Semen Collection, Evaluation and Processing in the Boar
Contents
1. temperatures with the semen Drastic temperature Total Sperm Numbers gt 15 x 109 sperm ejaculate fluctuation is detrimental to sperm quality Gross Motility Unextended Semen Evaluation SE 2 Good quality boar semen is essential to obtaining Table 2 Minimum values of fresh boar semen satisfactory fertility rates Standard tests currently processed and used for Al Source Althouse GC Compend used to evaluate boar semen quality include Contin Educ Pract Vet 19 3 400 404 1997 the 20 maxi i includ topl ic droplets includes both i sperm motility morphology and concentration iia a aan dei odor aa TA When used individually these standard tests have A itis limited usefulness in actually determining the fertilizing potential of an ejaculate These tests do however have the ability to identify ejaculates of overtly poor quality Minimum semen quality values for fresh unextended boar semen processed and used for Al are indicated inTable 2 Most commercial studs evaluate all ejaculates processed through their facilities For some on farm Al laboratories these same routine semen evaluations tend to be impractical because of limitations of equipment skilled labor or time At a minimum it is recommended that initial ejaculates on all new herd Al boars be examined by a trained individual to critically Figure 4 The edge of a microscope slide is used to assess the boar s semen quality Subsequently thinly spread t2. is not intended to be an endorsement to the exclusion of others which may be similar Persons using such products assume responsibility for their use in accordance with current directions of the manufacturer The information represented herein is believed to be accurate but is in no way guaranteed The authors reviewers and publishers assume no liability in connec tion with any use for the products discussed and make no warranty expressed or implied in that respect nor can it be assumed that all safety measures are indicated herein or that additional measures may be required The user therefore must assume full responsibility both as to persons and as to property for the use of these materials including any which might be covered by patent This material may be available in alternative formats PAGE 6 PIG 08 03 01
3. the number of sperm are counted within the defined surface area using a microscope at 200X to 400X A minimum of 5 large 80 small squares are counted in the center grid on each side of the hemacytometer Figure 6 Only sperm heads touching the top and left lines of the large square are included in the count while those touching the bottom or right lines are not counted Tails touching any of the lines are not counted The two counts are then averaged If the two counts vary more than 10 of each other prepare the hemacytometer again and count the two sides until within a 10 variation The number of sperm cells N are then determined by averaging the four counts This number N is then inserted into the formula supplied by the distributor of the counting chamber to determine the number of sperm cells per milliliter of semen The time and tediousness involved with hemacytometric counts make them PAGE 4 PIG 08 03 01 impractical for most Al laboratories Thus photometric analysis remains the most commonly used technique for determining sperm concentration per milliliter of gel free ejaculate Total Sperm Numbers Total sperm numbers are calculated by multiplying the total volume of the gel free ejaculate ml times the sperm concentration per ml Ejaculate volume is measured with a warm graduated measuring cylinder or by weighing the ejaculate assuming that 1 gram weight is equal to 1ml volume Semen Processing Semen extender Most por
4. cine semen extenders come packaged in a powdered form When buying powdered extenders in bulk they should be broken down and re packaged in tightly sealed containers that will make the desired volume of liquid extender If not mixed in the powdered extender preservative antibiotics should be added the day the powdered extender is reconstituted with water Purchased extenders should have production dates be kept in a frost free refrigerator and be used within six months of purchase Preparing extender Extender powder is reconstituted with Type or Il water and incubated at 37 C 99 F in a water bath for a minimum of 1 hour to allow for temperature and pH equilibration To prevent contamination it is best to prepare liquid extender in a plastic single use disposable bag Extending semen Total numbers of sperm per dose of semen tend to range from 2 6 billion sperm concentration of 25 to 80 x 106 cells ml A dose of semen should contain at least 60ml and no more than 120ml total volume 65 85mlI being the most common volumes for a dose of extended porcine semen The final dilution rate of sperm into extender should be dependent upon initial ejaculate quality extender type and anticipated duration of storage time Some facilities employ an arbitrary extension ratio of 1 part semen sperm rich fraction to 7 11 parts extender when storing and using semen within 24 72 hours Optimum extension ratios for each type of extender have yet to be establish
5. ed by the industry therefore this current practice remains questionable If boar semen is to be extended by the volume ratio method a conservative dilution of 1 part semen whole ejaculate to 4 parts extender should be followed with the extended product used within 24 hours of extension Problems that can occur when using the volume ratio method are 1 semen is underdiluted allowing for exhaustion of available energy substrates and buffers over a shorter period of time and 2 semen is overdiluted potentially causing reduced sperm viability and fertility In addition the optimum number of doses of semen is not obtained therefore an economic and genetic loss occurs because the use of sperm cells is not maximized The freshly collected semen and extender should be at similar temperatures for mixing The mixing of semen and extender can be accomplished by adding either semen into the extender or visa versa Semen is diluted with extender using either a 1 i e add all of the calculated volume of extender at one time or 2 i e adding one half the calculated volume of extender to semen allowing it to equilibrate for 5 to 10 minutes then adding the remaining extender to achieve final volume step technique Since the one step process is easier and less time consuming it is the method preferred by many laboratories Pooling semen Mixing or pooling semen from different boars is a popular technique for processing semen for Al The benefits of pooli
6. extender same temperature as the semen can be dropped on the sample before overlaying with a coverslip Sperm motility is then estimated to the nearest 5 by viewing groups of sperm in at least 4 different fields on the slide at 200 or 400X these readings are then averaged Only ejaculates with at least 70 gross motility should be used for further processing This is especially important because sperm motility and viability normally decrease during storage If ejaculates are used shortly after collection samples exhibiting at least 60 motility can be used PAGE 3 PIG 08 03 01 Examination of Sperm Morphology Several stains are commercially available and are essential for examining boar sperm morphology using dry mounted slides Stains accentuate the outline of the sperm when using a light microscope allowing for easier visualization by the observer Higher resolution and more expensive phase contrast or differential interference contrast microscopes have internal components that generate their own contrast Consequently wet mount samples can be used for morphological estimation PREISS ti CEES Ls BASS Se i Saal VT To make a stained slide equal volumes e g 10 ul of stain and sample are adjacently applied to a microscope slide The drops are mixed together using the edge of a second slide The edge of the second slide is used to draw the mixture across the flat slide to produce a thin layer which is allowed to air dry F
7. factsheet Pork Information Gateway Authors Gary C Althouse University of Illinois Donald G Levis University of Nebraska Semen Collection Evaluation and Processing in the Boar John Diehl Clemson University Reviewers Wayne Singleton Purdue University Semen Collection Tom Carr University of Illinois Boars generally show an interest in mounting stationary objects Therefore an estrous female is not required when collecting semen to be used for artificial insemination Al Adjustable height mounting dummies can easily be made or purchased from a supplier of Al equipment Basic requirements for a good mounting dummy include appropriate height for mounting and straddling of the boar s forequarters structural stability and durability Good footing around the dummy is essential to aid the boar in mounting and thrusting and in the semen collection process Rubber matting material with openings is a popular choice because it provides for good footing resiliency to constant use non absorbency and ease of cleaning between uses A separate semen collection area is incorporated into the design of commercial boar studs and most on farm studs The semen collection pen Figure 1 should have at least two or three of the perimeter walls constructed of 2 inch diameter galvanized pipe The pipe should be 36 42 inches in height and placed at 11 to 12 inch intervals on center thus a 9 to 10 inch space is provided between pipes These
8. he mixed semen stain drop across the surface a monthly screening of semen quality from of another slide The slide is then allowed to air dry and then all Al boars should be done during their use sperm morphology is assessed using a microscope Routine examination of Al boar semen quality is very important because it s impact on herd reproductive efficiency is increased many fold when compared to natural mating This examination is insurance against a reproductive catastrophe The costs from using poor quality semen become quite high when considering its effect on herd farrowing rate litter size non productive days and inventory of sows and gilts A record of semen quality should be kept on each boar Table 3 Estimation of Sperm Motility Visual assessment of the percentage of motile sperm by light microscopy is still the preferred Figure 5 A photomicrograph of normal boar spermatozoa method Accuracy of this technique is largely dependent upon the technician s experience and natural ability Sample preparation i e dilution rate type of diluent temperature must be standardized to reduce laboratory error and variation among examinations To estimate motility a small drop of fresh semen is placed on a warmed 37 C 99 F microscope slide overlaid with a coverslip When viewed under a microscope the sample should be thin enough to visualize individual sperm motility If individual spermatozoa cannot be seen a small drop of
9. igure 4 Under oil immersion a minimum of 100 sperm are then assessed and catagorized into one of 3 categories 1 normal sperm Figure 5 2 sperm with abnormal heads and 3 sperm with abnormal tails including cytoplasmic droplets If a large number of sperm in an ejaculate are morphologically abnormal it indicates that a disruption S Wl SNe Torr LN fal 2 Sn ies esra el ae Sat iS G LS of i Center grid Figure 6 Diagram of both top and of some type occurred during the development or maturation of side views of a hemacytometer and the sperm or that the semen was improperly handled Ejaculates of the center grid area where sperm are the general boar population usually exhibit less than 20 abnormal counted in order to estimate sperm sperm Therefore ejaculates accepted for Al use usually contain concentration greater than 80 normal sperm cells Determination of Sperm Concentration The most common way of estimating sperm concentration in gel free boar semen is by measuring the degree of sample opacity Sample opacity is estimated most commonly using a photometer an instrument that measures the percentage of transmittance or absorbency of light through a sample In boar semen sample opacity is dependent upon the number of sperm cells and other ejaculate components which interfere with the movement of light through the sample Boar semen is normally too opaque for light to pass readily through i
10. l from the fluid during ejaculation is important because the gel coagulates into a semisolid mass which interferes with harvest of the spermatozoa semen evaluation and processing Actual time of ejaculation in the boar can vary considerably A minimum of 5 7 minutes is usually necessary for a boar to complete ejaculation Ejaculate volume can be quite large sometimes exceeding 400ml and is dependent upon such things as boar age size collection technique and collection frequency Figure 2 Schematic of an on farm semen ollection pen Notes 1 If young boars walk Attention should be made to protect boar spermatozoa from external insults during and after the collection process Chemical e g latex gloves water soap Figure 3 Application of pressure to the spiral portion residues alcohol etc light i e sun ultraviolet and of the penis for the collection of porcine semen temperature hot or cold insults are detrimental to sperm cells and should be avoided As a general rule anything which may come into contact with boar semen should be clean and dry Single use disposable products are preferred to minimize the risk of exposure to sperm killing compounds and to eliminate PAGE 2 PIG 08 03 01 the chance of cross contamination between boars When collecting and handling semen it Semen Variable is important that semen only come into contact Miley TO orgain y COMSISTENEY with materials extenders that are at similar
11. lable for packaging boar Gel free volume mt _ To J P semen fAbnomalcolr S _ panendina endieemender Conmel don a extended and packaged porcine Motility o oo Too To So semen should be stored at Concentration per mk J T T 14 C to 18 C 57 F to 64 F Normal sperm Storage temperatures above Proximal droplets 18 C for short periods of time lt 24 hours appear to not appreciably affect overt semen quality These higher Tail bent at midpiece temperatures however with droplet are conducive to increased Tail bent at utilization of extender products and bacterial growth Conversely if extended semen is exposed to storage sperm head temperatures at or below 10 C Entire tail coiled around 50 F irreversible damage to the sperm cell may occur in most of the popular extender s used today Stored semen should be gently agitated Distal droplets Abnormal midpiece midpiece Entire tail coiled under sperm head Tail loops under the head and extends from midpiece Short twin tails rotated at least twice a day to Tail without head resuspend settled sperm cells in Head without tail the extender Giant small pyriform elongated or twin heads Loosened acrosome Lost acrosome Knobbed bent over acrosome Loosened neck Table 3 Record of semen quality for fresh neat semen Reference to products in this publication
12. nd his penis out of the sheath Remove the outer vinyl glove from your hand Once the boar extends his penis out of the prepuce the corkscrew shaped penile tip should be grasped with the fingers with uniform pressure applied Figure 3 Some boars require a substantial amount of digital pressure whereas other boars require minimal digital pressure The boar is only responsive to pressure applied around the corkscrew shaped tip of the penis Pressure applied elsewhere on the penis will elicit a negative response causing most boars to dismount With the proper pressure applied the boar will extend his penis and cease thrusting After a brief pause the boar will start to ejaculate Walk alley t Semen Processing Laboratory Heated cabinet A pre warmed 38 C 100 F insulated thermos or styrofoam cup is a convenient and economical semen collection vessel The first few jets of an ejaculate function to flush out the urethra and should be allowed to go on the ground These jets are usually emitted while the boar is still thrusting After thrusting has between the escape pipe place larger ceased fluid and gel components are ejaculated by the boar diameter pipe over the smaller pipe until The gel fraction should be filtered out of the ejaculate during boars get larger 2 A pig board is kept in collection using gauze or a mesh filter which has been placed the escape corner over the mouth of the thermos cup Separation of the ge
13. ng semen include 1 increasing processing efficiency in the laboratory by allowing for a large number of boar ejaculates to be processed simultaneously rather than individually and 2 a means to reduce or even eliminate inherent differences in fertility between boars For pooling semen freshly collected ejaculates are examined for semen quality to eliminate overtly poor ejaculates The ejaculate is then added to a set amount of extender i e 500 ml diluted 1 1 with an extender or fully extended When pooling semen all liquids i e raw semen extender should be at similar temperatures The number of boar ejaculates to be pooled should not exceed the volume capacity able to be processed at one time 3 6 pooled boar ejaculates are sufficient in most production systems After adding the final ejaculate to the pool the entire sample is diluted to its final calculated volume with the remaining extender or fully extended samples are mixed together The pooled fully extended sample is then further processed handled stored using standard protocols Packaging semen Choices for the packaging of the extended semen product are in either bags bottles or tubes Currently there does not appear to be any distinct advantages between the different packaging PAGE 5 PIG 08 03 01 systems with respectto either Baridentifeation eredate sperm ongeviy oriei AgeofBoarinmonths asoras automated systems are Date of Examination f J o Jo T T T avai
14. perimeter pipe walls are a safety feature that allow the handler ease and availability of sites to enter or exit the collection area without opening a gate or scaling a wall but still hold the boar within the pen area The collection pen and its surrounding area should be void of distractions that may divert the focus of the boar away from the collection dummy It may be useful to position the dummy within the pen in such a fashion that boar movement is limited around the dummy and to aid the handler and or collector in directing the boar to the dummy This can be done by placing the dummy in the corner of the pen or attaching it to a wall The recommended width of the collection pen is 6 8 feet and the recommended length is 8 9 feet When using a diagonal escape corner s on one side of the pen a width of 8 feet is recommended Figure 2 A smaller collection pen other than Figure 1 A semen collection pen demonstrating properly con structed perimeter pipe walls good placement of the mounting dummy and use of rubber matting to provide for good footing for the mounting boar PAGE 1 PIG 08 03 01 those described is helpful when training young boars to mount a dummy Table 1 Minimum Contamination Techniques for Preparing and Collecting Semen Sufficient time should be allocated for collecting semen primarily so that personnel do not feel compelled to hurry 1 Periodically trim hair from the preputial opening If needed clean prepu
15. t Therefore a small sample of boar semen is usually diluted into an isotonic solution before taking a measurement The photometric measurement is then converted into sperm numbers ml either internally by the photometer or by the producer using a conversion chart which accompanies the instrument For this photometric measurement to be relatively accurate it is necessary that the instrument be calibrated specifically for boar semen Because of inherent differences between instruments photometric conversion charts are not interchangeable between instruments Periodic recalibration of the instrument is necessary to maintain accurate readings Inaccuracies in photometric measurements can occur if readings fall outside the optimum operating range of the equipment human error e g incorrect dilutions improper warm up time solution mishandling and innate differences among boar ejaculates It is important that manufacturer recommendations be followed on the use of the instrument for determining sperm concentration in boar semen Another method of directly determining sperm concentration in boar semen is by using a counting chamber e g hemacytometer The surface of the counting chamber is etched to outline a defined surface area After diluting a portion of semen to a 1 200 ratio a very small portion of this mixture is transferred onto the counting chamber Avoid overfilling After allowing 5 minutes for sperm to settle onto the surface of the chamber
16. tial opening and surrounding area with a single use disposable wipe i e diaper wipe the boar to mount beyond his comfort Aggressively evacuate preputial fluids from the prepuce level Introduce the boar to the collection manually prior to grasping the penis for semen collection area and let him investigate Once the boar Have the semen collector wear disposable vinyl gloves or identifies and investigates the dummy use an evaporative hand cleanser between boars to mini he should readily mount After the boar mize contamination of semen and reduce risk of cross has become interested and mounted the contamination dummy approach the boar from the rear Hold penis perpendicular to the boar to minimize the Semen should be collected using sanitary chance of preputial fluids to run down the penis and into t chni t wo tamination Tabl the semen collection vessel ecnnigqueS t0 MINIMIZE contamination yanig Allow the first few jets of an ejaculate i e pre sperm frac 1 With two vinyl gloves on your hand tion which contains urethral flushings urine to go on the gently reach around to the boar s prepuce ground rather than into the semen collection vessel and massage the penis through the prepuce Dispose of rubber band and filter gauze before passing This helps to evacuate preputial fluids and collected semen through to the laboratory for processing aids in stimulating pelvic thrusting The boar will then start to thrust and exte
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