FERTILITY ASSESSMENT IN MALE DOGS

FERTILITY ASSESSMENT IN MALE DOGS

 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 attain maximal fertility or daily sperm output

until mature. The normal male can breed once every 2 – 5 days and maintain adequate

sperm numbers.

Health of the genital organs and quality of the semen are directly related to the ability

to produce offspring, only healthy animals in excellent body condition should be used

for breeding. Impaired fertility may lead to no or lower number of puppies. A breeding

soundness evaluation (BSE) can be performed for evaluating the health of genital

organs and breeding potential of dogs. Therefore, breeders often require BSE which

include andrological examinations of the genital organs, in some cases measurements

of hormones, and semen analysis.

The stud dog is half the equation when evaluating potential causes of infertility

in a canine breeding. Assessing male fertility is often the first step because of the

ease of obtaining a semen sample for analysis. Unfortunately, the results of routine

laboratory tests may not always correlate with actual fertility. The most accurate method

of assessing male fertility is insemination of a fertile female; however, this is time

consuming and not practical for small breeding operations. The realistic evaluation is

completed with an appropriate assortment of laboratory tests. The three main areas to

consider in evaluation of canine semen quality are total sperm count; viability, assessed

as motility, progressive motility, live/dead ratio, and acrosomal membrane integrity; and

morphology.

Breeding soundness evaluation – The principle of a BSE is that features predictive

of good or poor breeding or fertilizing potential are assessed. Fertility is relying on the

proper functioning of the reproductive system, its anatomy and physiology of organs,

including the hormonal regulation of the hypothalamus-pituitary-testicles axis (Fig.

1). Before further examinations, a throughout anamnesis including the reproductive

history, medications and stays in different countries or regions in the world needs to be

documented.

A complete fertility evaluation in the male involves history, physical examination,

libido determination, semen collection and evaluation, hormonal evaluation, and prostatic

examination. The initial database should include a detailed history, a complete physical

examination, complete blood count, serum chemistry and urinalysis. History should

include travel, diet, past or current illnesses, medications, vaccinations, deworming

history and prior laboratory tests. Details of breeding history should be obtained,

including the dates of all known matings, type of breeding (natural vs. AI – vaginal,

transcervical or surgical; fresh, chilled or frozen semen) and the results of these matings

(including pregnancy rates and litter size). Breeding management of each bitch should

also be described.

Quality of semen reflects the health of the seminiferous tubules, epididymis,

prostate, and the dog’s general health. Sperm morphology, in particular, is determined

by the seminiferous tubules and to a lesser extent by the epididymis.

Spermatogenesis is the process through which spermatogonial stem cells

undergo mitotic and meiotic divisions to become spermatids. This process occurs in

the seminiferous epithelium. Spermiogenesis is the process, occurring in the lumen

of the seminiferous tubules, through which spermatids undergo multiple cytologic

transformations and mature into spermatozoa. The stages of spermatogenesis and

spermiogenesis occur at specific locations within the seminiferous tubule. Aberrations of

the testicular environment at any of these stages can result in the production of abnormal

sperm and possible infertility.

Significant maturational changes also occur as sperm pass through the efferent

ductules and epididymis. It is in these areas that sperm develop the capacity for motility,

the acrosomal membrane forms, the cytoplasmic droplet migrates distally and is shed,

and some defective sperm are eliminated. The most important functional changes occur

in the efferent ductules and caput epididymis; sperm collected from these areas are

not capable of fertilization. Sperm obtained from the cauda epididymis are capable of

fertilization.1 Sperm are stored and remain viable for a period of time in the cauda

epididymis before ejaculation.

PHYSICAL EXAMINATION

Physical examination of all body systems should be performed with careful

examination of the skin, eyes, heart, lungs, abdomen and musculoskeletal system.

Following a complete physical examination, a complete reproductive system examination

should be performed, including palpation of the scrotal contents, examination of the

penis and prepuce and palpation of prostate per rectum. Serology for Brucellosis should

be obtained.

Complete physical examination of the stud dog should be performed, to include

palpation of testicular consistency, measurement of testicular width, visual examination

of the penis and prepuce, palpation of the prostate per rectum, and ultrasonography

of the prostate and testes, if indicated. Normal testicular consistency approximates a

peeled, hard-boiled egg. It is common for one testis to be slightly larger than the other.

A typical testis-to-epididymis size ratio is appreciated with experience; change in this

ratio may indicate testicular atrophy or epididymal swelling. Any heat, swelling, pain, or

dermatitis associated with the testes and scrotum should be noted.

The scrotal skin should be evaluated for any thickening, signs of infection or

accumulation of fluid within the sac. Both testes should be palpated for size, consistency

and presence of any masses. The total scrotal width can be measured with calipers or

by ultrasound examination. Testicular volume can be determined by measuring length,

width and height of the testes via ultrasound. The epididymides (the tubules in which the sperm mature before being transported to the vas deferens) and spermatic cords

(vas deferens and testicular artery and vein) should be palpated for any thickenings,

enlargements, pain (due to inflammation or granuloma [sperm plugs]), or missing

segments (aplasia).

Ultrasound examination can further be used to visualize the testes, epididymides

and spermatic cords for masses, signs of inflammation, or abnormal fluid accumulations.

The penis should be evaluated for the presence of a persistent frenulum (remnant of

skin securing the tip of the penis to the prepuce which normally breaks down around the

time of puberty), signs of inflammation, abnormal discharge, reddening, or the presence

of any masses.

Rectal palpation of the prostate should be performed (may be completed prior to

and/or after semen collection). The prostate should be located within the pelvic canal

and should be small and symmetrical. In some cases upward pressure may be applied

to the abdomen to push the prostate further backward such that it can be palpated in

its entirety. Enlargement (either symmetrical or asymmetrical) of the prostate should

instigate further investigation as to the cause. X-rays of the abdomen may help

determine the size and location of the prostate if it cannot be palpated completely per

rectum. Ultrasound examination of the prostate may reveal masses, cysts (either within

or outside the prostate), inflammation, abscesses or generalized enlargement. Prostatic

fluid can be evaluated either after semen collection or following prostatic massage or

wash (if the male cannot be successfully collected).

Morphological and hormonal examinations

The morphological examination includes an evaluation of size, shape, symmetry,

consistency, and position of the sexual organs. The findings may indicate normal function

or lack of hormonal activation or enlargement because of pathological conditions.

Several specific diagnostic tests for clinical examinations have been developed in

combination with reference intervals for sizes and volume of the testicles, testicular tissue

consistency or appearance of genital organs in diagnostic imaging. Low testosterone

concentrations, for example may lead to small and soft testicles and a decline of the

volume and function of the prostate. However, despite the wide recommendation

for examinations of breeding dogs, there have hardly been comprehensive studies

evaluating differences in examination outcomes between fertile and infertile dogs and

potential confounders. Over the last years, several advanced ultrasound technologies

have been developed which provide additional information of the examined organs.

However, their clinical application in the field of small animal reproduction is still limited. Sensitivity and specificity for the detection of specific conditions or diseases have not

widely been subject of research until now. It has been shown that Doppler ultrasound

parameters resistance index (RI) and pulsatility index (PI) may be potential markers of

seminal quality in dogs, but these parameters did not differ between infertile and fertile

dogs. For the examination of the prostate, ultrasound is considered an excellent tool to

detect any change of their homogeneous and echogenicity pattern of the gland. These

features, however, are not pathognomonic of a specific disease. For instance, the loss

of normal ultrasonographic pattern and the increase in echogenicity can be observed in

almost every prostatic disorder.

Measurement of concentrations of FSH and LH in blood serum or plasma provides

good insights into the hormonal regulation [49] but not all laboratories provide reliable

quantitative tests for these parameters, yet. Even if for testosterone good assays are

available, interpretation of the findings from a single sample may be misleading.

Anti Muellerian Hormone, which is produced in sertoli cells and can be used to

proof the presence of gonadal tissue, has been discussed as a potential fertility marker

and has shown a sensitivity (86%) and specificity (63%) to predict canine semen quality

in a preliminary research project. Measurement of this hormone may also be used to

identify subfertility over previous treatment with GnRH analogs. Elevated concentrations

may be used as a biomarker for Sertoli cell tumors.

Timing of the examination

When planning a breeding soundness evaluation, several factors should be taken

into account such as age, size of the animal, time from last semen collection and

perhaps season of the year. Spermatogenesis in the dog lasts around 56 to 63 days but

medical history should include also any diseases or medication that may have impaired

spermatogenesis within the at least past six months before semen collection.

Semen collection procedure

The most common collection method is by digital stimulation of the penis. One of

the most important factors for a successful collection seems to be sexual arousal of the

dog. Under ideal conditions, this can be achieved with the presence of a bitch in estrus.

Unfortunately, the availability of teaser bitches and pheromones is frequently limited and

clinicians are therefore forced to collect semen without sufficient arousal which may lead

to lower semen volume and total sperm count. Less effective are pheromones in the

form of vaginal discharge from a bitch in estrus (preserved on a swab or other absorbing

material). LABORATORY EVALUATION OF SEMEN

Parameters to be Evaluated

Colour – Milky colour is normal. Any blood tinge is noted as blood may kill the sperm. If

the semen collected is clear, it may indicate poor quality or no sperm production.

Sperm Motility – The assessment is made by placing a drop of semen on a warm

slide and evaluating the percentage of sperm swimming actively under microscope. The

semen sample is examined immediately as motility decreases with time.

Sperm Morphology – Approximately one hundred sperm in the drop of semen sample

are examined and classified as either active, dead or abnormal. The dead and abnormal

sperm may show abnormalities in head, neck, mid piece and/or tail.

Sperm Concentration – A semen sample is diluted at 1/200 with water and placed in a

counting chamber. The number of sperm in specific squares of the chamber are counted

and the sperm concentration in the semen is then calculated.

Total Sperm Output – This is calculated by multiplying the concentration of sperm (per

1. ml) in the sample by the total volume of semen collected.

Results of Semen Evaluation

Sperm Motility – Sperm in the semen of most fertile dogs have motility of 90 – 95%.

Cases below 80% motility may be an indication of reduced fertility.

Sperm Morphology – Examples of abnormalities include the presence of many ‘knobbed

head’ sperm which indicates sterility, presence of ‘proximal cytoplasmic droplets’ in the

sperm neck region which indicates immaturity, and abnormal sperm tail which affects

penetration into the egg cell during fertilisation. Any less than 80% normal may be an

indication of reduced fertility.

Total Sperm Count – Anything more than 100,000 are compatible with good fertility.

Red Blood Cells in Semen – Red blood cells are commonly seen in semen samples

collected from dogs over 5 years old. They originate from the prostate gland (even when

there is no prostate disease). Large quantities of red blood cells do not appear to affect

sperm motility.

White Blood Cells in Semen – White blood cells in semen usually originate from the side

of the penis or lining of prepuce. They also indicate the presence of prostate disease.

Prediction of Fertility

Semen quality is usually evaluated to be ‘falsely’ poor if semen is collected

from an ill at ease dog, or the second collection on a particular day. Semen

evaulation must be repeated another day before fertility can be predicted.

Semen quality may be severely affected if the dog has been on medication at the time

of semen collection. A further sample must be collected following completion of the

medication before fertility is assessed.

Dogs with good semen quality on evaluation are fertile and if mated with a bitch

close to ovulation should produce offspring.

Dogs with poor semen quality (i.e. poor motility, abnormal sperms, low number

of sperm) are often still fertile but mated bitches may be expected to ‘miss’. Repeated

mating around the time of ovulation may increase the chance of the bitch conceiving.

Absence of sperm or presence of small quantities of dead or abnormal sperm in the

semen sample indicate the ejaculation has not been undertaken correctly or that the dog

may have been treated with anabolics. Normal ejaculate will be produced two months

after the anabolics are ceased.

Other cases that show no sperm or only a small quantities of sperm indicate that the

sperm production has been arrested. These dogs were usually initially fertile but since

have suffered a rapid asymptomatic degeneration of testicles resulting in the permanent

absence of sperm and hence fertility.

Semen collection

Semen collection should be performed in the presence of an teaser bitch in

heat (estrus) whenever possible. If a teaser bitch is not available, some males can

be stimulated to erection using estrus bitch vaginal swabs or urine, or commercially

available pheromone, applied to cotton balls or the vulva of a non-cycling bitch. Some

males may not require any external stimulus beyond manual massage to attain erection.

During collection the male should be observed for ease at which he develops an

erection, presence of a normal erection, normal thrusting behavior and normal pulsation

associated with ejaculation and prostatic fluid emission. Semen is ejaculated in 3 fractions: 1) pre-sperm – which arises from the prostate and urethral glands, thought to cleanse the urethra of contaminants (urine, bacteria and

smegma) prior to ejaculation; 2) sperm-rich – which arises from the epididymal stores

and vas deferens; 3) prostatic secretions – which provides volume to the ejaculate,

assists in pushing the sperm out of the vagina, through the cervix, and into the uterus,

and provide nutrients for the sperm while traveling to the oviducts. Semen should be

collected in fractions whenever possible to facilitate evaluation of each portion of the

ejaculate. The pre-sperm fraction is clear in color, is usually minimal in volume (less

than 5 ml) and is not usually collected. The sperm rich fraction is white – cloudy white

in color and usually 0.5 – 4 ml in volume. The prostatic fluid is normally clear in color

and may range in volume from 3 – 80 ml. Following collection, it is important to be sure

that the erection subsides, that the penis is drawn back into the prepuce and that the

prepuce does not roll inwardly when this occurs. Once the semen is collected, all equipment contacting it should be maintained at 37°C either until insemination or it is properly extended. Routine semen evaluation includes measurement of semen volume, semen motility, semen concentration, evaluation of individual sperm morphology (shape and form), and sometimes determination of the pH of the ejaculate. Semen volume is measured in milliliters. There is no minimal accepted semen volume since it depends on how well fractionated the ejaculate is and how much of each of the fractions is collected.

Semen motility is assessed by placing a drop of raw semen on a pre-warmed microscope slide and applying a prewarmed coverslip. Semen is then examined at high and low magnification (i.e. 10x and 40x). A regular light microscope can be used, but the use of a phase contrast microscope enhances the ability to visualize individual sperm movements. Both total and progressive motility are determined and expressed as a percentage of 100. Total motility is defined as the percent of sperm that are moving, while progressive motility is defined as the percent of sperm that are moving forward, progressively. Of the two, progressive motility is most important in determining the number of sperm potentially able to fertilize the oocytes (eggs). During motility assessment, sperm velocity is also assessed. Velocity of forward movement is rated on a scale of 0 – 5 (0 = no movement, 5 = rapid, forward movement). A normal ejaculate contains a minimum of 70% progressively motile sperm. Semen concentration is measured in millions of spermatozoa per milliliter of semen.

Semen concentration and daily sperm output are directly related to testicular volume. So,

the larger the testicles, the greater the daily sperm output. A normal ejaculate contains

a minimum of 200 million spermatozoa/ejaculate. On average acceptable numbers of

total sperm/ejaculate will be from 200 – 300 million for toy breeds; from 200 – 500 million

for small breeds; from 400 – 800 million for medium breeds; from 500 million – 1.5 billion

for large breeds; and from 600 million – 2 billion for giant breeds.

Individual sperm should be examined (for normal shape and structure) after staining

the raw semen. The most common stain used is eosin-nigrosin. This stain is a vital stain,

which means that it stains the live and dead sperm different colors. The sperm is divided

into 3 segments: head, midpiece and tail. The head contains the DNA and has a cap (the

acrosome) which contains the enzymes that allow fertilization to occur. The midpiece

contains the motor apparatus that propels the sperm. The tail provides the propulsion to

move the sperm forward. Defects may be classified in several different fashions: primary

1. secondary defects (primary occurring in the testicles, and secondary occurring

during storage, transport or handling); major vs. minor (major affecting the ability of the

sperm to fertilize and minor not affecting the ability to fertilize) or compensable and noncompensable (compensable defects can be overcome by providing access of the sperm

to the egg and noncompensable defects cannot be overcome by the sheer presence of

providing access to the egg). Primary vs. secondary is the most common classification

scheme used. A normal ejaculate contains >70% normal sperm. Sperm may also be

examined to assess for the presence of a normal acrosome using special stains. These

stains differentially stain the DNA portion of the sperm head a different color than the

acrosome. In some cases of infertility, the acrosomes may have already reacted prior

to the sperm reaching the oviducts. If this occurs these sperm will be incapable of

fertilizing the eggs when they reach the oviducts. The semen may also be stained with

Wright – Giemsa stain to assess for the presence of white blood cells or germ cells

(immature sperm cells shed by the testicle when testicular degeneration is present).

Semen culture may be submitted if high numbers of white blood cells are present in the

ejaculate. Culture for aerobic bacteria and Mycoplasma are commonly obtained. The pH of the prostatic fluid is usually 6.3 – 6.7. Either a combination of fraction 2 and 3 or fraction 3 alone can be tested. Alterations in pH may affect sperm longevity and motility. Increases or decreases in prostatic fluid pH are common with prostatic disease. Increases in pH may occur with use of excessive amounts of lubricant or improper cleaning and disinfection of collection equipment. When semen will be chilled and shipped for insemination, assessment of sperm longevity may be evaluated (following extension with semen extender), in order to determine the potential success with the use of this type of semen. Semen is collected and extended depending on the semen concentration, the type of extender being used and the type of insemination being performed. Generally, an ejaculate will be extended at a minimum ratio of 2-3:1. Semen should be chilled (slowly) to 4 – 5°C and held for a minimum of 48 hours at this temperature. A small sample of the semen is warmed to 37°C at 24 and 48 hours and total motility, progressive motility and velocity are determined. In some cases, motility may be so good at 48 hours to make evaluation at 72 and 96 hours (or longer) indicated.

In certain cases, where fertility issues are evident based on pregnancy rates but are not

evident based on normal fertility examination, advanced testing may be dictated. This

may include hormonal evaluation, karyotyping, testicular aspiration or biopsy, sperm

chromatin structure assay, electron microscopy, HOST (hypo-osmotic swelling test) and

acrosomal integrity tests.

Hormones that may be evaluated include testosterone, estrogen, prolactin,

LH (luteinizing hormone), FSH (follicle stimulating hormone), and thyroid hormones.

Dogs with testicular degeneration may have elevated estrogen concentrations and

decreased testosterone concentrations. They may also have elevated FSH and LH

concentrations. Prolactin concentrations may be increased or decreased depending on

normal pituitary (brain) function and feedback from the testes. Thyroid hormones are

commonly assessed when faced with infertility problems. There is little direct evidence

to substantiate that hypothyroidism directly affects reproductive function. However, it

is believed that through indirect mechanisms, chronic thyroid dysfunction may affect

the brain’s ability to either produce hormones or respond to hormones released by

the testicles in feedback loops and may thereby result in reproductive dysfunction and

infertility secondarily (in dogs with overt signs of hypothyroidism).

HOST or hypo-osmotic swelling test evaluates the integrity of the sperm plasma

membrane (the membrane that surrounds the entire sperm cell). The test is performed

by placing the sperm in a special solution which results in water being transported across

the sperm plasma membrane into the cell to try to equalize the osmotic pressures from the inside to the outside of the sperm. Water will cross the membrane and enter the

cell if the membrane is intact and the transport mechanisms are functioning normally.

If the membrane is not intact, then the transport mechanism will not function properly

and no fluid will enter the cell to equalize the pressure differences. If fluid crosses the

membrane, the cell will swell, which results in a bending/curling of the sperm tail.

Sperm chromatin structure assay is a method to assess the DNA content of the

sperm head. This assay compares the amount of DNA present in each sperm and how

much variation there is between individual sperm cells. Normal dogs have very little

variation in the amount of DNA present in each sperm head, while dogs with abnormal

sperm may have a wide variation in the DNA content of the sperm. Electron microscopy

is a special microscopic technique that allows for very detailed examination of the entire

sperm at very high magnifications. Cross sections and full length sections of individual

sperm may be examined to see if there are abnormalities of structure that are beyond that

seen by the light microscope. There are 2 types of electron microscopy, transmission

and scanning. Transmission EM provides a two dimensional view of the interior of the

sperm cell, while scanning EM provides a three dimensional view of the exterior surface

of the sperm. Transmission EM is typically more helpful in the assessment of infertility.

Acrosome function is required in order for the sperm to penetrate the egg during

fertilization. In some dogs, adequate sperm may reach the oviduct and surround the

egg, but the acrosome reaction may not occur normally resulting in failure of fertilization.

In these dogs, all other sperm testing may be normal. To assess acrosome function, a

drug called calcium ionophore is added to the semen. This substance will induce the

acrosome reaction in normal sperm. A fluorescent dye is then added and those sperm

whose acrosomes react will take up the dye, while those that don’t react, don’t take up any stain. This test is still being refined for use in the dog, but holds good promise for

future use.

Males should be evaluated for reproductive function prior to their first attempt at

breeding, if it has been several months or years between breeding, or if fertility begins

to decline or is questionable. In some cases, a routine reproductive examination will

suffice, but in others, advanced diagnostics may be required.

Factors affecting Fertility in the Male

COMMON QUESTIONS WHICH YOU MAY OR MAY NOT KNOW THE ANSWERS TO?

When can I start using my dog at stud?
Puberty is defined in male dogs as normal semen quality and exhibition of normal breeding behaviour. The average age of puberty in dogs is 10 months with a wide normal range varying from about 5 months to 2 years. Small breed dogs go through puberty earlier than giant breed dogs. All intact male dogs should have normal semen quality by 24 months of age. Normal breeding behaviour may or may not be present by 24 months of age; many nonmedical factors affect breeding behaviour.

I want to sell my dog and the new owner would like to know what the dogs semen is like?
Make an appointment to see your vet and we will collect and evaluate your dog’s semen.

How is semen collection carried out?
The canine penis lies within the prepuce.
The penis is made up of the distal glans, the pars longa glandis, which contains the os penis and the proximal bulbus glandis. The prostate encircles the urethra at the neck of the urinary bladder and is the only accessory sex organ in dogs.

Semen collection should be attempted in a quiet area with a nonslip surface. Consistent use of one room or one rug acts as a training aid if the procedure is to be performed more than once. Presence of a teaser bitch, especially if she is in oestrus, is associated with ejaculation of better quality semen.

If a teaser bitch is present, she should be restrained such that her hindquarters are on the rug, and should be muzzled if necessary to ensure that she does not endanger the dog. The male dog is presented to her, and is allowed to lick at her vulva and mount her if he so chooses. Massage the area of the bulbus glandis through the prepuce, briskly. As soon as erection begins, use the hand holding the prepuce to put pressure on the caudal bulbus glandis and push the penis out of the prepuce while using the hand holding the collecting cone to push the preputial skin proximal to the engorging bulbus glandis. Encircle the penile shaft tightly through the collecting cone just proximal to the bulbus glandis.

The dog ejaculates three fractions. The first fraction is ejaculated as the dog thrusts vigorously. This fraction is of fairly small volume and is clear; it is prostatic in origin. The second fraction may be ejaculated while the dog is thrusting or just after he stops the vigorous thrusting behaviour. Many dogs attempt to lift a leg to form the tie after thrusting is done. The second fraction is the sperm – rich fraction, and is white. The third fraction is prostatic in origin and is secreted in pulses that will be palpable in the hand holding the collecting cone. Concurrent anal contractions are evident. Once the third fraction is present no more sperm rich fluid will be ejaculated.

Afterwards it is best to walk the dog away from the teaser bitch and the collection area, if necessary gently cold – packing the penis with cool cloths, providing cool hydrotherapy, or feeding the dog will encourage detumescence. Make sure complete detumescence has occurred and that the tip of the penis is not protruding before kenneling the male dog.

SEMEN EVALUATION

So how do we assess your dog’s semen?

1. Colour
   Colour is assessed visually. Normal semen is milky white . Abnormal colours that may be seen include clear (no spermatozoa in the ejaculate). Bright or dark red coloration is due to fresh blood contamination, most commonly due to prostate disease or penile trauma. Brown discoloration is usually indicative of old blood, associated with prostate disease. Yellow colour is urine contamination. Green discoloration is indicative of prostate infection.

   2. Volume
   Volume varies depending on how much of the third, or prostatic fraction of the ejaculate was collected. Volume is not correlated with quality.

   3. Motility
   Motility should be assessed soon after semen collection. Place one drop of semen on a glass slide. Subjectively assess the percentage of spermatozoa that are moving forward; normal is 70% or greater.

   4. Morphology
   A higher percentage of morphologically normal spermatozoa is desirable as poor morphology is often associated with poor motility. Morphologic defects can be classified as primary (occurring during spermatogenesis) or secondary (occurring during storage in the epididymis or as an artefact of sample preparation).

   Examples of primary morphologic defects are double heads, double midpieces, double tails, bent midpieces, and proximal cytoplasmic droplets.
   Examples of secondary defects are bent tails, detached heads, and distal cytoplasmic droplets. If most defects are primary, this suggests that a problem lies within the testis and prognosis for return to normal semen quality is worse than if most defects are secondary.

   5. Total number of spermatozoa.
   Concentration varies with semen collection technique; if a lot of prostatic fluid was collected, the sample will be dilute with low concentration of spermatozoa. However, the total number of spermatozoa in the sample does not vary with technique and is the valued number in semen evaluation. Volume (milliliter per ejaculate) multiplied by concentration (spermatozoa per milliliter) yields total number (spermatozoa per ejaculate). Normal value is dependent on the size of the dog, ranging from 300 million to 2 billion. The minimum number of normal spermatozoa needed to impregnate bitches is about 200 million. A dog with 500 million spermatozoa in his ejaculate would probably be fertile even if only 50% were morphologically normal (500 million × 0 .5 = 2 50 million). However, if that same male only had 10% morphologically normal spermatozoa, he would have to breed a given bitch five times over her fertile period to be likely to achieve pregnancy.

   Alkaline phosphatase (ALP) in seminal fluid arises from the epididymes and testes. Samples of seminal fluid containing no spermatozoa should be assessed for ALP concentration. Low ALP concentration value suggests that no fluid was retrieved from the reproductive tract, implicating that apprehension or pain is preventing complete ejaculation, or obstruction. On the other hand, normal value ( > 5000 IU/l) suggests that fluid was retrieved from the reproductive tract and that spermatogenesis is not occurring.

2. Miscellaneous tests
   Cytology and culture. Cytologic changes and growth of bacteria from seminal fluid are not well correlated. If prostatitis or infection elsewhere in the reproductive tract is suspected, microbial culture should be performed even if cytology is noninflammatory.

So what is normal semen?
A. Normal Colour = Milky or opalescent
B. Volume = 1 to 30 ml Percentage of progressively motile spermatozoa 70% or greater
C. Total number of spermatozoa in the ejaculate = 300 million to 2 billion
D. Percentage of morphologically normal spermatozoa = 80% or greater
E. pH = 6.3 to 6.7
F Alkaline phosphatase of seminal fluid = > 5000 IU/l
G Cytology = Noninflammatory; low numbers of healthy polymorphonuclear cells may be present in normal semen.
H Microbiological culture = Fewer than 10,000 bacteria/ml is nonsignificant bacterial growth.

I want to send my dogs semen to a different country. How can I do this?
The two main ways to transport semen are chilled or frozen. I guess before you decide to send semen anywhere the dog should be certified free of heritable conditions and infectious disease. All dogs should be tested and proven negative for canine brucellosis before semen is collected for chilling and shipment abroad. Specific tests for hereditary conditions vary by breed. Most breeds require Orthopaedic certification for hip dysplasia and Eye evaluation for ocular defects among other tests before sending semen anywhere.

1. Chilled semen
   Chilled semen may be inseminated into the vagina or the uterus. Reported conception rates with vaginal insemination average 47% and with intrauterine insemination 81%.
   Chilled semen is made up of ejaculated spermatozoa suspended in an extender. Extender is a fluid medium containing nutrients, buffers, and anti – microbials. Recipes for homemade extenders exist. Most veterinarians use commercial extenders.

   Semen is collected and evaluated. If the total number of spermatozoa in a single ejaculate is low, several samples can be pooled and processed together. Techniques to increase number of spermatozoa in the ejaculate include serial collections at 30 – to 60 – min intervals and presence of an estrous teaser bitch. The semen is then normally centrifuged and the extender added. The tube of extended semen should be tightly capped and placed in bag to ensure that the semen is not lost if the tube breaks during transport. The sample is then wrapped and placed in the cool shipping device. Ideally shipment should ensure that the sample reaches the bitch within 24h after collection. If properly packaged, chilled, and maintained, the sample should maintain its quality for up to 48h and perhaps longer with some samples lasting up to 7 days. Packaged samples can be shipped at the airport or a commercial shipper can be used. The advantage of using airlines is that the sample can be collected shortly before a flight is due to leave and the bitch inseminated soon after its arrival, permitting a very short turnaround time from collection to insemination. The disadvantages are inconvenience, cost, and the ability of the airline to open or refuse to carry unaccompanied packages.

   The semen of some male dogs does not tolerate chilling and shipment, presumably because of a subclinical disease or because of the semen’s chemical incompatibility with the extender. It is recommended that dogs intended for use as a stud with chilled semen have semen collected, extended, stored in the refrigerator overnight, and evaluated the next day, to best mimic chilling and shipment. This should be done well before any bitches of interest are in heat. If semen quality is not maintained, problems with the particular dog can be addressed or another suitable male can be identified to perform the breeding if necessary. The breeder is encouraged to trace all package tracking numbers and to have a back – up plan in place should the sample be damaged or lost.

   B. Frozen semen
   Viability of canine spermatozoa is decreased after freezing and thawing. This is due to physical destruction of cells as they undergo osmotic change, dehydration, ice crystal formation, and induction of chemical reactions during the freeze– thaw process that mimics capacitation. Insemination directly into the uterus is strongly recommended. Reported conception rates with frozen – thawed semen for vaginal insemination average 45%, for transcervical insemination 70%, and for surgical intrauterine insemination 95%
   Frozen semen is made up of ejaculated spermatozoa suspended in an extender. Extender is a fluid medium containing nutrients, buffers, and anti – microbials and, for frozen semen, a cryoprotectant to minimize disruption of the spermatozoa cells as they freeze and thaw. Cryoprotectants most commonly used are egg yolk and glycerol. Egg yolk may also prevent premature capacitation of spermatozoa.

   Collect semen in a routine manner and perform a complete semen evaluation. Only very good – to excellent – quality semen warrants freezing. Samples with high percentages of proximal cytoplasmic droplets may not be suitable for freezing; presence of proximal cytoplasmic droplets has been associated with decreased fertilizing capability and poor survival after freezing and thawing. Semen samples containing 40% or greater blood and blood products are not suitable for cryopreservation; haemoglobin released by haemolysis decreases post – thaw viability.

   The semen then goes through a process of extenders and slow cooling before finally being ready to transfer into straws in a permanent tank containing liquid nitrogen. All samples must be maintained in fluid liquid nitrogen or liquid nitrogen vapor for storage or shipment until they are to be thawed for insemination.

   The semen of some male dogs does not tolerate freezing, presumably because of a subclinical disease or because of the semen’ s chemical incompatibility with the extender. It is strongly recommended that dogs intended for use as a stud with frozen semen have semen collected and frozen, and one straw or pellet immediately thawed to assess post – thaw quality. This should be done well before any bitches of interest are in heat. Because some males can be identified as having semen that is difficult to freeze, breeders should be reminded that their unique problem may have a genetic basis.

   Semen must be maintained in fluid liquid nitrogen or liquid nitrogen vapor at all times. Some shippers refuse to carry canisters containing fluid liquid nitrogen.
   “Dry shippers” contain liquid nitrogen vapor and will remain charged for 1 to 3 weeks, permitting shipment; either the dry shipper should be replenished with fluid liquid nitrogen on arrival or the straws transferred to a more secure liquid nitrogen container upon arrival.

What is Infertility (Male)?

Possible symptoms of infertility include fever, bloody discharge from penis, failure to ejaculate, and pain/discomfort. Treatment varies depending on the underlying cause, but can range from medicinal to surgery. Prognosis depends largely on the underlying cause, some cases have a complete recovery while others have no viable treatment options at this time. Male dogs who should be evaluated for infertility are those who impregnate less than 75% of female dogs who are known to be fertile, provided that correct breeding management and ovulation timing has occurred. Infertile dogs may be consistently infertile or may have been fertile previously but have experienced a decline in successful impregnation.

Infertility in male dogs may result from a variety of causes. These causes can be broken down into the following categories: infectious or inflammatory, physical, traumatic, immune mediated, chemical, behavioral, genetic or chromosomal, endocrine, neurologic, tumors, and metabolic.

Symptoms of Infertility (Male) in Dogs

• Fever
• Uneasiness or discomfort
• Bloody discharge from penis
• Painful ejaculation
• Swelling/pain of testes
• Depression
• Pain in lumbar region
• Retrograde ejaculation (sperm is expelled into the bladder instead of the urethra)
• Changes in libido
• Failure to ejaculate
• Refusal to breed

Types

Cases of male fertility can be broken into categories by cause. The following categorizations can be used to identify male fertility:

• Infectious or inflammatory: This may affect specific parts of the reproductive system or be more general.
• Physical: Infertility may be caused by anatomic defects. These defects can be congenital (at birth) or acquired.
• Traumatic: Traumatic events that affect the scrotum may affect fertility.
• Immune mediated: This can affect the production of sperm if antibodies are produced against the testicle, essentially leading to self-destruction.
• Chemical: Exposure to certain chemicals or use of certain medications may impact fertility.
• Behavioral: In many behavioral cases, a change in libido or failure to ejaculate can be observed.
• Genetic or chromosomal: These regularly result in sterility but may occasionally cause subfertility.
• Endocrine: Endocrine causes are related to hormone imbalances that cause infertility.
• Neurologic: Infertility at the hand of neurologic causes has to do with changes in nerve transmission.
• Tumors: These can affect fertility by changing temperature regulation or taking up space and affecting functionality.
• Metabolic: These causes may affect the reproductive system secondarily through their impact on other organs.

Causes of Infertility (Male) in Dogs

• Infectious or inflammatory: Infection or inflammation may affect the testes, epididymis (a duct behind the testis through which sperm passes), spermatic cords (cord-like structure formed by the vans deferens), prostate, penis, and prepuce. Inflammation of the prostate is the most common site of inflammation in the male. Infection may be caused by bacteria, including staphylococcus, streptococcus, Escherichia coli, Proteus Pseudomonas, Brucella canis, Pasteurella, and Mycoplasma.
• Physical: These abnormalities may include missing segments of the reproductive tract, which could cause an absolute sperm deficiency if both sides are affected, or a decrease in sperm count if only one side is affected. Granulomas (plugs of sperm) may cause blockage to the ejaculatory tract. Obesity may cause overheating of the testes, affecting fertility. Fluid accumulation in the scrotum as a result of peritonitis (inflammation of the stomach) or leakage of fluid can also affect fertility.
• Traumatic: Trauma can alter testicular function or sperm production and ejaculation as the result of thickening of the scrotum, fluid accumulation in the scrotum, or bleeding in the testes or spermatic cords. Penis trauma can also have scarring which can affect ejaculation.
• Immune mediated: Breakdowns in the reproductive system can cause the reproductive system to turn itself, ultimately resulting in a form of self-destruction. With time, normal testicular function may return if the body is able to recover.
• Chemical: Environmental toxins, medications, anabolic steroids, and pesticides or insecticides may affect fertility.
• Behavioral: Some behavioral causes of infertility include fear or anxiety, inappropriate discipline for male sexual behavior, timidity or confusion, or territorial issues.
• Genetic or chromosomal: There are really no treatments for genetic infertility, though related members of their bloodline should be evaluated and heavily considered when determining breeding purposes.
• Endocrine: Possible hormone imbalances that affect fertility include steroid hormones (such as testosterone and estrogen), pituitary hormones, thyroid hormones, or others.
• Neurologic: Nerve transmission can be affected by problems with the spine or vertebral column, affection ejaculation.
• Tumors: Types of tumors in the testes include seminomas, Sertoli cell tumors, interstitial cell tumors, lymphoma, and teratomas. Castration is typically suggested.
• Metabolic: Types of metabolic diseases that may inadvertently affect the reproductive system include diabetes, hypothyroidism, and epilepsy.

Diagnosis of Infertility (Male) in Dogs

Your veterinarian will ask you to detail your dog’s medical history and any symptoms or problems that you’ve noticed. Your veterinarian will also rule out poor breeding management as the cause of presumed infertility. This may be evaluated by determining if the female has had reproductive success before, and if they have not, possibly by breeding to another female to see if this female becomes pregnant. Additional diagnostic tests may include:

• Physical exam
• Reproductive exam
• Endocrine profile, which will measure hormones to determine a cause of the infertility
• Ultrasound, used to identify abnormalities and examine the prostate gland
• If prostate infection is the cause, palpation of the prostate can aid in diagnosis by revealing enlargement of the prostate and pain.
• Breeding soundness exam, in which a sperm sample is collected and sperm count and characteristics are evaluated.
• Microscopic examination and bacterial culture of sperm and urine
• Testicular biopsy

Treatment of Infertility (Male) in Dogs

• If infection is the cause, antibiotics can usually treat the problem. In some cases, antibiotic treatment may be long term. In severe cases, removal of the infected reproductive part may be necessary.
• If sperm plugs are to blame, regular collection may be necessary.
• If obesity is to blame, weight reduction measurements should be taken.
• Retrograde ejaculation can be treated through medication to close the urethral sphincter prior to breeding/collection.
• If the cause is chemical, removal of the chemical or discontinuation of the medicine will likely return your dog to normal function.
• If a hormonal balance is to blame, administration of hormone supplements such as human chorionic gonadotropin, human menopausal gonadotropin, gonadotropin releasing hormone, thyroid replacement hormones, or others, may return your dog to normal functionality.
• Anti-inflammatories may aid in neurologic causes resulting from spine-related issues.
• Artificial insemination may be an alternative for breeding, should the male be unable to regain vitality.
• If a tumor is the cause, castration of the testicle is the most common course of action. The remaining testicle may still be able to produce sperm.

Compiled  & Shared by- This paper is a compilation of groupwork provided by the Team, LITD (Livestock Institute of Training & Development)

 Image-Courtesy-Google

 Reference-On Request