Semen Collection Methods in Various Species PDF

SEMEN COLLECTION METHODS IN DIFFERENT SPECIES  Various methods of collection of semen have been devised from time to time. The older unsatisfactory methods have gradually been replaced by the newer modern techniques. The three most common methods are: (1) Use of an Artificial Vag...

SEMEN COLLECTION METHODS IN DIFFERENT SPECIES  Various methods of collection of semen have been devised from time to time. The older unsatisfactory methods have gradually been replaced by the newer modern techniques. The three most common methods are: (1) Use of an Artificial Vagina (AV) (2) Electro-stimulation / Electro-ejaculation (3) Massaging the ampullae of the ductus deferens through rectal wall  Suitability of a particular method depends on the type and condition of a species, e.g., AV is applicable on most of our domestic animals but not on poultry, where the massage method is the only practical solution to get ejaculation. The ideal method of semen collection will be the one which is – safe for the sire and collector – the semen obtained is really a representative of a normal ejaculate – free of contamination and – it is protected from thermal shock Age When Semen Can Be Collected: Bull 12-14 months Boar 6 - 8 months Ram 6 - 9 months Stallion 20 - 24 months Dog 8 - 12 months  Volume of semen Species Semen volume (ml) Cow bull : 3-6 Buffalo bull : 2-4 Stallion : 50-150 Ram : 0.5-1.5 Boar : 150-350 Dog : 3-4  Artificial Vagina Method: Semen collection in Cattle and Buffalo: It has been proved to be the best method and is now most commonly used. Several types of artificial vagina are used for semen collection from the bull. Most commonly used cylinders are:- Danish or American model. The AV is consists of (i) An outer heavy rubber cylinder (ii) Inner sleeves of rubber / Latex liner (iii) The semen receiving cone / Latex cone (iv) Semen collecting vial made of glass or plastic which may be graduated (v) Protecting cover  Prior to collection all these parts are cleaned, sterilised and assembled into artificial vagina. Between the rubber liners fill hot water, to bring the inner temperature of 40 to 46 0C.  Semen collection procedure by AV: When teaser is secured properly in the crate the bull should be led to the collection yard. The bull apron should be tied to prevent direct touching of the penis to the hind part of the dummy and possible contamination by it. After the animals are brought to collection arena, before taking collection, the prepuce shall be cleaned externally with a sterilised napkin / disposable sterile paper napkin soaked in normal saline to remove any sand or dust particles. For each bull a separate napkin shall be used. The semen collector should wear apron, gumboot and disposable gloves, which is change at each collection.  The bull with normal libido should be given 3 false mount and restraint for 2 minutes before collection. This will exhibit a strong stimulatory response in bulls, which in turn will donate a good quality and quantity of semen ejaculate. Check temperature of the artificial vagina before collection. Preferably 10 or 12 inch size AVs shall be used for cattle and 8 inch size for buffaloes to ensure semen ejaculated in cone. For buffaloes goat AVs can also be used. The cone shall be of top quality so that semen can get drained quickly in the collection tube.  At the time of semen collection the collector should stand on the right hand side of the bull with holding the A.V. in the right hand at an angle of 450 with ground, keeping valve towards the ground and should stand near the hind quarter of the teaser bull, facing the bull. After excitement, when the bull mounts with fully erected penis, the sheath containing erect penis is drawn sidewise towards the semen collector by cupped fingers of his left hand holding the sheath, thus deviating the erect penis into the artificial vagina. The erect penis should never be grasped with the hand; because the touching of the penis by the collector causes shying of the bull and the bull will withdraw the penis or may give out incomplete ejaculate.  The tip of the glans penis is then just touch to the proximal end of A.V. that is held tilted alongside the buttocks and flank of the teaser. This downward and backward tilting on A.V. will create the needed pressure and warmth in the proximal end of A.V. to stimulate ejaculation. The bull should be allowed to thrust his penis on its own into the A.V., since a strong thrust is necessary for complete ejaculation. Immediately after thrust, when bull dismounts, A.V. should be removed from the penis and its cone is folded and after labeling pass the collection tube to the processing laboratory for further processing. Immediately after collection dip the AV’s in a container having non-spermicidal neutral detergent solution for cleaning.  Care/Precautions to be taken at the time of semen collection: The semen collector should understand the individual bull’s sexual behavior and physiology. The semen collector and bull attendants should not be changed frequently. On the day of collection, before taking semen collection, the bulls shall be properly washed and cleaned. Good quality rubber mat shall be put into concrete groove for adequate cushioning effect during semen collection. Separate AV should be used for each bull and for each ejaculate and even if the bull inserts the penis in the AV without ejaculation the AV should be changed. The same AV shall not be used twice.  To reduce possible loss of sperm do not remove the artificial vagina from the penis too quickly after the bull has ejaculated. Avoid contamination of semen by water, lubricating jelly or other harmful substances. Be certain to identify semen collection tube with the correct bull number. Report any abnormal events to laboratory personnel (for example: bloody semen, leak in inner liner, loss of ejaculate). Both the semen collector and bull handler should avoid sudden moves at the time of semen collection. Avoid presence of a crowd or unknown personnel near the collection site at the time of collection.  To harvest more quantity of semen, interval between two ejaculates shall be kept for 50 to 60 minutes. Wherever it is not possible to give rest for such a long period, the semen station shall keep a gap of 15 minutes between two ejaculates, depending upon the refractory period of the bull. After collection, the area must be thoroughly cleaned and odour-less disinfectant solution (Colloidal iodine) be sprayed. A dusty floor shall be avoided to prevent dust falling on the AV / semen samples. Wherever preputial wash is given the person responsible to carry out preputial wash must use disposable gloves and separate sterilised nozzle for each bull to avoid transmission of IBR infection from one bull to another. Before every collection, the semen collector shall either wash his hands with 0.1% Savlon solution or use disposable gloves or do both.  The open end of sterilised AVs shall be covered with aluminum foil which would be removed at the time when bulls are ready for giving semen. The AV shall not to be shaken after ejaculation; otherwise lubricant and debris may mix with the semen samples. Protective clothing (barn coat) and footwear shall be used by the Veterinarians and personnel coming in contact with the bulls. Footwear and barn coat should be washed daily immediately after completion of semen collection work.  Semen stations shall follow the norm of two ejaculates per collection and two collections per bull per week for taking annually at least 90 collections and 180 ejaculates from each bull. Never mistreat or abuse a bull at the time of semen collection. Too high temperature of AV, forcing of the AV on penis, grasping of the penis instead of sheath are painful conditions leading to sexual suppression. Components of AV American model Danish model  False mounting to arouse bull Semen collection by AV Semen collection on a dummy  Sheep and Goat: Collection of semen may take place in similar manner as in the bull by means of A.V. It is of smaller dimension. Correct temperature of A.V. is 43-440C. Semen collection on a dummy:  Stallion:  Use of an artificial vagina is far and away the most common method for collecting semen from stallions. The AV used with stallions is similar to that used for species such as bulls, except larger.  A popular design of a stallion AV (disassembled).  Due to the large ejaculate volume, a collection bottle is used instead of a tube. The blue cone is an insulating cone that is placed over the end of the AV to shield the semen collection bottle from thermal shock. Stallion semen often contains a large volume of "gel", a gelatinous secretion from the seminal vesicles.  Gel is ejaculated after the sperm rich fraction. To minimize contamination of the semen with this gel, a nylon filter can be inserted into the collection bottle when the AV is assembled; the gel is trapped in the filter and discarded.  Certain combinations of tranquilizers have been used to induce ejaculation from stallions. This technique is useful for collecting semen from stallions that cannot mount a mare or phantom due to musculoskeletal disease. Electroejaculation should not be attempted with stallions  Several models of A.V. have been developed for stallions. The Missouri model is commercially available. The internal temp. for A.V. of stallion should be 45- 500C. A filter on the Colorado model A.V. permits the fluid portion of ejaculate to pass and retains the viscous gel portion. Mares in oestrus and nymphomaniac mares have been the most satisfactory teaser animals. The disadvantages of this practice are that it can be dangerous for the collection team, the mare and the stallion, and there is a good possibility that sometimes the mare will be bred by the stallion. The mare’s tail should be wrapped. A pad may be placed over the mare’s neck to prevent harm from the stallion that bite during copulation. Collection from stallion on alternate days is recommended. An alternative to use teaser mare is to use a "phantom" mount - an inanimate mare look-a-like.  COLORADO STYLE MISSOURI STYLE Retains water temperature longer in Is cheaper to purchase initially cold weather Is lighter and therefore easier to Is more durable under extreme use handle when filled with water Liner is cheaper to replace when worn Will allow for the addition of air to or damaged make the liner tighter If not too long, allows the stallion to Allows a less direct manual ejaculate well clear of the heated stimulation of the penis, which can liner, thereby avoiding danger of be preferable with some stallions sperm damage by heat shock  HANNOVER STYLE ROANOKE STYLE Somewhat of a Nishikawa - Extremely light weight - can be Colorado hybrid. held with one hand. Outer case is made of hard Allows for great bell pressure rubber. on glans penis, which is Has a partially closed distal end preferred by some stallions. for the stallion to push the end Shortness permits manual of the penis against. stimulation of the upper shaft Disposable liners, filters, of the penis at the sensitive collection bottles are available, pressure point located close to similar to the Colorado. the preputial ring. Japanese (Nishikawa): This is basically an aluminium Colorado. It has a small hole in the cap that is designed to vent water and automatically adjust the pressure as the stallion's penis enters the AV. There is also a rubber ring in the end of the AV that is designed for the stallion to push his penis against and supposedly feel more natural.  Collection on a Live Mount: Always wear Helmet Restrain mare properly  Semen collection in stallion on phantom All the involved people should wear helmets. Stallions are large, aggressive, unpredictable and dangerous. They frequently strike, kick and bite. Hence, personnel safety should be given constant attention.  Estrous mare Phantom When a phantom is used for semen collection it is still useful to have a live, estrous mare to tease and arose the stallion. Stallion visiting the tease mare Washing the penis to minimize contamination Stallion ready to mount Stallion Mounting Collecting semen with the AV Stallion resting on mount Semen Evaluation in stallion As for any species, semen should be evaluated immediately following collection for gross abnormalities such as blood. Normal values and ranges for stallion semen are presented below. Parameter Normal Values Ejaculate volume 70 ml (range 30-250 ml) 120 million/ml (range 30- Sperm concentration 600 million/ml) Total sperm per ejaculate Typically 7-10 billion Progressive motility Greater than 60% Morphology Greater than 60% normal  Dog:  Semen is collected from dogs for breeding soundness exams, as well as for artificial insemination. Semen collected for insemination can be used fresh, or can be cooled and shipped to another location. Canine semen can also be frozen, allowing long term storage. Another indication for collecting semen is to obtain prostatic fluid for culture or cytology in cases of suspected prostatic disease.  Semen samples from dog can be collected by A.V. or a rubber directing cone. The cone is an affective method of collecting semen from dogs and is suitable for clinical practice. The cones are placed over bulbus glandis. When penis begins to erect ejaculation is obtained by digital manipulation. The canine ejaculate is delivered in three fractions. – The first fraction is relatively small and contains few sperms. – The second fraction is sperm rich and, – The third fraction is mainly prostate secretion which increase the volume of the ejaculate. The total duration requires 3 to 30 minutes  Semen ejaculation fractions in dog First Fraction : Clear, nearly sperm free Second Fraction : Sperm-rich, white, viscous Third Fraction : Watery, Sperm poor (Prostatic secretion)  Semen can be collected from most males without the need for a teaser bitch, particularly if the male has had semen collected previously. However, use of a bitch will allow more sperm to be harvested. Ideally the teaser should be in estrus, but considering the length of the canine cycle, that is often difficult to arrange, and a friendly, non-estrus bitch will often serve the purpose. If a bitch is used, she should be controlled with her rear quarters facing the male. An alternative means of stimulating the male is to present him with a vaginal swab from an estrous bitch - for convenience, a number of such swabs can be prepared from an estrous bitch and stored frozen until needed.  Swine: The A.V. of swine approximately 12 cm long, 40-42 0C warm and relatively high pressure by air. Dummy sow is used for semen collection. The boar is led to the dummy sow in a collection area with nonslip surface and the dummy sow should be stabilized by fixing to the floor or wall.  Allow the boar to mount the dummy sow and start thrusting without interference so that he commences emptying fluid from his prepuce. The boar has prepucial diverticulum the secretion of which is highly contaminated. Penis of the boar is caught in the funnel of vagina with one hand and rhythmic pressure with the fingers to stimulate the cervical folds of the sow. The ejaculation takes place within 5–10 minutes.  Once the boar settles and starts to ejaculate maintain a grip which allows semen flow. Note the emission of the first gel fraction and pre-sperm clear fraction and allow them to discharge onto the pen floor. Squeeze the penis periodically to further stimulate the boar. Attempt to collect only the milky white sperm rich fraction of the ejaculate in the collection vessel. Ensure a very firm grip as the boar reaches the end of the sperm rich fraction and large quantities of gel are produced, As gel starts to be produced hold the collection vessel to one side, in a position to avoid contamination.  Allow the remainder of the ejaculate to discharge onto the pen floor. Maintain the grip on the penis until ejaculation is complete and the boar has started to retract his penis and dismount from the dummy sow. Carefully remove the filter from the collection vessel and discard it taking care not to contaminate the semen. The volume of ejaculate is 100 – 250 ml when the gelatinous mass is removed. Most boars can be used 2 – 3 times a week at about 100 kg weight. The ejaculated semen is greyish – white and the consistency is lime milk.  Semen ejaculation fractions in boar First Fraction: Watery, sperm free (10 ml quantity) Its importance: Rising the urethral tract which usually has a high bacterial count Not advisable to mix it with semen Second Fraction: Dense, milky, contains 80 % of spermatozoa (40-80 ml volume) Third Fraction: Thin fluid with low sperm concentration This fluid originates from the secretions of seminal vesicles and prostate gland. 150-200 ml quantity Fourth fraction: Gelatinous and secreted by bulbo-urethral glands - Pre-sperm fraction is not used, whereas the 2nd and 3rd fractions are used for insemination.  AV method Advantages: Practically the whole ejaculate is collected in uncontaminated and natural stage. It is free from the extraneous secretions. A sterile condition of the apparatus ensures disease control. The viability of the sperm is better. No female is needed if dummy is a success. Disadvantages: Occasionally it is difficult to get the males to serve the artificial vagina. The apparatus involved is slightly costly and requires technical hands.  Electro-ejaculation method: This method is most useful method of semen collection in bulls and ram in which the A.V. method of semen collection is not possible due to old age or leg injury. This can be also used for untrained bulls in field condition or when the bull is extremely slow in serving the AV. The method is used on males of certain species where the use of AV is not possible or not practical.  By this method ejaculation of semen is brought about by inserting a probe or electrode in sire's rectum and stimulating nerves of the reproductive system by gradually increasing voltage in rhythmic fashion with a rheostat for a short period. Successful use requires skill, experience, patience and the knowledge of individual requirement of the stimulation. At present there are a number of electroejaculators in the market of the world, which are either operated only on battery, or battery-cum-electric transistorized circuits.  Modern models of electro-ejaculators consists of a single rectal probe equipped with bipolar electrodes capable of producing ejaculation by electrical stimuli of the Vesicular glands Ampulae of the vas deferentia Accessory sex glands and The hypogastic plexus The method of semen collection in bull is as follows: At the beginning the rectum is washed with 6 per cent sodium chloride solution. The probe is then inserted up to about 12 inches and held in a position of rectal floor. Alternate current increasing in voltage gradually from zero to 5 volts and returning again to zero within every 5 to 10 seconds is initially passed. The subsequent stimulation made progressively higher so that at about fifth stimulus a maximum of 10-15 volts is reached. Erection and ejaculation occur at 10 to 15 volts when 0.5 to 1 ampere current is passed. The source of electric current is AC/220-250 volts/single phase/50 cycles.  Sheep and Goat: Electrical stimulators with a bipolar rectal electrode that gives a 10- 15 Volt output. The ram is restrained in a lateral position. Straightening of the sigmoid flexure, extends the penis and the glans penis grasped and hold with a piece of gauze swab. The rectal probe is lubricated and inserted into the rectum to a depth of 15-20cm, taking care to avoid injury. The rectal probe is applied towards the floor of the pelvis and short stimuli are applied with intervals. The glans penis shows congestion and with the correct stimulation semen is collected in a prewarmed and sterile tube.  Electro-ejaculation method: Advantages 1. Semen can be collected from males that are too young or old or unable to mount due too weak or injured legs. 2. No female or dummy is required for collection. Disadvantages 1. The methods are highly technical and need considerable skill and practice. 2. The semen generally gets contaminated with urine. 3. Some males resist too much to this method and refuse collection. 4. Sciatic nerves are temporarily affected during the operation but is relatively minor if the electrodes are kept over the ampullar region.  Massage method: The method involves the simplest technique of semen collection by massaging the seminal vesicles and ampullae. Undoubtedly the collector should have a considerable training to adopt the skill. This method is commonly used to collect semen from cock, turkey (tom) and dog and bulls. Ampulla: The terminal part of the vas deferens is enlarged and is called as ampulla. There are no ampullae in dog and cat. The ampullae open in the cranial portion of the pelvic urethra through a rounded prominence called “colliculus seminalis”. Vesicular glands / Seminal vesicle: Vesicular glands are located on the pelvic floor cranial and lateral to the ampullae. They are paired and have distinct lobulations. The duct of the vesicular gland and the ampulla may share a common ejaculatory orifice into the pelvic urethra. In bulls the secretion of vesicular gland is yellow due to high riboflavin contents. These glands are absent in dog and cat.  Technique consists firstly to pickup seminal vesicles with fingers per rectum and than massaging the gland gently and carefully. Massages done centrally & backwardly. Than ampullae are milked one by one from ant. to post. End. Milking of the ampullae gently for 3 -5 minute results in to discharge of semen at the preputial end. Collection tube should be held at the prepuce to collect semen. There is no protrusion of penis during massage and release of semen.  Bull: The second and the simplest method of semen collection is the massage of the seminal vesicles and ampullae. This technique needs the training of bulls for the massage of these organs. Bulls which refuse to mount or are lame due to leg injury can easily be used for semen collection with massage technique. The gloveseminal vesicles are located hand is inserted in the rectum and the. They are massaged gently and carefully from centre to the backward portion. Then the ampullae are milked one by one from anterior to posterior end.  Milking of ampullae gently for 3-5 minutes results into discharge of semen at the preputial end. Collection tube should be held at the prepuce to collect semen. This technique was first used by Miller and Evans(1934). Semen collected from massage technique is not as good as collected in artificial vagina because it contains less concentration of spermatozoa and more bacterial infection.  Dogs: Many males can be induced to ejaculate by applying pressure and massage to the penis. This is the simplest and cheapest method of semen collection in dogs.  Method of semen collection in dog by digital manipulation Grasp the prepuce and pull/push it back to expose the tip of the penis. Slide the collection cone over the protruding penis and slide it over the penis, pushing the prepuce back over the bulbis glandis Lock your fingers in a ring around the penis, essentially holding the bulbis glandis inside your fist. Apply pressure with forward and backward movement; in most cases, the male will begin to thrust back and forth. Watch for semen to flow in the collection tube. Most dogs stop thrusting as they begin to ejaculate. Continue to apply pressure until you observe a crystal clear fluid (prostatic fluid) begin to flow into the collection tube; at that time gently slide the collection cone off the penis. Retraction of the prepuce Collection cone on and pressure being applied After collection. Note the engorged bulbus glandis Normal ranges for seminal characteristics in dogs Parameter Normal Values Ejaculate volume (without 1-6 ml prostatic fraction) Sperm concentration 100-500 million/ml Total sperm per ejaculate 300-2000 million Progressive motility Greater than 70% Morphology Greater than 80% normal  Semen collection in birds Digital massage is the preferred method for collecting semen from birds. Semen is collected by massaging the back of the bird, beginning behind the wings and stroking gently toward the tail with the thumb and forefinger making a slight upward movement at the base of the tail. This motion stimulates erection of the copulatory organ. Repetitive stroking may be required in untrained birds.  An alternative method is to massage the bird’s abdomen. The erection varies from bird to bird. Rough handling may inhibit the reflex and also increase the chances of urination and defaecation. The quantity and quality of semen obtained is depend on the degree of pressure that has been applied to the copulatory ducts. On stimulation, the ejaculatory ducts swell up and then it is possible to obtain semen with very light pressure of the fingers. Fowl spermatozoa are more resistant to temperature shock than those of the bull or ram.  For AI, it is best to use undiluted semen immediately after collection. Best fertility results have been claimed when neat semen is used for insemination within half to one hour after collection.  Massage method: Advantages: - Simplest method - Bulls which refuse to mount or lame can easily be used for semen collection/ reduced libido bulls. Disadvantages -The operated should be experienced with knowledge of location of ampullae and seminal vesicle. - Techniques required skill & practice -Semen collected by this method is not generally so good as in AV method - Semen will be poor in sperm conc. and has poor keeping quality. - It is painful to bulls.