Artificial insemination in Dogs : A Useful Tool in Breeding and Conservation

 

Artificial insemination in Dogs : A Useful Tool in Breeding and Conservation

Artificial insemination can provide many benefits to breeding both in canines and other species. It allows the use of semen from stud dogs around the world without the requirement to transport the dogs, thereby opening up the possibilities of genetic diversity within a breed. The techniques required to perform insemination are complex but an invaluable tool in the breeding world.Canine breeding is a rapidly growing industry and there is influx of exotic breeds of dogs into India for breeding purposes (Singh et al., 2019). Among different assisted reproductive techniques, artificial insemination (AI) is one that involves collection of semen from a stud male and introducing it into genital passage of a female or female reproductive tract, so that fertilization can occur in the absence of natural mating (Mason, 2018). In animals and humans, AI is one of the earliest techniques employed for assisted reproduction. It took longer period to be implemented and standardised in pet dogs due to species specific particularities. Abbe Spallanzani performed first successful AI in dogs in 1780. By 1950’s, the use of stored dog semen for AI was practiced. Later by 1990’s, the technology of frozen semen was developed for breeding practice in dogs (Foote, 2002; England and Millar, 2008). Currently, there has been a huge development in reproductive biology and biotechnology. However, initial efforts to improve AI failed, mainly due to the two limitations i.e., unexplained reproductive physiology of dog and unresponsiveness of dog sperm to freezing as opined by Hermansson and Forsberg (2006). The most common reasons attributed for AI in dogs are that the male or female may be shy, inexperienced or too aggressive. There may be presence of obstructions in the genital passage such as vaginal band, small hard mass at vaginal floor. Other possible reasons may be that both male and female would have grown together and know each other to aid in controlling the risk of sexually transmitted diseases on either gender (Johnston et al., 2001).

Artificial insemination (AI) is a method of introducing semen, previously collected from the male, and depositing it into the bitch’s vagina or uterus to achieve a pregnancy.There are many reasons that AI in the dog may be required (Figure 1). For example, it could be that a planned mating has not resulted in a ‘tie’, the male or female are inexperienced or the male, while still fertile, is not physically able to mate the bitch, or it could simply be that semen has been imported from another country to enable breeders to keep their gene pools diverse.

Figure 1. Guide dogs will use artificial insemination as a method of conception only when all attempts to achieve a normal mating have been tried. Conception rates are higher when a normal mating has been achieved. This litter will be monitored for their health and temperament throughout the first 12–14 months of their puppy training, to assess their suitability to become a guide dog, at which point, all being well, they will enter their formal training period.
Occasionally, and particularly if the reason for insemination is due to the inexperience of the male, it may not prove straight forward to collect a good semen sample. Therefore, ideally, before insemination is considered, the male should be proven fertile either by means of a fertility test or from a previous successful mating, and the dog should have been collected from at least once so that the process of collection is familiar to him. Unfortunately, fertility testing is not usually offered as a service in private veterinary practice, therefore, if a fertility test is required it is likely this would need to be arranged with a reproductive veterinary specialist.

AI must always be performed by an experienced professional. Incorrect collection techniques could cause pain to the male and could prevent a collection being successful or future collections being successful. A painful experience could also lead to difficulties in future matings. Improper insemination technique could cause pain to the bitch or result in damage to her vaginal tract. Improper insemination techniques could also result in failure to conceive in that breeding season. In the UK, in order to register a litter born from AI, the Royal College of Veterinary Surgeons have advised that AI is a veterinary procedure and therefore should only be performed by a veterinary surgeon (Kennel Club, 2012).

Any breeder wishing to register a litter of puppies as the result of AI should first refer to the Kennel Club’s regulations surrounding the use of AI. The Kennel Club will not register a litter resulting from AI unless certain criteria are met. For example, the General Committee will not normally accept an application to register a litter if the donor male is alive and domiciled in the UK, with one exception, namely that Irish Wolfhounds of 8.5 years or older and domiciled in the UK may be used as donors in AI (Kennel Club, 2012). Refer to the Kennel Club’s website for further information (http://www.thekennelclub.org.uk/item/478).

Methods of AI

There are three main methods of AI: vaginal insemination; trans-cervical insemination [TCI] also commonly known as uterine insemination; and surgical insemination. In addition, and depending on the time delay between collection and insemination, the semen may need to be preserved. Therefore, there are also three semen types: fresh semen; chilled semen (preserved with an extender and kept chilled); and frozen semen (preserved with an extender and cryoprotect-ants and stored, frozen in liquid nitrogen) (Table 1). The extender is a liquid that contains buffers, sugars and cryoprotectants to nourish and protect the sperm while they are kept chilled or frozen.

Table 1.

Survival time of semen once collected and where applicable, stored with an extender.

Semen type	Survival time (approximately)
Fresh semen	12 hours at room temperature
Chilled semen	4 days (kept chilled at 5oC)
Frozen semen	Theoretical indefinite life at a temperature of -196oC

Due to the varying survival times of sperm within the semen types (Table 2), some methods of insemination will be unsuitable (Table 3).

Table 2.

Survival time of semen in the reproductive tract after insemination

Semen type	Survival time In reproductive tract (approximately)
Fresh semen	7 days
Chilled semen	2 days
Frozen semen	(post thaw) 6 hours

Table 3.

Type of Insemination suitable depending on semen type.

Semen type	Type of insemination suitable
Fresh semen	Vaginal insemination
Chilled semen	Vaginal insemination
Frozen semen	Trans-cervical/ uterine or surgical insemination

The timing of the insemination should also be carefully considered depending on the type of semen used and the method of insemination chosen. For example, due to the relatively short survival time of chilled and frozen semen, it is imperative that the bitch has already ovulated within the preceding 24–48 hours, thus ensuring the ova are available to be fertilized immediately. There are several methods available to enable the optimum mating time to be detected. One of these methods is blood hormone levels, the most common type of testing available is serum progesterone levels, most commonly the qualitative test. This test detects a colour change in the sample which gives the user a result of low progesterone, intermediate progesterone or high progesterone levels. These will give an indication of the level of progesterone in the blood and therefore detect ovu-lation — at high progesterone levels (usually 4–8 ng/ml) ovulation occurs (two matings are preferable, 48 hours apart). Vaginal cytology is another method available. By looking directly at the cells that line the vagina and the changes that happen, it is possible to detect the optimum mating time.

Semen

A dog ejaculates in three distinct fractions. The first fraction is clear fluid and originates from the prostate gland; its function is to flush any urine or cell debris from the male’s urethra (Simpson et al, 1998).

The second fraction is the sperm rich fraction, usually cream in colour, containing millions of sperm and the fraction that is required for insemination (Simpson et al, 1998).

The third fraction is again clear fluid, originating from the prostate gland and its function is to wash the sperm through the bitch’s cervix into her uterus and forwards into her uterine tubes where fertilization will take place (Simpson et al, 1998). However, while the second fraction is used for insemination, the third fraction is not used as a flushing fraction. This is replaced with sterile saline, to ensure there is no chance of any detriment to the sperm.

Semen collection:

The most commonly employed methods of AI are artificial vagina, manual collection and electrical stimulation (Payan-Carreira et al., 2011). In the present study, the semen collection was performed using manual collection. For semen collection, male was placed in quite, non-stressful environment and adequately spaced, non slippery surface for footing. Semen collection was performed using digital pressure and massage technique as recommended by Jahangirbasha et al. (2018). In those dogs which did not show sexual interest, semen collection using digital manipulation was followed using the she dog in estrus being positioned infront of male dog and aiding it to mount the female. When the male dog exhibited sexual interest, using a gloved hand, the preputial skin was pushed caudally, exposing the glans penis. The base of the penis behind the bulbous glandis was grasped through prepuce and firm and constant pressure was applied by the palm and fingers. Pressure was continuously applied with backward and forward movements until erection was brought (Fig. 1). The ejaculated semen was collected in a sterile test tube for further evaluation.

Things to remember

There are a number of things to remember when carrying out AI:

• The chemicals present in tap water can be toxic to canine sperm and will kill them. Ensure any equipment is free from contamination with tap water.
• Syringes with rubber stoppers should be avoided since some types of rubber can be toxic to sperm.
• All equipment should be warmed to body temperature. Sperm are susceptible to heat changes and their motility will slow as semen cools.
• After collection, handle the semen carefully but inseminate as soon as possible (see Table 1) to prevent cooling of the semen and subsequent slowing/ reduced motility of the sperm, thus ensuring the semen is in the best possible condition.
• One ejaculate is required for each insemination.

Step-by-step guide to artificial insemination

Equipment required

The following equipment are required for AI:

• Heated plate
• Semen collection vessels (clean, dry funnels and test tubes)
• Syringes, 5 ml x 2
• Sterile quill
• Insemination catheter
• Sterile saline
• Surgical spirit on cotton wool swab
• Gloves.

Procedure of Artificial Insemination:

Female was placed in quite, non-stressful environment and adequately spaced non-slippery footing surface. In order to facilitate easy insemination, the she dog was positioned as per the method described by Jahangirbasha et al. (2018). The hind quarters of the she dog was elevated to an angle 45 to 60 degree from the surface of the examination table with the help of an assistant (Fig. 2). Perineal area of the vulval lips was cleaned with antiseptic solution and vaginal speculum was inserted aseptically. Sterile AI sheath was then introduced as much as possible to the level of external os of the cervix. Another assistant was directed to connect the free end of the AI sheath with the syringe pre-loaded with the semen. The semen was then injected slowly to the level of external os. The she dog was maintained in the required position for 10 minutes to prevent the back flow of the semen.Insemination should be repeated after 48 hours, providing the bitch is still in oestrus. This will maximize the coverage of the fertile period in the bitch and maximize the chances of conception (Simpson et al, 1998).

AI is used for a number of reasons, and it is essential that it is carried out by an experienced professional/practitioner in order to be successful and without problems. The correct timing is key to the success of AI, and survival times of semen and timing of ovula-tion should be carefully coordinated.

Key Points

• Artifical insemination (AI) is a method of introducing semen, previously collected from the male, and depositing it into the bitch’s vagina.
• There are three main methods of AI: vaginal insemination; trans-cervical insemination; and surgical insemination.
• There are three types of semen: fresh semen, chilled semen, and frozen semen. In AI only the second fraction of the dog’s ejaculate is used.
• The timing of insemination should be carefully considered and only carried out by an experienced practitioner.

Practicalities of canine AI

There are some practical reasons why dog breeders may prefer to use AI as a breeding method instead of natural mating methods (this list is not exhaustive):

• AI allows the use of semen from stud dogs worldwide. And eliminates the need to transport stud dogs across the globe to mate a bitch. As the stud dog would not need to travel, this would eliminate transport-associated stress.
• AI is sometimes elected as a conception method when there have been unsuccessful natural matings, failure to conceive naturally, or if the bitch or stud dog is inexperienced.
• Frozen semen provides an opportunity to pass on ‘superior’ genetic traits from stud dogs who are no longer with us; allowing their desirable traits to be passed on to future litters.
• AI reduces (although does not completely eliminate) the spread of sexually transmitted diseases. Because the AI method eliminates physical contact, it will prevent the spread of infections and devastating diseases; such as Brucellosis, canine herpes virus (CHV) and canine transmissible venereal tumour (CTVF).
• Practically, AI can lead to widening of gene pools as genetic traits are no longer restricted to narrow geographical regions.

Weaknesses/disadvantages of canine AI

There are also many welfare and ethical concerns with canine AI; and there are disadvantages with this method of breeding (this list is also not exhaustive!):

• There is a risk of direct physical or psychological trauma from the AI process. Inexperienced or incorrect AI methods may lead to complications and infection. Surgical AI in particular poses a high surgical procedure risk. Some argue that this procedure does not consider their best interest.
• Given that semen can be frozen and used to pass on certain genetics for many years, there is a risk of passing on undesirable traits and genetic disorders for a very long time.
• AI is sometimes a chosen method of breeding in bitches who are unable to conceive naturally due to anatomical or breed conformational reasons. Breeding these dogs with suspected hereditary traits using AI is a major welfare and ethical concern as there are clearly undesirable traits that are likely to be passed onto their offspring, potentially affecting their health in the future.
• Animal welfare remains hugely topical because AI poses risks of the transmission of hereditary diseases and conditions.

 

Introduction and quality criteria of native semen

The first recorded and successful artificial insemination (AI) was performed in the dog in 1785 by Spallanzani, the first successful use of frozen canine semen was reported by Seager in 1969. However, only in the last two decades AI in the dog became a rather widespread and generally accepted breeding technique. It may be applied for medical reasons, to prevent infections, to overcome travel and quarantine restrictions and to avoid long, expensive and stressful travels with the animals. In conjunction with cryopreservation AI is especially indicated to build up stocks from excellent studs and for conservation of semen from rare breeds. Only semen meeting the quality criteria outlined in table 1 should be used for conservation. The same quality criteria apply when studs are examined for breeding soundness.

Semen collection

The most common method for semen collection in dogs is by manual dissemination, needing no further elaborated equipment. It is generally no problem in young and possibly hypersexual and in trained dogs; it may be problematic in experienced males used for natural breeding. In these cases the presence of a teaser bitch at the optimal stage of oestrus may be essential and efficient stimulation may only be obtained when the stud is allowed to mount. To our experience the preparation of bitches not in heat with methyl, a component of vaginal secretions of female dogs in oestrus, has no satisfying effect on male dogs not responding to manual stimulation. Semen collection should be performed in a quiet and non-distracting environment. Typically, canine ejaculates are collected as three fractions: a small pre-sperm fraction, the sperm rich fraction and a more voluminous post-sperm fraction mainly provided by the prostate. A lack of the milky sperm rich fraction may be due to hypo-/azoospermia, obstruction of seminiferous ducts, retrograde ejaculation or an irregular course of the ejaculation reflex due to insufficient stimulation. Retrograde ejaculation may be confirmed by the detection of numerous spermatozoa in urine sediments after mild centrifugation. First results suggest that in dogs alkaline phosphatase activities in the sperm rich fraction can be used as a marker to monitor depletion of epididymal sperm stores during semen collection, as this enzyme is highly expressed in the epididymal tail (Gobello et al., 2000). In the dog semen quality in general is not improved in a short termed second ejaculate; however, 70% more spermatozoa may be obtained compared to a single ejaculate (England, 1999). Alternatively, canine semen can be collected applying electroejaculation under general anesthesia, and pregnancies have been obtained after the use of cryopreserved epididymal sperm, even after postmortem extraction (Marks et al., 1994, Hori et al., 2004).

Semen preservation and dose for insemination

Except for transfer of native semen only the sperm rich fraction should be used for semen preservation.

Native Semen

Native semen may be transferred due to medical or behavioural reasons without any further handling. The ejaculate may be split, however, transfer should be performed as soon as possible after semen collection and evaluation, although fertilization rates of 70% have been observed with unextended canine semen after two days of storage at 4°C (Tsutsui, 2002).

Table 1. quality criteria for dog semen (Hoffmann, 2003)

forward motility	> 75 %
pathomorphology	< 20 %
pH-value	6.2-7.2
total number of spermatozoa	> 0.3-1.0 x 106 (1)
volume	0.5-2.0 (2)

• depending on breed; e.g., Dachshund >0.3; Dobermann >1.0
  2. Sperm rich fraction

Chilled Semen

The use of chilled semen requests the availability of the donor on a short call and semen transport within a short term. A number of semen extenders has been described; generally accepted is a tris-buffered solution containing 20% egg yolk, 12.5% fructose and an admixture of an antibiotic. The cooling down to about 5°C is over a period of 2 hours. Depending on the concentration of spermatozoa, the ratio (volume) of ejaculate to extender may vary between 1:3 to 1:5. Mild centrifugation might become necessary when a selective collection of the sperm rich fraction was not possible. The total dose is stored in an appropriate sealed vial. The whole sperm rich fraction may be extended and used as one dose. If the ejaculate has to be split, one dose should at least contain 100-200×10⁶ spermatozoa (Linde-Forsberg, 1991). The fertilization capacity of chilled semen decreases progressively, and it may be difficult to estimate the time after which the use of a certain semen portion may become unpromising, as after 5 days of storage motility may be still high, when fertilization capacity has considerably decreased (Tsutsui et al., 2002).

Frozen Semen

Whereas pregnancies may be obtained with fresh semen of inferior quality, albeit with smaller litter sizes, insemination of frozen semen of poor quality is unpromising. Thus, raw material of adequate quality and appropriate preservation procedures are indispensable for acceptable results. There are various protocols for cryopreservation of canine semen. Basically the same extender as used for the preparation of chilled semen may be used. The concentration of the cryoprotectant glycerin is at 6%. In our clinic semen meeting the criteria given above is diluted and packed in 0.5 ml straws. Following cooling to 5°C over 2 hours they are left for 10 min at-140°C (vapor of liquid nitrogen). Final storage is at-196°C. It is an absolute requirement to test the quality of each individual batch by thawing (37°C; 20 seconds) and reexamining a frozen aliquot, as the suitability for freezing may vary considerably between different ejaculates of an individual stud. Loss in forward motility should not exceed 20%, pathomorphology may be increased up to 30% (Hoffmann 2003). For insemination, a number of straws corresponding to approximately 1.5×10⁸ progressively motile spermatozoa is thawed and pooled.

Insemination regime

As with natural mating, precise determination of the optimal time of insemination is less critical using native or chilled semen compared to cryopreserved semen due to the longevity of fresh canine spermatozoa in the female genital tract which is between 5-7 days, yielding a broad biological leeway for pregnancy even under suboptimal insemination management (Jeffcoate, Lindsay, 1989). Thus, using native or freshly provided chilled semen, a single insemination around the time of ovulation may be sufficient, although significantly higher pregnancy rates have been found with two inseminations (Linde-Forsberg et al., 1993). In contrast, the limited life-span of frozen-thawed spermatozoa in the female genital tract, which is estimated to be shorter than 12-24 hours (Concannon et al., 1989), requires a precise definition of the time of insemination in the individual bitch, which is about 2-5 days after ovulation due to a comparatively long postovulatory process of oocyte maturation. Repeating the AI after 24-48 hours may result in significantly higher pregnancy rates and litter sizes (Tsumagari et al., 2003) probably due to the occurrence of an extended ovulation and maturation period. Indications for the appropriate time of insemination may be obtained by observation of behavioural changes, vaginal cytology, vaginoscopy and determination of the electrical impedance. Repetitive examinations are necessary and the variability inherent to these approaches and the underlying parameters is high. It is therefore recommended to directly determine the underlying hormonal changes by applying a reliable quantitative assay for progesterone. Progesterone levels closely reflect the pre and postovulatory luteinization of granulosa cells with about 1-2 ng/ml during the LH-peak and about 5 ng/ml at ovulation. Termination of oocytes maturation is more difficult to assess due to the variability in the increase of progesterone levels with formation of the corpora lutea. However, progesterone levels between 10-12 and even up to 20 ng/ml are generally considered as optimal for insemination with cryopreserved semen (Linde-Forsberg et al., 1989).

Technique of semen transfer

Due to the anatomical structure of the vagina intrauterine insemination in the dog is more difficult than in other species and requires quite some experience. This is largely due to a dorsal median fold of tissue that extends caudally from the vaginal portion of the cervix. At vaginoscopy the caudal portion of the fold and the constrictions of the lateral and ventral vaginal walls give a distinct appearance, referred to as pseudocervix. The true vaginal portion of the cervix is cranial to the pseudocervix. This makes intrauterine cannulation difficult, also because the cervical canal is nearly perpendicular to the longitudinal axis of the vagina and the uterine body.

Currently four methods are used for semen transfer in dogs: deep intravaginal insemination, transcervical insemination using an rigid catheter (Norwegian Catheter) with transabdominal fixation of the cervix, transcervical endoscope-aided insemination and intrauterine insemination using a laparoscopic approach. Freshly ejaculated spermatozoa and spermatozoa from chilled semen may readily penetrate the cervix and reach the site of fertilization in the oviduct. Hence deep intravaginal insemination with this type of semen is common practice. As a substitute of the vaginal plug by the erected bulbus glandis it has been generally recommended to elevate the rear end of the bitch at a 45° to 60° angle for 5-20 minutes after AI. However, pregnancy rates and litter size was not different comparing elevation times of 1 and 10 min (Pinto et al., 1998). In respect to cryopreserved semen it was widely accepted that semen deposition must be intrauterine for optimal results. However, data from literature are contradictory as in some studies no significant difference was found between precervical and transcervical insemination using frozen thawed semen (Rota et al., 1999). Yet up to now the statistical basis is very weak not allowing any definite conclusions (see below). Hence we still recommend intrauterine semen deposition using the transcervical approach since the laparoscopic approach is not accepted by many pet owners, which consider this method as unethical and too risky for the bitch. Yet it must be anticipated that the transcervical approach may be difficult in maiden dogs and in those cases with a high discharge of cervical mucus, blinding the optical system. In these cases the semen should be placed as close as possible to the orificium externum of the cervix.

Pregnancy rates and litter sizes

Success of AI in dogs depends on various parameters such as type and quality of semen used, sperm dosage, the method and site of semen deposition, insemination regime and breed.

Provided semen of adequate quality is used, success of AI using native or chilled semen is equal to that after natural mating (Pinto et al., 1999).

Several studies are available reporting pregnancy rates and litter sizes after AI with frozen-thawed semen in bitches using intravaginal and/or intrauterine insemination. However, results vary substantially obviously due to the high variability of the conditions for insemination. However, in a relatively large comprehensive study using semen of defined quality, pregnancy rates/liter size after a single insemination per cycle were 84.3%/5.0 ± 3.0 puppies after intrauterine semen deposition, but only 34.8% / 2.5 ± 1.3 puppies after intravaginal insemination. These data show that–provided a good method of cryopreservation and intrauterine semen deposition are applied–results with AI using frozen-thawed semen may be comparable to those obtained with natural mating. In those studies yielding no difference between intravaginal vs. intrauterine insemination, the benefit of intrauterine insemination may be overridden by higher doses of spermatozoa or by higher numbers of AI’s per cycle. Accordingly, in the above mentioned study whelping rate/litter size after intravaginal deposition of frozen-thawed semen increased to 63.9%/5.1 ± 3.1 when three inseminations per cycle were applied (Linde-Forsberg et al., 1999).

 

FREQUENTLY ASKED QUESTIONS

1) How frequently can a dog be used for breeding?

The usual recommendation is neither to use a dog for breeding nor to collect semen more often than every other day. Many of the spermatozoa in an ejaculate come from storage in the epididymis. Whenever a dog ejaculates, many of those stored spermatozoa are released. If the dog is used more frequently than every other day, the number of spermatozoa in the ejaculate declines with each collection as that reserve is depleted. Most dogs also show a decrease in libido if they are used or collected very frequently.

2) What is a “rusty load”?

A rusty load is an ejaculate containing many old spermatozoa from storage, many of which are misshapen and have poor motility. If semen is collected from a dog that has not been used for a while and many of the spermatozoa appear abnormally shaped, semen should be collected again before that dog is evaluated critically.

3) How do you get a semen sample from a dog?

Semen is collected from dogs by manual ejaculation. The dog does not need to be sedated. You will get a better-quality sample if there is a teaser bitch present, but often you can get an adequate sample with no teaser present. The dog’s penis is stimulated, and as he becomes erect, the penis is directed toward a collection vessel. I like to use a rubber collecting cone that fits over the penis and prevents loss of the semen sample onto the floor, but you can collect semen into a cup.

• SEMEN COLLECTION

Manual ejaculation of dogs is a vital component of a breeding-soundness examination, the definitive diagnostic test for male dogs presenting for subinfertility or infertility, and the technique used to collect semen for artificial insemination or semen preservation. Electroejaculation, in which a rectal probe is used to stimulate ejaculation neurologically, is not performed in domesticated dogs.

Semen can be collected into any container. I have heard of people using cups, sandwich bags, and other household items. Any container used must be very clean and contain no soap residues; any contaminant may kill spermatozoa collected into that vessel. I prefer to use a rubber collecting cone with a clear plastic centrifuge tube attached to the end (Figure 23-1) because it is a self-contained unit that does not allow semen to be spilled or ejaculated outside of the collecting instrument and because it best mimics natural service, presumably yielding a better quality ejaculate. Again, the equipment must be kept scrupulously clean. Rubber should not be cleaned with soap at any time because soap residues easily adhere to the rubber and may kill spermatozoa subsequently collected with that equipment.

Most male dogs can be collected in the absence of a teaser bitch. However, it has been demonstrated that the libido of the male and quality of the semen are improved in the presence of a teaser bitch, especially if she is in heat. Some very experienced male dogs require not just a bitch in heat but one that is at the optimal time for breeding. If a teaser bitch is not available, there are several techniques that can be used to entice the male: (1) use of a commercial product mimicking the pheromones produced by a bitch in heat, (2) allowing the male to sniff at swabs collected from an estrous bitch and frozen until needed, and (3) administration of a drug that stimulates contraction of the smooth muscle in the epididymis. In my experience, the first two techniques have not proven very successful. The last technique involves administration of prostaglandin (Lutalyse) 10 minutes before attempted semen collection. This technique has been demonstrated to increase number of spermatozoa in the ejaculate to the same extent as does the presence of an estrous teaser bitch.

The floor should be a nonslip type. If semen collection is to be performed with any regularity, a consistent surface, such as a rug, should be used. This provides secure footing and trains the dog as to what is expected of him when the rug is present.

If a teaser bitch is used, a handler should hold her with her hindquarters on the rug. If the bitch is not in heat, she should be muzzled or otherwise securely restrained. The male is allowed to investigate her hindquarters and to mount if he so wishes. The bulbus glandis is palpated through the prepuce. The area over the bulbus glandis is manipulated briskly and enthusiastically. As soon as the penis starts to become erect, the prepuce is pushed behind the engorging bulbus glandis. This is best accomplished by using the rubber collecting cone to push the prepuce back while “squirting” the penis out of the prepuce with the other hand. A better-quality ejaculate usually is obtained if the prepuce is pushed proximal to the erect bulbus glandis. Once the rubber collecting cone is advanced completely proximal to the bulbus glandis, the penis between the bulbus glandis and body wall is encircled tightly with the fingers. This mimics the tie. At this point, most dogs thrust vigorously and ejaculate a clear presperm fraction and then the cloudy sperm-rich fraction of the ejaculate (Figure 23-2). The dog stops thrusting and steps down. He may try to step over the collector’s arm. If the bulbus glandis is completely free from the prepuce, the collector should swing the dog’s penis 180 degrees in a horizontal plane and continue to hold. The final fraction, the prostatic fraction, is ejaculated as distinct pulses of clear fluid that will be visible filling the tube attached to the collecting cone and will be palpable in the hand holding the collecting cone and penis. The anus contracts rhythmically as prostatic fluid is ejaculated. The amount of prostatic fluid collected depends on the purpose of the semen collection (Table 23-1). At the conclusion of semen collection, the penis is released and the rubber collecting cone gently peeled off the penis. Allow the male to lick at the penis, walk him away from the collection area, or gently massage the engorged penis with towels moistened with cold water (not ice) to hasten detumescence of the penis. Ensure that the penis is completely flaccid and within the prepuce and that the prepuce is not rolled in on itself before kenneling the male dog.

 

Purpose of Semen Collection	Amount of Prostatic Fluid to Collect
Breeding soundness exam (complete semen evaluation)	One to two good “squirts,” enough to ensure the entire second (“sperm-rich”) fraction has been collected
Semen collection for culture and cytology testing or other diagnostic tests (prostate disease, subfertility, or infertility)	As above or enough to provide sufficient volume for tests to be conducted
Artificial insemination with fresh semen (see Chapter 21)	Dependent on size of bitch to be inseminated. For bitches weighing less than 10 lb, total semen volume should be 1.5 to 3 mL; for bitches weighing 10 to 50 lb, 3 to 5 mL; and for bitches weighing more than 50 lb, 5 to 8 mL
Semen to be chilled and shipped overnight	One good “squirt”—amount of prostatic fluid collected should be minimized as incubation of spermatozoa with the dog’s prostatic fluid is associated with decreasing motility of those spermatozoa
Semen to be frozen	One good “squirt,” enough to ensure the entire sperm-rich fraction has been collected. Sample will be centrifuged and the spermatozoa removed for further processing

• SEMEN EVALUATION

The components of a complete semen evaluation include measurement of volume, assessment of color, subjective assessment of percentage progressively motile spermatozoa, determination of concentration and total number of spermatozoa in the ejaculate, and assessment of spermatozoal morphology (shape).

• VOLUME

Volume is not an indicator of semen quality because it is dependent on the amount of prostatic fluid collected. However, the value for volume must be recorded to allow determination of the total number of spermatozoa in the ejaculate. A normal value for volume has been reported at 1 to 30 mL (Figure 23-3).

• COLOR

Color is a superficial indicator of semen quality and possible contamination of semen. Semen should be milky or opalescent. All milky fluid collected should be evaluated microscopically because occasionally dogs will ejaculate fluid that contains no spermatozoa but has many fat droplets that give it a milky appearance. Yellow is indicative of urine contamination. Brown or red indicates that blood is present in the semen. Brown discoloration is due to old blood and usually is associated with prostate disease. Red discoloration is due to frank blood and may be associated with prostate disease or with trauma to the penis. Occasionally young male dogs bleed from the surface of the penis the first time they become erect because small blood vessels rupture on the engorging penis. Green discoloration may be seen in animals with reproductive tract infection. Finally, clear fluid indicates lack of spermatozoa in the ejaculate (azoospermia) (Figure 23-4).

• MOTILITY

The percentage of progressively motile spermatozoa is assessed by evaluation of a drop of semen on a glass slide, examined microscopically at 40 to 100÷ magnification. Evaluation should be immediate because the percentage of progressively motile spermatozoa declines quickly as the microscope light heats the drop of semen on the slide. This is a very subjective assessment of what percentage of spermatozoa are moving forward. Some investigators evaluate total motility, progressive motility (those moving forward), and speed of motile spermatozoa. Normal percentage progressively motile spermatozoa is 70% or greater.

 The normal male dog attains puberty at approximately 6 – 8 months of age. Sexual maturity is generally attained at 18 – 30 months. Males may successfully breed bitches prior to sexual maturity but they will not achieve maximal fertility or daily sperm output until mature. The normal male can breed once every 2 – 5 days and maintain daily sperm output.

A complete breeding soundness examination in the dog includes history-taking, general physical examination, reproductive system examination, libido determination, semen collection and evaluation, hormonal evaluation, and prostatic examination and testing for disease (eg. Brucellosis).

Semen collection

In addition to artificial insemination breeding, collection of semen is indicated for evaluation in conjunction with a breeding soundness examination, in the diagnostic workup of potentially subfertile or infertile dogs, in the diagnostic workup of reproductive tract disease (infectious, degenerative or neoplastic disease), or for short-term (fresh-chilled extended semen) or long term (frozen semen) storage of gametes to be used in the future. When very young (< 7 months) or aged (>12 years) sires are used for breeding, the American Kennel Club (AKC) requires documentation, including a semen evaluation, of the male’s ability to sire a litter.

The collection process – tips for success

• Ambiance is important. In the typical setting, ejaculation in the dog is a voluntary process and cannot be forced or rushed. The dog needs to be relaxed and comfortable with the place and the people involved.
• Be prepared. Have all supplies and adequate personnel available. You will need a dog handler, a bitch handler, a estrous teaser bitch (optimally), collection cones, nonspermacidal lubricant, 15-ml conical centrifuge tubes, a microscope, microscope slides, coverslips, bulb pipettes, stain, a means to determine sperm cell concentration (hemacytometer or Spermacue™) and other supplies depending upon the purpose for the semen collection. Some males may not require any external stimulus beyond manual massage to attain erection. If a teaser bitch is not available, many dogs will respond favorable to swab scent from an estrous bitch. Swabs can be collected and saved ahead of time.
• Perform the collection on a non-slip, dog friendly surface (I have non-slippery epoxy flooring and my “magic carpet”).
• With the bitch adequately restrained, the dog is allowed investigate the bitch’s hindquarters. He may sniff or lick her external genetalia or rear limbs. He may or may not mount the bitch. Signs of readiness include salivation and “chomping” of his teeth. During collection, the dog should be observed for ease of achieving an erection, presence of a normal erection, normal thrusting behavior and normal pulsation associated with ejaculation and prostatic fluid emission.
• It is important to be aware of the onset of penile erection. Some males will achieve erection without any manual manipulation. Other males require brisk massage of the bulbus glandis through the prepuce to elicit erection. As soon as erection begins, the prepuce is moved proximally such that the entire prepuce is proximal to the exposed bulbus glandis. Failure to reposition the prepuce at the correct time may result in an inability to slide the prepuce proximally over the bulbis glandis. The collection process may then become painful to the dog as the skin of the prepuce tightens over the engorged bulbis glandis. It is important to roll over the edge of the cone such that the edge of the cone does not come into direct contact with penis. Failure to do so may result in a “paper cut” type of injury to the penis.
• Concurrent with prepucial positioning, the collecting cone is introduced over the engorging penis. The collecting cone covers the penis to the level of the bulbis glandis.
• The collector uses his or her thumb and finger to form a ring proximal to the bulbis glandis and pressure is applied to the penis circumferentially, thus simulating the copulatory lock or “tie.”
• With the onset of ejaculation, dog will thrust vigorously for several minutes. During this period, the first and second seminal fractions are ejaculated. The first or pre-sperm fraction is of prostatic origin and is clear. The pre-sperm fraction arises from the prostate and urethral glands, and is believed to cleanse the urethra of contaminants (urine, bacteria and cellular debris) prior to ejaculation The second or sperm-rich fraction originates in the cauda of the epididymis where spermatozoa are stored.
• After a brief period of rest, the dog will ejaculate the third seminal fraction. The third fraction is comprised of prostatic fluid and is normally clear. Prostatic secretion provide volume to the ejaculate, assist in propelling the sperm out of the vagina and into the cervix/uterus, and provide nutrients for the sperm while traveling to the site of fertilization in the oviducts. During third fraction collection, the dog may try to step over the collector’s arm. If this occurs, the collector should lift the dog’s rear limb over his or her arm and redirect the penis caudally to simulate the copulatory lock or “tie.” It is important to redirect the penis caudally while maintaining the horizontal plane of the penis. Do not redirect the penis caudally in a vertical “pendulum swing” plane as this may cause injury.
• During ejaculation of the third fraction, the collector can feel and visualize the rhythmic pulsations in the penile urethral and perineal muscles. There is also an audible spurting of prostatic fluid into the receptacle.
• For breeding purposes, usually only the second, sperm-rich fraction is collected. If all three fractions of the ejaculate need to be collected, it is advisable to collect the fractions into separate receptacles. Fractionating requires the use of funnels or additional cones, dexterity and quick hands.
• Once semen collection is completed, the collector releases the penis and removes the collection cone. The dog often will continue to ejaculate pulses of prostatic fluid. Detumescence of the penis may require several minutes and often takes a similar amount of time as would recovery from a natural mating. Care should be taken to ensure that no injury occurs to the penis at this time.
• Make sure that subsidence has occurred and that the prepuce is positioned normally over the penis before allowing the dog to be discharged. Occasionally the prepuce rolls inward as penile engorgement wains leaving the unprotected glans of the penis vulnerable to dessication and trauma. With the aid of lubricant, the prepuce can readily be manipulated over the tip of the penis.
• Number of spermatozoa collected is optimized in the presence of an estrus teaser bitch. As much as a four-fold increase in spermatozoa number can be realized when using a teaser bitch. Alternatively, swab the perineum of any tractable female with frozen-thawed swab scent from an estrus bitch.
• For reticent dogs or when a teaser bitch is not available, semen collection may be facilitated by the administration of prostaglandin F2 alpha (Lutalyse®) at a dosage of 0.1 mg/kg subcutaneously 15 minutes prior to collection. Side effects commonly associated with prostaglandin administration (salivation, emesis, etc.) are minimal at this dosage.
• For breeding purposes, usually only the sperm-rich second fraction of the ejaculate is collected.

Semen handling

Sperm are motile and vigorous cells, but are also fragile and susceptible to damage and demise by environmental conditions. When collecting and handling semen it is critical to avoid exposing sperm cells to two types of insults – toxic chemicals and thermal insult. Keeping collection equipment clean and disinfected is important, but soap and disinfecting chemicals are quite potent spermicides. For this reason and for their convenience of use, disposable polypropylene collection cones are often preferred over their latex counterparts. When using latex cones, take great care to rinse thoroughly deionized water to remove all soap and disinfectant residue. It is usually best to use new, sterile collection tubes. Finally, be certain to lubricate collection cones with a sterile lubricant known to be non-toxic to sperm. If the tube of lubricant is not clearly labeled as being nonspermacidal then you cannot presume that it is safe to use.

Sperm are sensitive to both heat and cold. Rapid chilling of semen results in a phenomenon called “cold shock” that is often manifest by abnormal sperm motility and morphology. Short periods of exposure to temperatures just a few degrees above body temperature will usually kill large numbers of sperm. To avoid thermal stress, the collection cone should be pre-warmed to body temperature. Additionally, microscope slides, coverslips, stains, extenders and pipets used to handle and examine sperm are best maintained on a warming plate prior to use. If you are performing a large number of analyses, a heated microscope stage is also a valuable piece of equipment.

Semen from most species is not damaged by exposure to room temperature (20-22°C) for an hour or two. If longer periods of storage are required, it is best to dilute the ejaculate in a buffered nutrient solution, called an extender, and cool it slowly to refrigerator temperature (4-5° C). A large number of extenders have been developed and are commercially available for use in short term and long term storage of semen. They are similar in having an energy source (eg. glucose), buffers to maintain pH (eg. Tris or citrate) and a source of protein (eg. egg yolk or skim milk).

Semen evaluation

Putting the evaluation into perspective

Ultimately, the goal of any semen evaluation is to predict the likelihood for a dog to be successful in siring a litter. Routine semen evaluation in the dog includes determination of semen volume, color and pH, spermatozoa motility, velocity, concentration, total number of spermatozoa in the ejaculate, and sperm cell morphology. In humans, The World Health Organization (WHO) has developed strict criteria for semen collection and evaluation. WHO protocols recommend that semen be collected when the man has had sexual rest for at least 2 but no more than 7 days, and that two separate samples, collected 7–21 days apart, be evaluated before any recommendations are made. Similarly with morphologic evaluation, “A normal human sperm has a specified size and shape, with a smooth outline, an acrosome that comprises 40-70% of the sperm head, has no neck, midpiece or tail defects, and has no droplets more than ½ the size of the sperm head.” Use of these criteria makes morphologic examination of the semen more standardized and allows for easier comparison of research studies. To date, there is no “WHO equivalent” for the dog. No such criteria have been adapted to any of the domestic species, including the canine, beyond the guideline that a dog should not be condemned on the results of one semen evaluation.

Male factor infertility accounts for up to 50% of failed pregnancy attempts in humans. Compared to human medicine, little is known in canine medicine regarding specific findings on semen evaluation and their correlation with fertility. No estimates have been reported for the canine, but experiential evidence supports the notion that male factor infertility does account for a significant portion of conception failure, early embryonic death and small litter size.

Veterinarians routinely perform semen analysis on dogs. Unfortunately, there is little data associating the parameters measured with the issues practitioners really need to evaluate, i.e. testicular function, fertilizing capability of spermatozoa, and likelihood that puppies will develop normally. With the current methodology, semen analysis may only be reliably predictive of fertility if the semen quality is either very good or very bad.

A big problem is that in vitro analysis ≠ in vivo function. Ejaculated spermatozoa examined in vitro do not exhibit the characteristics they will take on as they traverse the reproductive tract of the female. For a spermatozoon to fulfill its role, it must:

• develop properly in the testis
• undergo maturation in the epididymis
• pass through the cervix and uterus
• undergo capacitation and the acrosome reaction as it passes from the seminal fluid into secretions from the female reproductive tract
• bind to the epithelium of the uterus or distal uterine tube
• detach at the correct time and move into the uterine tube
• penetrate the cumulus cells surrounding the ovum
• bind to the zona pellucida
• and for that lucky “one,” penetrate the zona

Another problem is that evaluation techniques are difficult to standardize. Results of tests that may be performed in a semen evaluation are influenced by sample collection technique and timing, concentration of spermatozoa in the sample, amount of time from sample collection to evaluation, temperature at which the sample was held, equipment used, and many other factors, not the least of which is the subjectivity of the evaluator. Another impediment to standardization of semen evaluation in the canine relates to the enormous variability present as a result of breed. Along with the obvious extremes in testicular size, there may be other significant influence of breed on semen parameters.

Acknowledging the problems encountered and limitations inherent with making functional conclusions on the basis of in vitro testing, multiple parameters are used in performing a semen evaluation.

Color and appearance

Color evaluation is subjective but informative. The turbidity or opacity of semen provides a rough indication of concentration. Commonly, the turbidity of a sample is graded on a subjective scale of 0 to 5, with 5 being the most opaque as in the whole milk. A clear sample contains no spermatozoa. Although cloudy or milky samples almost always contain spermatozoa, they must still be checked microscopically because occasionally a dog with azoospermia will shed excessive numbers of fat droplets into the sample, giving the appearance of normal semen. Yellow semen is indicative of urine contamination and is also seen in humans with icterus or after ingestion of certain vitamins. Brown discoloration is indicative of old digested blood and red discoloration is indicative of fresh blood. The most common causes of hemospermia in dogs are prostatic disease and penile trauma. In humans, hemospermia also may be associated with urinary calculi, sexually transmitted diseases such as syphilis and gonorrhea, spermatocele and hydrocele, and treatment with anticoagulant medications.

pH

The value of pH measurement of canine semen is debatable. Values for normal pH in non-fractionated canine semen vary from 6.4 to 6.8. Seminal pH may change in the presence of disease, such as prostatitis, or if the semen sample is contaminated with urine. There are no published data comparing pH analysis techniques for canine semen or specifically describing the clinical value of pH measurement. Alterations in pH may affect sperm longevity and motility. Decreases in prostatic fluid pH are common with prostatic disease. Alterations in pH may occur with use of excessive amounts of lubricant or improper cleaning and disinfection of collection equipment. Standardization in the determination of semen pH with respect to timing and method of measurement (pH meter versus “dipstick” pH paper) would be useful.

Volume

Dog semen is ejaculated in three fractions. The first, pre-sperm fraction is small in volume (less than 5 mls) and contains few to no spermatozoa. The pre-sperm fraction is believed to cleanse the urethra of contaminants (urine, bacteria and cellular debris) prior to ejaculation. The pre-sperm is not usually collected. The second, sperm-rich fraction comes from the epididymes and testes. The sperm rich fraction is cloudy white in color and usually 0.5 – 4 ml in volume. The third fraction consists solely of prostatic fluid and contains few to no spermatozoa. Prostatic secretions lend volume to the ejaculate, assist in pushing the sperm out of the vagina and into the cervix/uterus, and provide nutrients for the sperm during their transit to the oviducts. The volumes of the first and third fractions are variable. In particular, the volume of the third fraction is controlled by the person collecting the sample, as they can choose to collect more or less of the cell-free prostatic fluid. Prostatic fluid is normally clear in color and may range in volume from 3 – 80 ml. Volume is not an indicator of semen quality in dogs. However, the measurement of semen volume is important in the calculation of total number of spermatozoa in the sample.

Motility

Sperm motility is essential for fertilization because it allows or at least facilitates passage of the sperm through the zona pellucida. Without technologic intervention, a non-motile or abnormally-motile sperm is not going to fertilize. Hence, assessing the fraction of a sperm population that is motile is perhaps the most widely-used measure of semen quality.

For canine semen, motility is better maintained if samples are kept at room temperature than at body temperature. Warmer temperatures increase sperm cell metabolism. Delay in evaluation of warmed samples may result in errors due to sperm cell energy depletion. Rapid temperature fluctuations should be avoided. Ambient temperature may affect the motility assessment of a sample if the evaluation room is excessively hot or cold. Percentage of progressively motile spermatozoa from a given dog is not affected by frequency of semen collection.

In evaluating motility, sperm cells are classified as immotile, progressively motile or non-progressively motile. Both total and progressive motility are determined and expressed as a percentage of 100. Sperm cells showing motility of any kind are included in the calculation of total motility. A progressively motile spermatozoan moves forward in an essentially straight line, whereas a non-progressively motile sperm cell moves, but with an abnormal path, such as in tight circles (tail chasing), fish flopping, and when normal movement is prevented by sperm head agglutination. In the dog, the normal percentage of progressively motile spermatozoa is 70% or greater. Speed or quality of motility also may be assessed; a canine spermatozoon with normal motility should traverse the microscopic field of view in 2–3 seconds.

Progressively motility is positively correlated with normal morphology in dogs. Just as is the case with the whole dog “on the hoof,” think function follows form. If the sperm cell is put together normally, then it should move normally. Immotile sperm cells that are morphologically normal may have experienced “iatrogenic immotility” as a result of technical errors in semen collection or handling. Exposure to rapid or extreme temperature fluctuations, any kind of residue on collection equipment, or the wrong pH or osmolality of an extender can adversely affect motility. Motility is also affected by periods of sexual inactivity – males that have not ejaculated for prolonged periods often have poor motility on the first ejaculate, but much better motility for a second ejaculate collected soon thereafter. This is the so-called “rusty pipe” condition.

In humans, age is a factor in motility; motility has been documented to decline at rate of 0.27%/year in men > 45 years old. In protocols for humans, two samples of 200 spermatozoa are evaluated separately and graded as A (=rapid progressive motility), B (slow or sluggish motility), C non-progressive motility), and D (complete lack of motility). If results for the two samples don’t “jive” (ie., the difference is greater than expected by statistical random variation), two additional slides are evaluated from that semen sample. 

Conclusions

By now AI in the dog must be considered a well established breeding technique with the use of frozen semen gaining constantly more importance. Techniques for semen preservation, the quality assessment of semen and the dosages used are fairly standardized. Appropriate semen quality and AI management provided, results with native, chilled and even frozen-thawed semen may be comparable to natural mating. It is predicted that the use of AI will continue to rise in dogs with more AI centers becoming available, increasing acceptance by breeders associations and the abolition of import restrictions.

References