Semen collection, freezing, and shipment (Proceedings)

Collection of semen

Fresh or shipped chilled semen

The male is prepared for semen collection by placing him in a quiet area with minimal disturbances. Some males may require the presence of a bitch or even a bitch in heat in order to cause sufficient stimulation to permit semen collection. Semen can be collected with a latex or plastic artificial vagina with a graduated sterile tube attached. The latex collection device used is one sold as a small inner liner for a bull artificial vagina. These can be easily cleaned and sterilized for reuse. It is important to be certain that no soaps, detergents or sterilizing agents remain on the latex since these will be spermicidal. Although prolonged contact with latex rubber has been shown to be detrimental toward sperm in some species, the influence of the latex in this situation does not appear significant since the ejaculate does not remain in contact with the rubber. Sterile nonspermicidal lubricant used in minimal quantities is utilized to lubricate the latex rubber in order to reduce the possibility of injury to the penis and prepuce. The primary injury occurring during collection is the rupture of small blood vessels on the penis and/or prepuce. A graduated tube is superior to unmarked tubes in that one can determine the quantity of the ejaculate without the need to transfer semen to another vessel for volume determination. This information will be necessary in order to calculate the total number of sperm in the ejaculate.

The penis should be prolapsed from the prepuce before erection is complete in order to reduce the possibility of pain from complete erection within the prepuce. The penis is caused to become erect by grasping it proximal to the bulb and applying pressure by placing the fingers and thumb around the penis in this area. Once erect, the penis is firmly squeezed in this same area and then relaxed. This procedure is repeated over and over until ejaculation has occurred. It is unnecessary to vigorously manipulate the penis to cause ejaculation and doing so may cause rupture of superficial blood vessels. Once the urethra begins to pulsate, the first several jets of semen are permitted to go onto the floor. This permits the flushing of the urethra from contaminating the remainder of the ejaculate. The collection device is then placed over the penis and the ejaculate is collected until the seminal plasma becomes clear and adequate volume is present for an insemination. The volume is recorded. The motility is evaluated immediately since this is the seminal characteristic most subject to change. A morphologic examination is performed followed by determination of concentration.

Semen is placed in extender being certain the extender is not detrimental to the semen. It may be necessary and is advisable to collect and test this before collecting to ship. Extended semen is cooled slowly in the shipping container to approximately 6-8° C. Semen is shipped in a sealed tube, which has been placed in plastic bag. It is important that the tube never come into contact with the ice packs. The ice packs are placed in a styrofoam shipping container which is sealed, labeled and contains all necessary paperwork, including information on the female and male, this includes the semen evaluation report and instructions on insemination technique. After receiving the shipment, the semen is warmed to near body temperature before insemination.

Insemination of fresh or chilled semen

After determination of a sufficient number and quality of sperm for insemination, the semen is drawn into an Air-Tite brand syringe that already has 2 cc of air present. The reason for this is that other syringes with a rubber plunger have been shown to be spermicidal and the air is present in order to prevent part of the ejaculate from remaining within the syringe or rod. The bitch is positioned with her rear quarters elevated approximately 18 inches above that of the head. An insemination rod of the In Fu Zee brand is attached to the syringe and inserted into the anterior vagina of the bitch. Although not necessary, it is recommended in some situations to insert a finger into the anterior vagina and use it to guide the rod into the proper position. A speculum can also be utilized to assure proper placement of the insemination rod. The bitch's rear quarters should be maintained in an elevated position for approximately 5 minutes following insemination. During the first 1 to 2 minutes of this time the vulvar lips are massaged to cause the release of oxytocin and thus enhance the transport of sperm.

Cryopreservation of semen

1. Induce ejaculation by means of a digital massage of the penis.

2. Collect semen via a small inner liner into a 15 ml plastic graduated tube.

3. If possible tease the semen donor with an estrus bitch for a few minutes prior to collection.

4. Only use the sperm rich fraction for extension.

5. Commercial extenders are available and should be used.

6. Add extender at 30° C to the sperm rich fraction.

7. Cool semen to 5° C by placing the tube containing semen in a beaker or rack and then transfer to a refrigerator set at 5° C.

8. Two hours after extension and cooling, invert semen three times mix the contents of the tubes.

9. Draw the semen into 0.5 ml straws at room temperature.

10. Remove a small quantity of semen from the end to be sealed.

11. Seal straws with sealing powder and lay horizontally on a polystyrene rack.

12. The nitrogen should be 2-4 cm deep in a polystyrene container.

13. Wait until the vapor clears after pouring the nitrogen before adding the tray.

14. Place rack into liquid nitrogen so that the straws rest 4.0 cm above the nitrogen.

15. Close container immediately after positioning the rack into the nitrogen vapor.

16. Twenty minutes later rapidly plunge the straws into the liquid nitrogen by inverting the rack.

17. Freeze samples for at least 24 hours before thawing a test straw.

18. Thaw semen at 35° C for 2 minutes for examination.

Timing insemination and insemination technique

The timing for insemination is critical since the survivability of frozen sperm once thawed is much shorter than freshly collected sperm. Due to the reduced probability of sufficient sperm passing through the cervix and an increased conception rate, surgical inseminations are indicated. The bitch is prepared for surgery as personal preference dictates and anesthetized. An incision is made on the ventral midline immediately anterior to the pubis. The uterus is located and brought to the incision. The semen in the straws can be withdrawn with a #3.5 French end opening tomcat catheter and a 3 cc AirTite syringe. The blunt end of a large suture needle or a balled end feeding needle is used to put a hole through the uterine wall of one of the horns near the body of the uterus. Immediately upon withdrawal of the needle, the tomcat catheter is inserted toward the ovarian end of the horn. The semen is slowly placed into the uterine lumen with care given to the degree of horn distention. If the volume of semen to be placed into the horn exceeds the capacity of the lumen, the catheter is redirected the opposite direction through the same hole in the uterus. The blunt end of a feeding needle is used to more easily and surely determine the location of the lumen of the uterus. Laparoscopic inseminations can be performed but the technique requires additional equipment, time, and experience and has no advantage other than the size of the incision and elimination of anesthesia. The hole in the uterine wall is pinched for a few seconds before releasing the uterus; no suturing of the uterus is performed.