION Semen collection is an integral part of any artificial insemination and cooled/frozen semen transport program as well as breeding soundness evaluations. Semen collection can be defined as the successful recovery of a sperm rich (SR) fraction of semen that can survive in vitro storage and examination while providing quality gamete capable of surviving at least 24 hours in the mares reproductive tract to successfully capacitate and fertilize the ova. Properly utilized, collection and processing of semen can dramatically reduce infection, increase semen quality, and help in pinpointing fertility problems common to some stallions. According to Blanchard et al, the artificial insemination program, when utilized effectively, can improve reproductive efficiency and service a greater number of clients in the industry.l When considering the idea of collecting semen, you must keep in mind that it is intertwined with numerous aspects of a breeding operation. It relates to reproductive efficiency of the mare, allows for more effective management in the problem stallion, and more importantly, allows for effective management of a stallion allowing him to service multiple mares in a days time without seriously afiecting libido or seminal characteristics. As with all Standard Operating Procedures (SOP’s), there are both advantages and disadvantages of the procedure in question. In semen collection, SOP’s are primarily established for ” repeatability and 2) ensuring quality control that provides an appropriate environment for sperm survival. Construction of an AV is at the heart of the collection process. Pressure, weight, and stimulatory processes for individual stallions should be established. Improperly constructed AV’s can impart a negative affect on the breeding stallion resulting in indifference or complete refusal to the collection process. Furthermore, exposing semen to adverse environments can seriously affect the survivability of semen in vitro as well as in viva, affecting quality of collection. Therefore, controlling quality of the collection apparatus and post-collection procedures are of the utmost importance. ADVANTAGES AND DISADVANTAGES OF SEMEN COLLECTION When looking at the advantages and disadvantages of semen collectiOn, it must be observed that it is often strongly correlated with artificial insemination, due in part to the fact that at the heart of artificial insemination lays a healthy sperm rich fraction. Semen collection allows for the division of the ejaculate into doses allowing intensive management of a stallion‘s reproductive efficiency, thus allowing him to service more mares per calendar year. Furthemiore, collection via an AV reduces direct biological contact and contamination between mare and stallion. With addition of an antibiotic to the sperm rich fraction. the potential for uterine infection and disease transmission can be further reduced. In addition. an appropriate extender will provide Lu b.) NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL lNSEMlNATlON PROCEDURES a supportive as well as protective mixture that further enhances NOTES pregnancy rates. There are few disadvantages to the Al and semen collection program; but nonetheless they do exist. Probably, the most important disadvantage to collection via AV is the knowledge required to properly handle Spermatozoa for successful insemination and maintenance of reproductive efficiency. The process requires extensive knowledge of cooling and environmental effects on spermatozoa, knowledge of equipment involved in achieving and maintaining an environment capable of sustaining sperm life which involves the purchase of expensive equipment as well as disposable costs involved in the AI program for insemination of mares, and the increased risk of placing personnel under the stallion for collection increasing the likelihood of injury. Advantages Increase the reproductive efliciency of the stallion. Increase the number of mares that can be impregnated per calendar year. Allows for control of infective agents through antibiotics. Addition of seminal extenders provides Disadvantages Requires increased knowledge of environmental effects on spermatozoa. Expense related to purchasing AI and Collection Equipment. Increase risk of human injury during the collection process with the AV. supportive and protective agents enhancing sperm motility and fertility. Allows for the use of a phantom increasing the safety of mares and handlers. Collection allows for scrutiny of the sperm rich fraction indicating reproductive problems early. Table 9: Advantages and disadvantages of semen collection as discussed.l v) Lu PRINCIPLES OF EQUINE REPRODUCTION EFFECTS OF SEMINAL PLASMA, FRACTIONATION, AND DILUTION EFFECT In order to understand some of the more complex concepts involved in storing and insemination of an ej aculate, you must have some idea of the ejaculatory process and although complex, the effects of seminal plasma and accessory fluids added during the ejaculation. According to McKinnon and Voss, the process of ejaculation involves three sequential processes: erection -) emission 9 ejaculation. All three components are serious factors that effect a successful collection and viability of that collection, if conducted and handled improperly. l. Erection —— is defined as the lengthening and stiffening of the penis, resulting from increased blood to the corpus cavemosum and spongiosum penis. Teasing plays the critical role for appropriate sexual stimulation for this stage of ejaculation and initiates minor muscular contractions that prepare the reproductive tract for transport of sperm out of the tail of the epididymis into the vas deferens. 2. Emmision — is the muscular contractions that actually move and deposit the sperm and accessory fluids into the vas deferens. Mixing of these fluids (termed seminal plasma) and sperm together make-up semen. 3. Ejaculation — is the actual expulsion of semen through the urethra for collection or deposition into the uterus During emission and ejaculation, usually involving 3 - 6 pulsatile jets, sperm are mixed into a diverse environment of factors directly affecting their survivability. Sperm — Accessory secretions are very complex. Mature sperm stored within the cauda (or tail) are basically immobile. And, it is theorized that the addition of seminal plasma components from accessory sex glands react with certain proteins found in the membrane structure of the sperm that initiates motility during ejaculation. However, we will primarily focus our attention on the seminal plasma, examining its effects, fractionation, and dilution in terms of fertility. Reproductive physiology of the ejaculatory process will be discussed in lecture. NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL INSEMINATION PROCEDURES Fractionation NOTES During the ejaculatory process, semen is voided from the reproductive tract in a sequence of fractions. The first fraction passing into the tract is a clear, watery type secretion originating from the bulbourethra and is termed the Pre-Sperm fraction. Its function is to clean the urethra of urine, bacteriological, and other debris found in the urethra of the penis. The sperm rich (SR) fraction directly follows the Pre-Sperm and is composed of a high concentration of sperm cells from the tail of the epididymis and secretions from the PrOstate Gland. The final fraction consists of secretions almost entirely from the vesicular glands and contains a limited number of motile sperm cells. The final combination of sperm with these fractionated components generate the fertilizing ejaculate termed semen. Seminal Plasma The entirety of the secretions from the accessory sex glands compose the fluid, non-gamete fraction of an ejaculate and is the transport system for sperm originating at the tail of the epididymis.9 According to Varner et a1. seminal plasma does not play a critical role in capacitation and fertilization capability, and it has been shown that mature sperm cells directly extracted from the tail of the epididymis have the same fertilization capability as do normally ejaculated sperm. Further, research has shown that sperm quality is higher in the sperm rich fraction referencing the fact that almost all of the toxic components of the ejaculate are found in vesicular fluids. In fact, the first three pulses during ejaculation represent only ~43% of a total ejaculate; however, seventy-seven percent of the sperm cells will be voided from the reproductive tract during this time. Research has shown definite increases in quality when the post- sperm ejaculate (sperm poor) is fractionated out of the ejaculate, either during the collection procedure or by centrifugation. Results of a Varner et al. study are shown in Table 1. Seminal plasma has many more inhibitory factors than it does supportive factors that increase sperm quality. As previously mentioned, vesicular fluids are the main source of repression. These fluids inhibit capacitation reversibly, interfere with sperm motility and ovum penetration, and reduce sperm oxygen consumption and respiration. On the other hand, prostatic fluids have been identified as providing a supportive function increasing sperm motility and protecting sperm from vesicular fluid effects for a short period. PRINCIPLES OF EQUINE REPRODUCTION Ejaculate Type 0.5 Sperm Rich Ej aculate 81 75 Total Ejaculate 83 74 Table 10: Effect of Semen Fractionation on Mean Total Spermatozoal Motility in the Stallion? While fractionation has been shown to be beneficial in increasing sperm quality, it is notnecessarily beneficial in breeding operations. In most breeding management situations, semen is collected, diluted with an appropriate extender, and inseminated within a short period, usually within a half to three hour time window. This time frame does not benefit management in this situation. However, the means of increasing sperm quality becomes an essential component when decisions are made for extended storage where semen may be cooled for a 48-hour transport period or frozen for indefinite storage. Dilution Eflect As should be evident, seminal plasma is not suitable for sperm and should be immediately diluted with an appropriate extender. However, one must be careful as not to over dilute a collection, since sperm longevity was shown to be proportional to the extent of semen dilution (V arner et al. 717). Vamer et al. found that a concentration of 25 x 106 Sperm/mL optimized survivability. However, sperm concentrations below 20 x 106 sperm/mL induce the dilution effect. A state characterized by reduced viability of spermatozoa and excessive dilution and low concentrations. Dilutions are largely based on type of application. Collections for artificial insemination programs use a 500 x 10‘5 sperm/mL concentration to determine dose, where as cooled/frozen semen programs utilize a fresh semen concentration of 50 x 106 sperm/mL pre-centrifugation and resuspension to a final 400 x 106 sperrn/mL insemination dese. Calculation of dilutions is based on the chemistry equation MiV; = MrVr, where M is concentration and V is volume. However, not all sperm ina collection are necessarily viable and a breeder may or may not need to consider this in his/her calculations. Therefore, dilution calculations may be based on total sperm count, % total motile sperm (TMS), or % progressively motile sperm (PMS). NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL lNSEMlNATlON PROCEDURES How TO CALCULATE DILUTIONS ,7 1. Semen was collected and found to have a concentration of 291x106/mL, with a total motility of 75%, a PMS of 60%, a gel-free volume of 35-mL, and extended 1:1 bringing total volume to 70- mL. A a Dose calculation of 500 x 106 TMS I mL per insemination dose. i. Calculate the concentration of sperm cells when extended 1:1 or in this case to 70- mL. 6 a _ _ (MI-)5 IML] = (Diluted Concentration( 70 lmL] m Doing the math, this gives a 145.5 x 106 sperm / mL extended concentration. ii. Using the above concentration of 145.5 x 10‘5 sperm / mL, calculate how many of these cells should be motile based on the 75% TMS. 109.125 sperm mL 145.5 sperm iii. Now, using the ( mL dilution formula again, calculate the target dosage of 500 x 106 sperm / mL. W (“J-mi _ W (V—mLofDose) mL 1 1 ' )x75%= Doing the math, you should have come up with a dose of 15.278-mL. J You can double check your calculation by doing the following: 0 Calculate Total Sperm in the Container by multiplying the extended volume by the extended concentration as follows. 6 (109.125x10 sperm x( L 70 — mL)=763 8.75 x106 Total Motile Sperm In Total Sperm = 0 Now, using total sperm, calculate concentration in a single 20.3- mL dose by dividing total sperm by dosage in mL, as shown below. 7638.75 3:106 sperm , 2500x105 sperm/"IL 15.278 — deose PRINCIPLES OF EQUINE REPRODUCTION PROCEDURES FOR COLLECTING SEMEN FROM THE STALLION The collection procedure varies according to management scheme and performance of the stallion, but the most proficient means of collection is via the artificial vagina. Other means of acquiring an ejaculate include the use of condoms for a natural intromission experience as well as pharmacological inducement of ejaculation using drugs such as xylazine. The following outlines the procedure utilizing the artificial vagina and mount as the primary means of stallion collection. The procedure can be sub-divided into three components: pre-collection, primary collection, and post-collection procedures. Pre-Collection Procedures Before a collection is allowed to proceed, several observations and preparations should be made prior to any involvement in a breeding situation. These will include: 1. Properly washing all equipment with a non-spermicidal agent capable of disinfecting all components. This should be done at least 3 — 4 hours prior to collection time, but 24+ hours prior is preferable. 2. All lab equipment that may potentially be exposed to the seminal fraction of the collection should be thoroughly warmed to 37°C. 3. Immediately before breeding/collection: a. Tease mares should be placed in a secure teasing pen, or if collecting off a dummy, an estrus mare should be placed into the stocks. Placement in the stocks is a personal management decision. The mare should be placed in the first or second stock providing the stallion some access to the mare. This method provides some protection from overly aggressive stallions and/or mares, however this is used to primarily encourage manners from the stallion. b. The stallion should be brought to the teasing/breeding area by a pre-determined route on breeding days. Remember, stallions are creatures of habit. They can distinguish halter types and routes in regard to the activities that day. c. Time of AV construction will vary according to teasing and collection method. Are you collecting from a dummy where time will be limited for teasing and prepping of the stallion? Or, will you utilize a group teasing practice allowing ample time for AV construction? The AV should be constructed quickly. and in such a way as to allow for minimal exposure to 38 NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL INSEMINATION PROCEDURES d. ambient temperatures to prevent cooling of the lumen NOTES of the AV as well as rapid cooling of the collection vessel. Further, consideration should be observed when collecting from a dummy to prevent excessive periods before collection. This will help prevent frustration from the stallion. Remember, in a natural situation, breeding occurs without delay when receptivity is determined. Delaying intromission after sufficient teasing may create resentment and frustration from the stallion. If group teasing, the stallion can remain in the teasing pen/cage until the AV has been constructed and collection is to proceed. e. Selection of a collection mount should be pre- determined based on individual stallion experience and age. Younger stallions may be trained to mount the dummy, but older stallions are difficult to transition from mare to dummy. There is little to no preparation required of the dummy before collection; however, if an estrus mare is to be used as the collection mount, several preparatory procedures should be observed before collecting of the stallion. o Estrual state and receptivity should be determined to prevent a negative breeding experience and a dangerous situation for handler and collector. o The mare’s tail should be sufficiently wrapped with a tail wrap or OB sleeve to prevent hair from contacting the shaft of the penis. Tail hair is capable of paper like cuts or abrasions on the penis creating a negative breeding situation. 0 In case of explosive reaction to mating, the mare should be hobbled during teasing and collection to help prevent injury to the stallion and/or collector. 0 The perineal area should be washed with a good disinfecting soap before collection to help reduce bacterial contamination from fecal matter, urine, and extraneous debris. As part of some operating procedures, the penis is washed with warm water and a mild disinfectant removing excessive dirt and pathogens. However, this procedure interrupts natural microfloral growth leading to increased chances of virulent bacterial growth not commonly found on the penis. This procedure is not commonly utilized in the breeding management of TSU stallions. They are allowed to naturally cleanse themselves. With time and regular collection, the penis will cleanse itself and not be a major problem in its affects on the ejaculate or the mare. PRINCIPLES or EQUINE REPRODUCTION Primary Collection Procedures Once the stallion, mare (if needed), and AV have been prepped, the collection process can proceed as described. At this time, the tease or mount mare and AV should be in place and properly prepped before the stallion is introduced to the tease/mount mare. Once the area is secured to insure safety of those present, the collection may proceed as follows. 1. 40 The stallion handler will introduce the stallion to the tease and/or collection mare at the shoulder to mimic natural courtship. At this time, the collector and AV will offset themselves behind the stallion handler to insure safety of him/herself from being kicked or struck by the stallion during this short teasing period. At this point, the handler is only there to maintain discipline and control. It is now the collector’s responsibility to follow and read the stallion for a successful collection. The collector should effectively read the stallions intentions and move out from the handler as the stallion moves toward the flank area. As the stallion approaches the flank and perineal area of the mare, the collector should now be in a position to fall in under the stallion to guide the penis into the lumen of the AV. If collecting off of a dummy, experienced stallions will turn and look at the dummy when they have sufficiently teased. The handlerwill bring the stallion to the approximate shoulder position of a mare for the mounting process to begin. i This places the collector in a vulnerable poSition. Remember stallions are agile and quick. You can be kicked and/or struck in a matter of seconds. Safety is your responsibility at this time. Never place yourself in a compromising situation where you have no way out. . As the stallion lightens in the front and begins to bring the front feet over the dummy or the mares back, the collector will move in and guide the penis in an open palm into the AV lumen. .0 Never grasp the penis in a closed hand. The pressure is uncomfortable for the stallion and can illicit unfavorable reactions compromising the collector’s safety as well as those in the general area. Once the penis has been guided into the lumen. the AV should be securely braced against the dummy or mare, the lumen angled slightly downward as to mimic the squatting posture of a receptive mare, and the free hand should grasp the collar of the lumen with the index finger placed on the urethra to aid in the detection of ejaculation. NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL INSEMINATION PROCEDURES 5. The stallion handler should now relax the lead and allow the NOTES stallion to commence intromission on his own. Discipline at this point becomes very difficult if we are to avoid imparting a negative breeding experience. As long as the stallion does not become overly aggressive, he should be allowed to proceed on his own with as little interference from the handler as possible. 6. As the intromission process smoothes out, stimulation of the AV may be require and is beneficial to the quality of an ejaculate. Movement of the mare during intromission provides natural stimulating motions. This is achieved with a combination or single use of the following techniques: a. The free hand that is grasping the collar of the lumen can gently apply and release pressure by squeezing the collar intermittently while keeping the index finger on the urethra. b. As the mating act progresses and initial pulses of ejaculate are felt at the index finger of the free hand, the collector can gently bump the glans penis with the hip at the container end of the AV. 7. Once ejaculation occurs, the collector should allow the AV to remain in place for a few seconds allowing the penis to somewhat soften before removing it and directly proceeding to the lab for processing. 4! PRINCIPLES OF EQUINE REPRODUCTION Post-Collection Procedures 1. 42 An optional procedure directly following collection is washing of the penis with warm water and disinfectant to further aid in controlling infective agents. However, in most cases, the natural microflora are left as if they were in a natural mating situation. This option is at the discretion of individual breeding praCtices. The stallion should be allowed to dismount the mare on his own as long as the mare is tolerable of the stallion. Insuring the stallion’s safety from an aggressive mare is still very important. The stallion should be allowed to rest in the general area for a few moments allowing him to think about and recover from the collection process. The handler may want to reward the stallion for a good collection by letting him graze on the way back to the stud quarters. The AV and semen should immediately proceed with the following afier the ejaculate has been recovered: ‘ a On the way into the lab, water from the bladder should be released to allow any trapped semen in the pressurized lumen to drain into the collection vessel. b. The container should be immediately removed from the AV and placed into an appropriately warmed incubator (warmed to 370C). And initial analysis completed and an extender added preferably within 5 minutes of collection time. c. Once this is completed, the AV should be dismantled and the rubber liner allowed to soak in a NovelsanTM (chlorohexidine) solution and washed according to the procedures outlined in the previous lab when convenient. NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL lNSEMINATlON PROCEDURES INSEMINATION PROCEDURE UTILIZING FRESH SEMEN NOTES PreQaration Procedure [or Insemination 1. Using a tail wrap or masking tape, wrap the mares tail down to the tail head. 2. Once the tail is properly wrapped, tie the tail up using a bungee strap or have someone hold the tail up out of the way. 3. Preferably, using warm water, thoroughly wash the perineal area of the mare using a clear ivory soap and Betadine mixture, removing any visible debris and/0r fecal material from the area. 4. Rinsing with warm water, thoroughly wash away any soap and other material lathered during the soaping process. 5. Taking dry, clean paper towels, begin drying the perineal area in vertical strips starting at the mid-line working your way out. Never use the same towel to re-wipe a previously dried area, as this will recontaminate the area with the dirty towel. 6. Once wiped down, you are now ready to inseminate the mare. Figure 8: The perineal area being washed and Figure 9: Perineal area being rinsed with scrubbed with soap.5 ‘ water.S Figure 10: Perineal area being dried with clean paper towels.S PRINCIPLES or EQUINE REPRODUCTION Insemination Procedure 1. 44 Pour an appropriate dosage of extended semen into a warmed syringe and place back in the incubator temporarily. DO NOT pull the semen up through the syringe nozzle, as this will damage sperm cells. Put a warmed OB sleeve on and get a pre-warmed individually packaged insemination pipette from the incubator. DO NOT remove the pipette from the plastic casing, as this will increase contamination. Rather, limit exposure to air and pathogens by only opening the end that is to be attached to the syringe. Now, mate the pipette and filled syringe together, and fill the pipette with semen approximately to the tip to remove excess air. Once the syringe and pipette have been mated and air removed, lube the back of the hand and OB sleeve with warm obstetric lube (either KY Jelly or OB lube #2) and quickly proceed to the mare. Figure 11: A properly held syringe and pipette 'filled with semen.5 When ready to inseminate, remove the plastic casing covering the pipette and place the tip of the pipette into the palm of your hand clasping it to protect it as much as possible from dirt and debris that may cause infection (See Figure 4). Wiping the back of the hand containing the OB lube vertically down the vulvar lips, lube the vulva for insemination. Making a wedge shape with the hand clasping the pipette tip, pass the pipette through the vulva into the vaginal cavity, proceed to the anterior vaginal wall and stop. To find the cervix. follow the anterior vaginal wall down to the floor of the vagina where a rose bud like protrusion should be felt. This is the cervix. Depending on the state of estrus. the cervix may vary in position vertically on the wall. Deep estrual mares will have a relaxed cervix on the floor of the vaginal cavity. NOTES TECH 104: SEMEN COLLECTION AND ARTIFICIAL INSEMINATION PROCEDURES 9. When the cervix has been located and still clasping the NOTES pipette, maneuver the pipette tip just beyond the base of the index finger and point the index finger. 10. Push the point of the index finger through the cervix. Depth of the cervix varies depending on the mare. Some are shallow allowing the tip of the finger to pass into the lumen of the uterus and others are thicker not allowing complete passage. 11. Pass the tip of the pipette up along the index finger and through the cervix just beyond the tip of the finger. 12. Once the pipette breaches the uterus, pull the index finger out leaving the pipette in the uterus and allowing the cervix to seal around the pipette. 13. Using the hand in the vaginal cavity, stabilize the pipette by gently massaging the cervix and holding the pipette in place. Massaging the cervix stimulates the cervix and causes a tighter seal around the pipette. 14. Using the free hand, depress the syringe plunger at a moderate, steady rate. Forcing semen out of the syringe may cause damage to the sperm cells. Figure 12: A mare being inseminated with the pipette passed into the uterus} 15. Once the syringe is empty, remove it from the pipette leaving the pipette in place. The uterus may or may not pull the remaining dose in the pipette into the uterus. If the uterus does not pull the remaining dose out of the pipette, pull 1 — 2 cc of air into the syringe and place back on the pipette, depressing the plunger and moving the remain semen into the uterus. 16. When all semen has been passed to the uterus. pull the pipette from the cervix and gently massage the cervix again, stimulating it to seal. 17. Finally, remove your hand and pipette simultaneously from the vaginal cavity. 18. You have now successfully inseminated a mare. ...
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This note was uploaded on 01/16/2012 for the course ANSCI 310 taught by Professor Donhenneke during the Spring '12 term at Tarleton.

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310 Semen Collection - TECH 104: SEMEN COLLECTION AND...

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