Artificial Insemination With Fresh Semen...

 
Semen Collection
Ideally, several days of sexual rest should be allowed prior to semen collection and evaluation.
Canine semen is most easily collected using masturbation with a gloved or bare hand, although some operators find the process distasteful and prefer to use an artificial vagina (AV). However, most artificial vaginas are made of latex and most latex is spermicidal. In addition, these artificial vaginas cannot be gas sterilized because the residues of gas sterilization are also toxic to spermatozoa. If an AV is used, it should be cleaned by washing and chemical disinfection then rinsed several times with distilled water and air dried.
The artificial vagina and attached plastic tube are warmed to body temperature and lubricated with a small amount of sterile aqueous lubricant.


It is much easier to obtain an ejaculate when an estrual bitch is present; therefore owners should be asked to bring in teasers when appointment are made. Alternatively, a non estrous bitch of the same breed or size may be used. A commercially available pheromone (methyl paraben "Eau d'estrus, Synbiotics corporation phone 18584513771, fax at 18584515719 ) may be used to stimulate the male but we have no experience in its use. It is sometimes very difficult to collect semen if no bitch is available at all can it can be done.


Optimally, the male and female are brought together on leashes in a quiet room with nonslip flooring. As the dog sniffs at the bitch's vulva or mounts her, the collector quickly moves the prepuce back, behind the bulbus glandis and directs the tip of the penis into the AV, held in the left hand. Once the artificial vagina is slipped onto the penis, the right hand is used to hold the artificial vagina onto the penis while exerting firm pressure around the back of the bulbus glandis. Once this occurs, the dog will usually show pelvic thrusting and normal ejaculation. An AV is certainly not essential to collect semen from dogs. Excellent ejaculates can be obtained by hand collection alone.
If a gloved or bare hand is used instead of an AV, the dog is masturbated rapidly for a few seconds until he gains a full erection. In the process, the prepuce is slipped behind the bulbus glandis. Masturbation ceases and the hand held behind the bulbus glandis using very firm pressure, until ejaculation is complete. The other hand is used to hold a plastic bag over the end of the penis.


Almost any warm receptacle can be used to collect the semen but most commonly sterile "Whirlpak" bags are used. Syringe casings and other hard objects should be avoided as the penis is very easily traumatised during collection and substantial bleeding may occur into the ejaculate. This does not seem to decrease fertility in dogs (Cf horses) but it interferes with semen evaluation and of course, alarm owners.
Semen can be collected when the bulbus glandis expands within the prepuce but some dogs object to this. Therefore, it is usually best to be sure that the bulbus glandis is out of the prepuce before it expands.
The reader can see therefore than the term "masturbation" is somewhat misleading. Most of the contact time consists of pressure exertion behind the bulbus glandis; a process identical to that used with an artificial vagina!


Ejaculation occurs intermittently over a variable period, perhaps five to 15 minutes, usually just long enough to deprive the collector (squatting on the floor) of all blood flow and feeling to the legs.
If pressure is maintained firmly around the bulbus glandis, pulsations can be palpated in the urethra. The anus will also be observed to contract in a rhythmic fashion. The dog may stop ejaculating for several minutes then pulsations will resume.


Initially, a few drops (one to 3 ml) of clear to slightly cloudy pre sperm fraction are ejaculated, followed by a whitish spermrich fraction (0.1 to 6.0 ml) but most often these fractions are mixed and only a homogeneous light greyopalescent ejaculate is obtained. The collector should try keep one hand around the collection vessel to keep it near body temperature. This is easiest when a plastic bag is used as a collection vessel.


Soon after the dog begins to ejaculate, he will often lift his hind limb as though attempting to step into the rumptorump position that occurs during natural breeding. If this is observed, the collector should allow the dog to step over his/her arm so that the penis then extends out caudally from the dog. Soon the clear, third fraction of the ejaculate (mostly prostatic fluid) is ejaculated increasing the volume to as much as 60 ml. If the semen is being collected for artificial insemination as well as evaluation, enough prostatic fraction is collected to bring the total volume to three to 10 ml so large numbers of sperm are not lost in the insemination process and the insemination volume is comfortable to work with. Frequently, only a few ml of semen are collected but total sperm numbers, not semen volume, is what is important in A.I.
After collection is complete, the male is observed until his erection subsides. Paraphimosis may occur following collection, so the dog must never be kennelled or sent home until the penis is completely inside the prepuce. To prevent paraphimosis, one should lubricate the preputial opening liberally after semen collection.


Semen evaluation
Semen should be kept at a 35 to 370C until progressive motility has been determined, after which it can be allowed to cool to room temperature. Semen volume is not important, but it is necessary to record the volume of the spermcontaining portion so that the total number of sperm per ejaculate can be calculated. Sperm cell motility, morphology, and concentration are determined in the conventional manner.


Normal prostatic fluid makes a good diluent if one is required for motility estimates. It can also be pooled and frozen for future use as a diluent for per vagina insemination of frozenthawed semen (a relatively new approach to the use of frozen semen).


A normal canine ejaculate:
Colour: Opalescent to milky white with a clear prostatic supernatant or homogeneous greyish white.
Volume: Pre sperm fraction: 0.1 to 3 ml, Spermrich fraction: 0.1 to 6 ml Prostatic fraction: one to 50 ml Total volume: one to 60 ml
Progressively Motile Sperm: 60 to 90%
Number of Sperm per Ejaculate: 200 to 3000 X 106 (the population of the United States)
Morphologically Normal Sperm: 70 to 90%
Bacteria: Many; usually more than10,000/ ml. However, only the presence of many white blood cells is an indication for bacterial culture of the semen.


The presence of epithelial cells, red blood cells, inflammatory cells, and germinal epithelial cells are noted under low magnification. All cells other than sperm (COTS) are easy to see if a smear is stained with WrightsGiemsa or DiffQuick but difficult to differentiate using common sperm morphology stains.


Semen from males that have not ejaculated recently may contain more epithelial cells and debris than semen from a male that is used frequently but if large amounts of debris or dead sperm are present, a second sample should be collected 24 hours later.


Artificial insemination
Artificial insemination is performed when behavioural problems prevent mating (especially female dominance) when mounting is difficult or unlikely due to orthopedic problems or inexperience, or when transported or frozen semen is used.


Semen is collected from the dog using masturbation or an artificial vagina and only the spermrich fraction and a small amount of prostatic fluid is used (total volume three to 10 ml).
Semen collection should not be more frequent than once every two days. Daily ejaculation results in very low concentrations of ejaculated sperm after five to seven days.


Artificial insemination of the bitch is most easily performed by depositing the semen in the cranial vagina with a Cassou sheath shortened to about 25 cm. These sheaths are normally used to cover the rigid A.I. Cassou rods used for inseminating cattle. They are available from any veterinary supplier or A.I. cooperative. They are soft and flexible and therefore, far superior to the rigid plastic cattle inseminating pipettes sometimes used. In addition, they fit directly on a syringe and do not require adapters like the rigid pipettes. The Osiris apparatus from France is recommended by some operators but its superiority to other methods has not been demonstrated.


In all cases, the vulva is washed, rinsed and dried. The pipette is then inserted into the vagina, first dorsally for several centimetres, then cranially until the cervix is reached. If resistance is felt, the pipette is withdrawn one or two centimetres, then reinserted at a slightly different angle. The vagina of the bitch is long and an insemination pipette may have to be inserted to a depth of more than 20 cm in large bitches. The hindquarters of the bitch are then elevated so that the spinal column is at an angle of 45 60 degrees and held there for as long as possible, up to 10 minutes.


One excellent study demonstrated that this was advantageous for sperm transport. Some inseminators also stroke the dorsal wall of the vagina with a gloved finger (æfeathering') or massage the clitoris for about one minute when the hind quarters are elevated. This may promote semen transport within the uterus but it has not been objectively studied. Along the same line of thought, after insemination, the bitch should not be allowed to squat or jump for another 10 to 15 minutes.
It is usual to inseminate the total volume of undiluted ejaculate and prudent to use the ejaculate as soon as possible after collection. Insemination doses should contain at least 200 million motile sperm because fertility decreases with ejaculates containing less than 50 million live cells. Single estrus pregnancy rates up to 90% can be achieved with A.I. using fresh semen in fertile dogs. Optimal extension rates for dogs have not been studied but experience with horses suggests that it should be extended at a rate of between 1:1 to 1: 6 (semen: extender) for transport or if it is to be used more than an hour or two after collection. In most cases, a dilution rate of one part semen to two parts extender works well. It is also packed and transported in cooling containers like those used for equine semen transport. The "Equitainer" (Hamilton Thorne; sales@hamiltonthorne.com) cools the semen at 0.3oC/minute maintaining better motility than other cooling rates. Perhaps more important, this container has the best insulation on the market, a consideration when semen is to be transported through various temperature extremes.


Various semen extenders can be used for canine A.I. with satisfactory results. However a simple and effective extender can be made for shippedcooled semen by heating skim milk to about 95 o C for ten minutes in a double boiler. It is then cooled to 37 o C for use. The heating step denatures a spermicidal albumin component in milk. It can be frozen in 50 ml aliquots for several weeks but its exact shelflife is unknown. A specific canine extender called "Fresh express" is available from the Synbiotics corporation (phone 18584513771 or fax at 18584515719). Commercial extenders used for equine semen transport can also be used; for example Kenney's or "EZ mixin" extenders (arssales@dupreeinc.com or mntubcan@execulink.com) but no data exist to show that any of these extenders are superior to the others.
The extender is warmed to exactly the same temperature as the semen and slowly added to the semen until the final dilution rate is obtained. The semen is then packaged according to the instructions that come with the shipping system. Often this involves just placing syringes loaded with semen into a styrofoam shipper. The method used for the Equitainer is more involved.


Some operators recommend warming the chilled semen to room temperature just before insemination but this recommendation is not universal. It is a good idea to evaluate the motility of the semen when it is inseminated just so its status at the time of receipt is known. Occasionally all the sperm are dead! Motility should be evaluated in a drop of semen warmed to 370C.


It is recommended that a longevity trial be conducted with the semen of any male animal (any species) before it is shipped over long distances in the chilled state.
Pregnancy rates of 50 to 60% higher have been reported with the use of chilled semen