Breeding Soundness Examination of the Dog
287-298

 

Identification

• The identification is not only important for the medical records, but is essential in the case of a pre-purchase examination.
• Failure to positively identify an animal could cause legal problems in the future.

• Has the dog ever been bred?
• Has he ever sired a litter? If has bred, but never sired a litter, you may consider congenital infertility.
• If he has sired litters, and is now infertile you may consider acquired infertility.
• Frequency of use
• Every other day breeding is usually acceptable.
• Breeding more than that may be overusing the dog.
• Has there been any exogenous drug therapy (testosterone etc.)?
• What is the dog's habitat?
• Is he in a kennel?
• Is he housed with bitch? If so, the bitch may be dominant.
• Is he 'mommies little boy', who has not had any canine contact?
• Has he had any exposure to sex?
• Has he been punished for showing sexual interest?
• Has his libido changed?
• Has he ever been shown?
• Does he have any endocrine disease?

• Look for signs of endocrine problems.
• Check for any congenital disorders that would make him an unsuitable potential breeder.
• Reproductive Exam

• Check for shape, size, and consistency.

A bad way to measure dog testes.

A good way to measure dog testes.

• Testicular asymmetry can be indicative of testicular degeneration.
• The normal size of the testes can be estimated using the equation:
  • log Scrotal Width = 0.324(log Body Weight) + 1.249
• Check to make sure he is not cryptorchid. 
  • The testes should be present by 16 weeks. 
  • You can give them 6 months, but they probably will not descend. 
  • There is generally no acceptable treatment, but hCG is cited to help (100-1000 IU 2 times/week for 2 weeks) at less than 16 weeks of age
• Check this link out about testicular prostheses for those of you who are unscrupulous!

• Check for abnormalities, including:
• Blood
• Trauma
• Tumors
• Paraphimosis
• Short Sheath
• Phimosis
• Short Penis
• Stricture
• Priapism - Seen with Spinal Injury
• Os Penis -Fractures, Congenital Abnormalities
• B. canis - covered in bitch notes. The Brucella canis status of the male should be ascertained. The rapid slide agglutination test has many false positives and a true positive titer may wane over time.

• It is recommended that the dog have five days of sexual rest before obtaining a semen sample for evaluation. 
• Conversely, if the dog has not ejaculated within ten days, a higher than normal number of detached heads and distal droplets will be seen because of prolonged storage. 
• This problem will normally resolve if a second ejaculate is collected.

Method
Hand collection or manual stimulation

Click to see 'semen collection'

• Use a rubber cone and collection tube or a disposable plastic cone with end sealed.

• Slide the prepuce over the bulbous glandis. (Erection normally occurs after intromission into the vagina.) 
• Slide the cone over penis and apply a gentle 'stoke' pressure to penis, bulbous.

• Three portions to ejaculate
  • Pre-sperm is the clear, first portion seen during the initial thrusts.
  • Sperm rich - White portion seen during thrusts.
  • Prostatic portion - Clear portion seen when thrusting is done. It may be seen after the 'stepover', when the dogs are rear to rear.
  • The prostatic portion is not needed for fertility. It can be collected to evaluate the prostate.

• An estrus bitch is helpful for collection.

• Sperm numbers have been found to be greater if an estrual bitch is present.
• You can give estrogen to ovariectomized bitch, but there are potential problems doing this.
• You can use the dog pheromone, hydroxy benzoic acid, or ICG's 'Eau d' Estrus', or frozen swabs from an estrual bitch.
• Recent work from VPI shows that 0.1 mg/kg PGF_2alpha 15 minutes prior to semen collection significantly increased sperm output over that of saline or GnRH or oxytocin treated males. No comparisons were made with estrual bitches.

• The swabs usually give a better response than synthetic chemicals. These attempts may help, or may not.
• There may be technique problems in the collectors performance, such as being too vigorous or not vigorous enough.
• The dog may be in an unfamiliar environment, such as a clinic. The distractions at a clinic include the smells, noise, and 'gapers'.
• It may be better to not have the owner present, or it may be better to have the owner present, or it may make difference at all.
• Poor sexual experience, pain with sex resulting from orchitis, prostatitis, lumbar and stifle pain may hinder collection.

• At the 1992 Annual Meeting of the Society for Theriogenology, guidelines for the breeding soundness examination of the dog and a canine semen evaluation form were introduced.
• The first step in semen evaluation is to examine the color of the different fractions (Fraction 1 - F1, Fraction 2 - F2 and Fraction 3 - F3). F1 and F3 should be clear, whereas F2 should be cloudy.
• Yellow, brown or red samples may indicate the presence of urine or blood, respectively.
• Always remember to use warm slides and equipment when examining semen samples for motility.
• A cytologic examination of the non-sperm cells (F2 and F3) can be made by smearing a drop of semen and staining it with modified Wright's (e.g. DiffQuick). White cells indicate an inflammatory process and a culture may be indicated.
• RBC
• WBC
• Macrophage - associated with azoospermia

• The standards set forth by the Society for Theriogenology are not absolute and do not guarantee fertility or sterility, but are set forth as guidelines for the practicing veterinarian to use as a standard method to evaluate a dog's potential fertility.
• Canine semen evaluation forms are available from the Society for Theriogenology.

Breeding with Fresh Semen

• Artificial Insemination requires no special treatment of the semen

  • Always examine the ejaculate

    • Some dogs have grossly good looking semen that has no sperm cells!

  • The prostatic portion is not needed - you may want it to increase the volume

  • You may want to extend to have an easier volume to handle (usually do not want more that 5 to 6 cc.

  • Volumes as low as 2.2 ml and up to 3.6-3.9 ml  have been reported to yield good pregnancy rates.

  • Conception rates should be around 90% if the bitch is bred every other day while in estrus

    • At least  220 x 10⁶cells need to be inseminated to achieve optimum fertility.

    • Vaginal AI on 3 consecutive days after acceptance of the male by the female using 50 x 10⁶ spermatozoa in fresh semen extended 1:4 for each breeding resulted in lower fertility (20%) than AI on 3 consecutive days with 200 x 10⁶  cells of fresh semen extended 1:1 (80%) or natural mating (80 %).

  • Vaginal AI is suffcient

  • It has been shown that if AI is performed twice, rather than once, around the time of best fertility conception rate is significantly improved.

  • There also appeared to be a trend toward increasing pregnancy rates when the number of inseminations increased

  • Transcervical breeding when the dogs have normal fertility is not needed

  • Transcervical breeding has been shown to help when fertility is low

  Breeding with Chilled Semen

• AKC requires the proper paperwork to be completed, as well as DNA identification of the stud and the bitch before a litter can be registered.

• Ovulation timing in the bitch is critical

• It may take 1-2 weeks to train a male to ejaculate in a veterinary office without an estrual bitch present. Therefore, it is ideal for the dog to have had semen collected and to be familiar with the collection procedure long before any semen is actually needed. 

• It is also advisable to prepare a “test shipment” in advance. 

  • A semen sample should be extended, stored a minimum of 24 hours in the container that will be used for transport, and evaluated after 24 hours to ensure that extension and storage do not have detrimental effects on the semen quality. 

  • Not all males’ semen  will respond to extending, cooling and storage in a similar fashion, and this ‘test shipment’ will determine the viability of the sperm after extension and cooling. 

• Not only must the male be trained, but also shipping company schedules and venues to which they ship need to be carefully assessed well before the need arises.

  • Some destinations are serviced by shipping companies and some are best served by counter-to-counter airline shipments. 

  • In some cases shipments cannot be sent out on the appropriate day and in others shipments cannot be delivered on the appropriate day. 

• Semen is analyzed 

• Extension 

  • Appropriate semen extender is added to the ejaculate, usually 1:1. 

  • Semen extenders provide an energy source and buffers that enhance the survival of chilled sperm cells. 

  • Extenders can be obtained from commercial sources that are manufactured exclusively for extending canine semen (Synbiotics, San Diego, CA 92127; CLONE, Chester Springs, PA 19425; Camelot Farms, College Station, TX 77842; International Canine Semen Bank, P.O. Box 651,Sandy, OR  97055), 

  • Equine semen (Lane Mfg., Denver, CO 80231; IMV Intl, Minneapolis, MN 55430) work well and are much cheaper.

  • Homemade semen extenders can also be prepared, however proper laboratory techniques

  • The extender must be pre-warmed before adding it to the semen or the spermatozoa will suffer cold-shock. 

  • The prostatic portion of the ejaculate may have detrimental effects on the storage of canine sperm cells,

  • We found no detrimental effects on pregnancy rate or fecundity when whole ejaculates were extended 1:1 and inseminated after 24 or 48 hours of storage.

  • If the entire prostatic portion of the ejaculate is collected and the semen is extended, the volume of the resulting extended semen may be such that it cannot be completely inseminated without some vaginal reflux.

  • LSU research (EVSSAR 2007 Estoril, Portugal)

    • Semen collected from 6 dogs and extended in INRA 96 for horses

    • Diluted 1:1 and:

      • Not centrifuged

      • Centrifuged at 900 x g 10 min.

      • Stored in Bioflite 24 hr

      • Non-centrifuged sample centrifuged after storage

    • Prostatic fluid and centrifugation have no effect on the membrane integrity of the cells when semen is extended in a commercial equine extender.

    • Semen extended should have the prostatic fluid removed before storage and it should be stored at a relatively high concentration.

    • If a sample does arrive that has a large volume because the prostatic portion was included in the extended semen, centrifugation to remove the prostatic fluid yields progressive motility that is not different from removing the prostatic fluid before extension.

• Containers

  • Containers designed to ship semen can be obtained from many of the same sources that provide canine semen extenders. (Synbiotics, San Diego, CA 92127; CLONE, Chester Springs, PA 19425; Camelot Farms, College Station, TX 77842; Bio-Flite, Anaheim Hills, CA 92807; International Canine Semen Bank, P.O. Box 651, Sandy, OR  97055). 

  • The containers usually consist of a Styrofoam box, an ice pack, and container for the semen.

  • These commercially available semen containers maintain semen well enough that acceptable pregnancy rates result. 

  • Commercially available shipping containers offer a predictable, attractive and easy way to ship semen. 

  • Many of these containers are relatively expensive when compared to the disposable equine shipping containers. 

    • A commercially available (although no longer marketed) disposable equine shipping container allowed storage of extended canine semen for up to 48 hours and the resulting pregnancy rates were equivalent to AI with fresh semen.

    • We have used other brands of  disposable equine semen  shippers and have had good success in maintaining semen viability. 

    • The advantage of the equine shippers is their lower cost than the canine semen shippers

  

• The semen need not be warmed prior to AI. 

• It is recommended to save a small aliquot of the semen for evaluation.

• Results

  • Same as natural breeding if sufficient spermatozoa are inseminated at the proper time. 

  • Vaginal AI on 3 consecutive days  with 200 x 10⁶  cells/breeding with semen extended 1:1 and stored for 24 hours gave the same pregnancy rates as natural mating using the same breeding schedule (80%),

  • Using  50 x 10⁶ cells extended 1:4 and stored for 24 hours resulted in lower fertility (20%).

  • Reports from Sweden that summarized data from chilled semen inseminations show pregnancy rates can vary from 28-60% depending upon the type of extender used.

  • Pregnancy rates are lower when ovulation is timed and the number of breedings is limited compared to the 90-100% conception rates when an average of 3.9 breedings of 250-350 x 10⁶  cells/breeding were used by us.

Frozen Semen 

• AKC requires the proper paperwork to be completed, as well as DNA identification of the stud and the bitch before a litter can be registered.

• Semen FreezingA simple method to freeze canine semen that is currently used by the authors’ laboratory is as follows:

  •  After the semen is collected, a complete analysis is performed. 

  • During the evaluation process, the semen is diluted 1:1 using a commercially available semen refrigeration extender (Refrigeration Media - TEST Yolk Buffer (TYB) - 9972 - Irvine Scientific, Santa Ana, CA 92705-5588) and centrifuged for 10 minutes at 900 x gravity. 

  • After centrifugation the supernatant is removed and the pellet is resuspended to a concentration of 400 x 10⁶ cells/ml using the same refrigeration extender. 

  • This standardized semen sample is then placed in a 5^o C refrigerator for one hour. 

  • During the hour of cooling, an appropriate number of 0.5 ml French straws are labeled with all the data required by the dog’s registry (name, registration number, breed, date, collection facility, etc.). 

  • After one hour, a commercial freezing extender containing 12% glycerol (Freezing Medium - TEST Yolk Buffer (TYB) with Glycerol - 9971 - Irvine Scientific, 92705-5588) that has also been kept at 5^o C is added to the cooled semen solution  at 1:1, v:v, to make a final concentration of  200 x 10⁶ cells/ml containing 6% glycerol. 

  • In a 5^o C cold box, the 0.5 ml straws are then filled and the ends are sealed. 

  • The straws are then placed on a screen that is attached to a 3 cm thick Styrofoam frame, which is floating in liquid nitrogen. 

  • After 10 minutes, the straws are plunged into the liquid nitrogen before storage. 

   

• Frozen semen storage

  • Semen is stored at -196^o C in commercially available liquid nitrogen tanks.

  • Storage and inventory are critical aspects of frozen semen use. Meticulous records must be kept as to the location and number of straws frozen from each male. 

  • The liquid nitrogen must be routinely monitored to ensure that there is sufficient liquid nitrogen to maintain the temperature at  -196^o C. If frost starts to accumulate on the tank, the semen should be transferred immediately to a new tank and the damaged tank discarded.

  • Most dog breed associations are not as concerned about the quality of the frozen semen as they are about identification of the semen. The quality control of the final product is dependent upon on the integrity of the freezing facility. 

  • Frozen semen is usually shipped in ‘dry dewars’. 

    • These are small tanks that do not contain any liquid nitrogen, but keep the sample at -196^o C for a short (e.g. 1 to 2 weeks) period of time.

    • Since there is no liquid in the tanks, the airlines and shipping companies will allow their shipment, unlike liquid nitrogen tanks. 

    • If a liquid nitrogen tank is available at the shipping destination, the semen may be shipped well in advance and transferred to the liquid nitrogen tank upon arrival. 

    • If, however, a liquid nitrogen tank is not available at the insemination site, shipment should be timed in accordance with the intended date of insemination.

  

• Thawing

  • Thawing procedures for frozen semen vary as much as freezing protocols. 

  • We thaw straws at 50^o C for 10 seconds. 

  • Some facilities recommend thawing at lower temperatures for a longer time

  • Some recommend adding thawing media during the thaw. 

  • Best to use the freezing facility’s recommendation

• Insemination technique

  • Vaginal

  • Laparotomy

  • Lapasoscopy

  • Norwegian

  

  

  • New Zealand transcervical

  

  

• Fertility

  • Vaginal

    • Repeated days during the estrous cycle with adequate numbers of sperm cells conception rates of 50 and 60% 

      • 1-12 breedings with 9-300 x 10⁶ cells/insemination,

      • 2 breedings with 132 x 10⁶ cells/breeding,

      • 2 breedings with 200 x 10⁶ cells/breeding

      • 1-6 breedings with 183 x 10⁶ cells/breeding.

      • 100%  when as few as 21 x 10⁶ cells or up to 105 x 10⁶ cells were vaginally inseminated daily during cytologic estrus.

      • Increasing the number of breedings from 1 to 2 increased conception rate and fecundity from 34% to 60%, but a third insemination had no additive effect.

    •  Conception rates with frozen semen using vaginal AI have been less than or equal to those using intrauterine insemination.

  • Laparotomy

    • 90% when 100-300 x 10⁶ cells inseminated once during the fertile period 

    • 60% conception using one insemination of 200  x 10⁶ cells (The 60% conception rate was not different than the 100% conception rate attained by vaginal AI twice with  310  x 10⁶ cells)

    • Many private facilities in the US currently use surgical insemination and report success rates averaging 83% using a single timed AI,

  • Lapasoscopy - not common

  • Norwegian

    • 67% breeding once or twice with an unstated number of sperm,

    • 74% breeding twice with 132 x 10⁶ cells/breeding,

    • 83% breeding twice with 200 x 10⁶ cells/breeding

    • 84% breeding 1-3 times using 186 x 10⁶ cells/breeding

    •  Increasing the number of breedings from 1 to 3 did not increase the conception rate or fecundity

  • New Zealand transcervical

    • 100% breeding twice with 200 x 10⁶ cells/breeding

    • 85% using as few as 50 x 10⁶ cells/breeding

    • 57% using a total of 452 x10⁶ total cells in 2.4 breedings/cycle