SUCCESS WITH FROZEN SEMEN: SIRE MANAGEMENT

Dr John F Newell B.V.Sc.
Rocky Ridge Frozen Semen Facility
PO Box 1166 Warners Bay, NSW Australia

• Sire Management
• Sire Stress Syndrome
• Case Study – Big Daddy Cool (With permission from Nick Janjic)
• Extender Technology
• Semen Extremes
• References
  

We have discussed the essential considerations of brood bitch management based on the “Four Steps to Breeding Success” developed by Richard and Sharyn Conole of Camelot Farms, Texas USA. The important Sire considerations for frozen semen success involve three broad areas:-

• Sire management approach and Semen quality
• Sire Stress Syndrome
• Extender technology

Sire Management

Sire management procedures and understanding the peculiarities of each sire is essential for prolonging the active working life of a sire. It also facilitates the collection of quality semen which is more likely to sustain the rigors of processing to -196°C. Planned sire management is often not practiced, however a regular health and behavioural assessment as well as a usage plan will maximize semen quality.

This applies equally to the stud dog who may be collected only 2 -3 times per year as much as high demand greyhound sires capable of generating in excess of $A1 million service fees per annum. We must be acutely aware of the relationship between sire management, behaviour and the resultant physiological responses that ultimately affect semen quality.

With an elite group of high demand greyhound sires in the more temperate regions, the concept of 12 month availability needs re-thinking as this places depletion stress on the endocrine system and the consequences of such depletion may include up to six months to recover. Frozen semen allows us to better manage our sires. Quality collections can be stored at times of highest fertility during the cooler months for use when high temperature stress precludes satisfactory collection. The majority of bitches cycle at this time of year when the sire is less capable of “keeping up with the demand”.

The Advantages of Frozen Semen

• Breed to a sire when he is no longer capable of doing so
• Breed more than one bitch to the same sire on the same day
• Breed more than one bitch to the same sire in different locations on the same day
• Prove sire before he retires to stud
• Utilizes known semen quality – maximizes conception rates
• Provides less stress for aging sires – thereby prolonging reproductive life
• Less time and travel cost for the bitch
• Provides affordable insurance against loss of stud dog reproductive services
• Achieve more than one breeding from one collection
• Exert positive control of reproductive diseases
• Preservation of the sire’s gene pool after death
• Allows pre-purchase of breeding units facilitating a planned breeding program
• Provides easy access to the sire of your choice
• Allows affordable access to overseas sires
• Provides access to a wider gene pool encapsulating hybrid vigor
• Avoids trauma and mating injury with post coital haemorrhage
• Allows better sire management with less demands in summer when bitch demands are high
• Avoids sire – bitch behaviour incompatibility

How to Approach the New Sire

Proper sire management begins on day one and financial pressures from brood bitch owners wanting to use the sire as he “walks off the track” need to be resisted. A proper “let down” period to facilitate the transition from racing to stud duties needs to be enforced to minimize rebound sub-fertility.

• Avoid early negative conditioning experiences. The new sire is always inquisitive and usually keen. Make collection a positive experience – one that will be co-operatively sought next time. Always a routine in a relaxed and familiar environment with no extraneous noises or visual distractions of other males that may invoke dominance-submissive behaviour. The veterinary clinic may not be such a place to facilitate quality collections.
• Has the sire relocated from another stud? Need to inquire about prior routine and history if congenial terms exist with the previous stud. Is he more relaxed for collection inside or outside? Have any collection peculiarities or habits been noted? – Colour bias has been observed in one notable black sire in Australia who is particularly stimulated by black bitches irrespective of progesterone level.
• Allow time to settle in to the new kennel, gain his confidence and establish a new routine. Ideally the new sire must be treated so he quickly displays a likeness for his new surrounds and the new stud-master. A minimum four weeks settling in is recommended.
• It is advantageous to allow novice sires to observe experienced sires working from a distance.
• The collection technique should be a reinforcing positive experience based on routine which is repeated at each collection. By these means a Pavlov conditioning experience is established and ease of collection increases. Difficult or flighty teaser bitches should be quickly identified and removed during this early learning phase so as not to provide one or two negative experiences which may impact profoundly on subsequent collection attitudes
  
  A latex collection sheath (bull nose cone) is usually preferable to disposable plastic sheaths – these are noisy and quickly loose heat in the cooler weather. Collection sheath, collection tube and non-spermicidal lube are held in an incubator at 38°C immediately prior to collection.
  
  1. Ensure the sire is well exercised prior to introduction of the teaser bitch. Familiar trees in the collection pen will encourage frequent and complete urination – essential to not contaminate the collection which would render it unsuitable for freezing. The use of a defined collection yard presents residual pheromones essential in defining the positive collection reflex. It is recommended that a second collection sheath be held warming in the pocket so that it can be easily substituted should urine appear in the first few milliliters of the collection.
  2. The collection technique should simulate the comfort of a natural service. As penile engorgement progresses and at the time of partial engorgement of the glans-penis it is important to slide the sheath behind the engorging glans and apply annular pressure around this area. This simulates a coital lock and usually elicits a maximal second fraction release. Incomplete delivery of the second fraction can occur if the glans swells whilst still covered by the sheath causing discomfort and erection loss.
  3. The teaser bitch is introduced and allowed to urinate whilst the sire is restrained. Sniffing this pheromone laden urination is profoundly stimulatory to the male.
  4. In the absence of a teaser ensure that a supply of frozen urine from an peri-ovulatory bitch is available to be thawed as pheromone persistence in this frozen aphrodisiac elicits strong jaw champing and salivation from the sire and facilitates a successful collection without a teaser.
  5. If both sire and bitch arrive for collection ensure they have traveled in separate compartments to avoid sire exhaustion and masturbatory semen loss.
  6. Traditionally if the collection is for freezing then only the second sperm rich fraction is harvested whilst for direct artificial insemination some of the third fraction is also collected. Some nervous or inexperienced sires will continue to pass sperm well into the third fraction and we must avoid stopping the collection too soon if the clear demarcation between second and third fraction is less defined.
  7. Once a satisfactory sample is obtained ensure the erection subsides without painful inversion of the prepuce with consequent hair irritation to the sensitive penile mucosa.
     
• Collection frequency needs to be carefully considered guided by both semen quality and environmental factors. Most regulatory bodies state a collection frequency of fourteen times per month, however this is too frequent and will lead to primary semen abnormalities if this collection frequency is adhered to. In reality sires producing four splits or better will produce even more soundly if only collected twice weekly.
  Be aware of the client who presents a dog for freezing with a last mating 12 months ago. The spermatogenic cycle, although active is in hibernation, with low grade leakage of mature sperm into the bladder, and a better sample for freezing will be produced after at least two subsequent ejaculations.

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Sire Stress Syndrome

Commonly observed with high demand sires but also seen in some individuals who are only called upon for collection 3 – 4 times per year. Many factors need to be considered in an attempt to prevent or treat a sire presenting with this signalment.

Typically we see new sires initially responding positively to the stimulatory stud environment, with good libido and semen counts. Within 4 – 6 weeks semen quality may deteriorate. We see low counts of poor morphology and at times azoospermia (ejaculation of seminal fluid that does not contain spermatozoa). Testicles often soften but libido usually remains high.

The Sire Stress Syndrome represents a sub-fertility state and correct recognition and management in the early stages will stop progress to an infertile state. This needs to be conveyed to sire owners in strong terms regardless of the financial implications of such management decisions. Owners need to be informed that the impact of sub-fertility will not correct quickly as it takes on average 62 days for spermatozoon to form and as this is a gradual process total count and quality may not improve for two to six months. If stem cells have been destroyed then there may never be a return to sperm production.

As you scan through this investigative checklist it soon becomes apparent that many factors may act simultaneously to compromise semen production. The end result being the “Sire Stress Syndrome”.

Sire Stress Syndrome – Investigative Checklist

• History - Ensure a thorough history is taken of the sire, his recent breeding frequency, management routine at a previous stud and time in residence at the current facility. Ensure that the “Approach to the new Sire” principles and recommended collection protocol has been implemented.
• Clinical Examination – A thorough palpation and assessment of the reproductive track is essential to define any early abnormality. Testicles are palpated for symmetry, tone and size. Palpation for inguinal and scrotal hernias that may cause pain inhibition to normal function. The penis is extruded and examined for mucosal integrity, secretions, ulcerations. The prostate is palpated for size, symmetry, tone and pain. By using three collection sheaths a prostatic fluid sample can be obtained for cellular presence and bacterial culture. Normal prostate fluid is clear, acellular and with a pH between 6.0 to 7.2. Bacteria are cultured from the prostate fluid of normal dogs and the clinical significance of bacteria in prostate fluid still endures debate. Prostatic secretions, however have a bacteriocidal role, preventing ascending infections from the bladder. The prostate produces most of the ejaculate volume. Dihydrotestosterone facilitates the growth of prostate cells whilst Prolactin increases the uptake of testosterone and the synthesis of dihydrotestosterone. Prolactin levels rise with stress. Most dogs have developed some degree of prostatic hypertrophy by 5 years of age. This usually develops to benign prostatic hyperplasia and it is thought all entire dogs over 10 years of age will be so affected.
• Observation – Much information is to be gained by observing the stud master interact with the sire whilst collection is occurring and at other times. Particularly look for fear and apprehension by the sire which constitutes strong negative feedback and will quickly shut down endogenous GnRH. This behavioural interaction is critical and must be relaxed and accepting to get the best from the sire in the long term.
• Exercise Regimes – It is important not to provide added stress to the sire by focusing on a need to keep him fit. He is no longer a racing greyhound - a daily walk, free gallop and slip once a week does not make the transition from a track athlete to a sire an easy physiological task.
• Inbreeding Co-efficients – Individuals with inbreeding co-efficients between 0.125 and 0.558 have decreased reproductive performance compared to out-bred dogs
• Unilateral Cryptorchid Progeny – The sire progeny of single descended testicle sires have reduced reproductive performance. The greyhound industry seems to be the only administrative body that allows this fault to perpetuate and there may be serious implications for breeding in the future if we largely base our breeding industry on unilateral cryptochorid sires. Cryptorchidism is a heritable, sex linked autosomal recessive trait and the incidence is higher in closely bred or inbred greyhounds. Many authors in the canine reproductive literature refer to the hereditary trait of cryptorchidism and state that a dog with this condition should not be used for breeding.
• Testicular Biopsy – Is very much a last resort in my assessment protocol as it is an invasive procedure not without complications. Post surgical haemorrhage, infarction, fibrosis are possible sequellae. A special fixative must be used to minimize artifact development - Zenker’s Fixative is suitable. Given that definitive diagnosis is possible in only 15% of cases examined thus far and many of these are established without the aid of biopsy then the positive value of the procedure falls into question.
• Drug effects – A thorough and honest history must be obtained to determine the likelihood of residual drug effects on semen production. Anabolic steroids exert a negative feedback on the hypothalamus and adenohypophysis. As a consequence serum LH and intra-testicular testosterone will decrease even if normal testosterone levels remain in the blood as a result of exogenous testosterone administration. Intra-testicular testosterone concentrations, necessary for spermatogenesis, are usually 50 to 100 times that found in the peripheral blood. Intra-testicular testosterone declines after anabolic steroid therapy and spermatogenesis is adversely affected. Administration of Gonadorelin® as a performance enhancer has a negative feedback effect on endogenous LH release which may take many months to revert to normal cyclical rhythm.
• Functional Abnormalities – In cases of azoospermia functional abnormalities such as retrograde ejaculation need to be assessed as a diagnostic possibility. Cystocentesis may determine semen presence in large numbers in the bladder after ejaculation. Smooth muscle spasm directs the ejaculate to the bladder.
• Pathology Profiles
  (I) Full blood count as a standard screen assessing fitness, dehydration, organ function and general medical conditions that may impact on reproductive fitness. In conjunction a urine sample for standard Dipstix, Specific Gravity and microscopic assessment as well as a faecal flotation test for residual parasite determination.
  (II) Brucella canis screening for new sires is mandatory in those countries where the infection is endemic. As a precaution twice yearly testing is recommended. Frozen Semen only sires avoid the career threatening risks of such an infection
  (III) Semen Assessment
  Evaluate immediately after collection. It is often wise not to make decisions based on a single sample as external factors can impact on a particular collection. At least 2 – 3 samples should be assessed over a ten day period. Consider the sire’s age, experience, workload, nutrition and physical condition when making decisions about a semen sample.
  Motility:
  Assess the percentage of progressively motile (= moving along a straight path) spermatozoa with 20X and 40X objectives, ideally on a warm stage. Motility at collection should be greater than 75%.
  Concentration:
  Using the Unopette™ counting system and haemocytometer manual counting will provide an accurate result. This will determine the count per ml and a total sperm count is determined from the total volume of evenly suspended ejaculate. Counts vary from 150 million to over 3 billion dependent on the size of the dog, testicular size, frequency of collection and concurrent health and physiological status.
  Morphology:
  Assessment with conventional light microscopy or after staining with eosin-nigrosin or Diff-Quick®.
  Not only is it important to assess % normal v/s % abnormal but it is of critical importance to differentiate primary and secondary abnormalities.
  • Primary Abnormalities - these reflect altered spermatogenesis, often the result of orchitis, hormone therapy or physiological imbalances The common findings are macrocephalus, microcephalus, double heads, indented heads, pointed heads, thickened neck, eccentric insertion, coiled or kinked mid-piece, double or triple tail
  • Secondary Abnormalities – these occur if epididymal function is abnormal or if sperm has resided for a long time in the epididymis or sample artifacts during handling. Commonly found are bent heads, detached heads, cytoplasmic droplets, bent mid-piece, coiled tail, detached tail, folded tail.

  Semen Culture:
  May be indicated if white blood cells are evident on microscopic examination or if there is distinct pain at the time of ejaculation.
  Semen assessment is of course an essential element in frozen semen processing. It provides the mathematical basis for calculations that ultimately determine how many splits are possible from an ejaculate. Moreover it provides a window to view deleterious changes occurring at a cellular level within the testes before they progress to overt clinical “Sire Stress Syndrome”. There is however no direct correlation between % abnormal and conception rate.
  (IV) Free T4 is essential to determine. If low (less than 18nmol/l) then a thyroid supplement is indicated. Subsequent blood tests should occur six hours after dosing with the levothyroxine sodium. Hypothyroid sires generally show reduced libido, softening of the testicles and an increase in primary abnormalities with morphology assessment.
  (V) Canine Prolactin levels increase with stress. Elevated prolactin levels cause a negative feedback and both FSH and LH secretion is reduced. Prolactin may be a good indicator of stress levels and is highly significant because of its effect on FSH & LH when elevated.
  (VI) Cholesterol should also be specifically assessed as it is the pre-cursor of the major sex hormones. Fifty percent of the energy in the diet should be in the form of fat and during the digestive process animal fats are converted to cholesterol. It is a common error to not provide sires with sufficient dietary fat.
  (VII) The relevance and significance of hormone assays needs to be considered. For Follicle Stimulating Hormone & Lutenizing Hormone draw blood sample 10 minutes after GnRH administration. Serum testosterone levels are of dubious usefulness given its episodic release. Samples taken from a normal dog ranged from 0.5 to 9.0ng/ml depending on time of sampling relative to burst of testosterone release. (1) Gonadotrophins are also released in an episodic manner.
  (VIII) Assess Alkaline phosphatase (AP) in the seminal plasma – in a full ejaculate is >5000U/L and if this ejaculate is azoospermic then primary testicular failure is indicated and the prognosis may be poor. AP in the first fraction, or in an incomplete ejaculate is often less than 100U/L. Obstructive granulomas usually result in a seminal plasma AP less than 5000U/L. (2)
  

Sire Stress Syndrome – Therapy and Management Checklist
Investigation Plan
Therapy Considerations

I would urge a re-think of our approach to male infertility. It represents a multi-factorial cascade of varied aetiology and a classical cause and effect investigation rarely arrives at a definitive diagnosis. In fact a recent investigation confirmed that a definitive diagnosis was possible in only 15% of cases of male infertility. The problems with investigative hormone assays stem from variable excretion rates and values difficult to directly link to a clinical response plan. Testicular biopsies clearly define the resultant pathology but rarely lead to an effective clinical course of action. They are not without risk and extensive fibrosis has been observed as a result of such biopsy.

A more holistic approach has yielded good results at our facility. Just about every sire will at some point lapse into some level of Sire Stress Syndrome. Be aware of these changes and manage your sires according to the preceding protocols. Direct therapeutic approach involves providing raw materials to stimulate the body’s natural gonadotrophin releasing hormones and judicious rest and routine changes.

Of course specific conditions can be addressed as indicated and the management of these is often long term. For example chronic prostatitis is difficult to resolve as the organ undergoes fibrosis with age and blood supply to deliver effective medication is compromised. Culture and sensitivity of the prostate fluid is always useful. Many cultures reveal Enrofloxacin® 150mg – One tablet / 30Kg daily to be effective medical management, however it is prudent to combine this with adjunctive therapy.

• Saw palmetto inhibits 5-alpha-reductase and aromatase which are both significant in the development of Benign Prostatic Hyperplasia and it is very useful given such anti-androgenic activity.
• Zinc is essential for normal reproductive performance. With high demand sires dietary levels may be inadequate. The prostate contains more zinc than any other gland whilst sperm and seminal fluid contain a lot of zinc. Deficiency may cause prostate problems and reduced fertility.

There are, however many metabolic pre-cursors which are important to provide at times of sub-fertility or reproductive stress. These may not be in sufficient levels in the normal diet or by their presence help to stimulate GnRH production. As a guide supplements that may be indicated presumptively or directly by the lab work-up include:-

• Thyroid supplements at the dose of 0.1mg/4.5kg twice daily. Re-check bloods in 2 weeks ensuring bloods are taken 6 hours after medication dose. Combine this with Seaweed meal or kelp supplement for maximum effect.
• Safflower oil – depending on bodyweight – teaspoon daily for 5 days then tablespoon daily
• Arginine powder – _ teaspoon daily
• Methionine 500mg daily
• L-Lysine powder _ teaspoon daily
• Green lip muscle – Adult dose daily
• Vitamin A – D – Selenium supplement
• Vitamin C – 1000mg daily
• Thiamine mononitrate - 100mg daily
• Calcium pantothenate - 1g daily
• Tribulus terrestris – Horny Goat Weed – Several active ingredients including dioscin and protodioscin aid sexual energy level by increasing the strength of free testosterone. Sterols and saponins protect the prostate from swelling and are also thought to act to protect the prostate from cancer. An adult dose is used for a 30kg dog.
• Chaste tree (Vitex agnus-castus) has been used for maintenance of male sexual function through its effect on GnRH release.

It has been shown that three homeopathic drugs will increase mitochondrial activity in bovine semen when included with the extender. (3)
At Rocky Ridge Farm we have utilized these drugs systemically to assess the response in sub-fertile sires. Ubichinon comp.®, Coenzyme comp.® and Selenium Homaccord® (Heel, Baden Baden - Germany) were administered twice weekly to a total volume of 5ml and there is early presumptive evidence, on this small sample trial, that sub-fertile males showed a more rapid improvement in semen quality than similar untreated sires. The basis of this overt response may stem from the positive effects of these drugs on mitochondrial activity.

Generally direct administration of trophic hormones is to be avoided as initial therapy because of potential negative feedback effects. Serum gonadotrophin (PMSG) can be used at 500IU IM twice weekly for impaired spermatogenesis but best sustained results are achieved when administered after an appropriate spell and supplementation with the above regimes.

Libido difficulties can be treated with chorionic gonadotrophin – 500IU 12 hours before attempted collection. This is a direct effect by stimulating the production of testosterone but may ultimately worsen a sub-fertile state by negative feedback to LH production.

Multi-factorial therapy is essential to address a syndrome which is multi-factorial in origin. Simple cause and effect conditions do exist but are in the minority. Always a good dose of father time and stimulation of interest through behavioural variability is essential in the recovery plan.

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Case Study – Big Daddy Cool (With permission from Nick Janjic)

Big Daddy Cool won 14 of his 25 starts during 2003 before a major fracture ended his short but explosive career in January 2004. He underwent surgery in early February, and with high demand as a sire commenced limited stud duties in mid March where he served 14 bitches over the next month. Conception rates were less than 50% and an examination in Melbourne determined “he was 98% finished as a stud dog”. He exhibited azoospermia at this time. He was withdrawn from the sire list.

I saw Big Daddy Cool on the 27th May 2004. Clinically he had reduced testicular tone and size. Prostate was of normal dimension and symmetry. Coat was dry and muscle tone reduced. He had been relocated to a facility to reside and have semen collected eight weeks earlier.
Bloods showed a lowered T4 (< 6), PCV 52, Cholesterol low (2.4), High stress ratio (N:L = 75:20) No attempt was made to collect semen at the time of this examination as one week prior azoospermia was present and the clinical presentation and work-up suggested that this was likely to be unchanged.

The recent history, the combined effects of internal and external factors formulated an approach to management from a very holistic aspect. Firstly – don’t do a testicular biopsy!

Considerations & Actions:

• Traumatic stress – Insufficient time to recover & initiate the LH cascade
• Relocation stress – BDC was returned to where he was reared to gallop
• T4 supplements & Seaweed meal commenced
• Hippiron® (Iron III hydroxide-sucrose) 1.5ml IV weekly – 5 weeks
• B12/Folic acid 2ml every second day – 6 doses then 2ml twice weekly
• Increase dietary fat – mutton fat, safflower oil or maize oil
• Green lip muscle supplement – glucosamine
• Oysters (Vitamin E rich)
• Arginine powder – _ level teaspoon daily
• 3 weeks before first collection attempt Tribulus terrestris was commenced

Bloods re-checked every 3 weeks revealed a steady improvement in values. Muscle tone, coat health, energy and attitude all improved markedly with de-stressing and treatment. Regular visits from his familiar trainer were essential during the recovery. Only after 10 weeks was he re-introduced to oestrus bitches. He responded with high libido and a strong collection of 3ml with 95% motile sperm. At this time counts were 350M – 550M and he commenced serving and collecting for freeze on a limited schedule in mid August 2004

• Fertagyl® (A synthetic gonadotrophin releasing hormone – Gonadorelin) was commenced at this time at a dose of 1ml weekly

By November 2005 counts had risen to 850M – 1250M with excellent morphology. The owner now reports he ignores him when he yells at him rather than backing away as he did when he was unwell (depressed) and azoospermic. This ignorance is the sign of a happy, confident and playful dog. Ignoring the anthropomorphism, these behavioural observations are a convenient and essential way for the owner to assess progress with the behavioural traits he is familiar with but may not have carefully defined.

Big Daddy Cool has gone on to be booked out each month and with a conception rate exceeding 90% from this frozen semen only sire, now regularly producing 6 – 8 splits per ejaculate.

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Extender Technology

The principles of extender technology are well defined. They are based on reducing the energy consumption and increasing availably energy sources for the sperm cells. As semen is cooled sperm cell metabolism is slowed and this in turn reduces their energy consumption whilst added extenders have constituents that protect spermatozoa from cold shock and rapid thawing, provide energy for sperm metabolism and have correct pH and osmolarity to maintain the integrity of sperm cell membranes.

There are numerous commercial extenders and recipes available to extend and protect semen during the freezing and thawing process. They may utilize

• Cryoprotectants such as sugars, glycerol or DMSO
• Buffers – traditionally Tris-hydroxymethyl aminomethane
• Energy sources – fructose, disaccharide lactose, trehalose
• Detergents
• Antibiotics- to inhibit bacterial growth
• Egg yolk – an excellent cryo-protectant

In reviewing the current literature there is a general consensus that thawed semen when implanted survives for 12 – 24 hours.

It has been our experience that semen frozen using the Camelot system and extender will exhibit some motility for up to three days when held in a tube at room temperature. One of the keys to success involves extending the longevity persistence of frozen semen once thawed and the ability of Camelot frozen semen in this respect is far superior to other extenders observed thus far.

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Semen Extremes

It is interesting to review some of the extremes we may be faced with when attempting to collect and freeze semen.

Largest
My largest patient is “Mack”, an 85kg South African Boerbel. Quality semen is delivered in each of the 30ml collected and his total count exceeds 3.1 billion. It is prudent to distribute his collection over 3 tubes prior to centrifugation and treat each tube as a stand-alone freeze. By doing so we avoid the over-compaction that very concentrated ejaculates may tube pellet to after centrifugation. The compaction of such a large pellet in the centrifuged tube often reduces semen recovery and live sperm losses are proportionally higher in such circumstances. Such a collection regularly yields a 12 or 14 split.

Smallest
Eight pups from a dead dog! – Dr Ray Ferguson – A Camelot vet from Melbourne was asked to freeze semen from a rottweiler which had undergone euthanasia the previous day for bone cancer. Interestingly the euthanasia solution had not killed the sperm (which normally survives for 2 – 3 days after death of the male). Generally bone cancer is associated with testicular atrophy even in the early stages and semen quality is further compromised.

Nevertheless the chilled testicles were dissected and semen milked from the tubules and suspended in warm saline, centrifuged, combined with Camelot extender and subsequently frozen.

Four years later the semen was implanted to “Lucky” who subsequently produced 8 healthy pups. The recovered sperm from the epididymal tissue would only have been a few million but still sufficient for a normal litter! (4)

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References:

1. Freshman JL, Amann RP, Bowen RA, Soderberg SF, Olson PN, Clinical Evaluation of Infertility in Dogs. Sm An Comp.1988;Vol 10 No.4:443-460.
2. Johnston SD, Clinical Approach to Azoospermia in Dogs. WSAVA Proc. 2003
3. Aziz DM, Janowitz U, Schnurrbusch U, Enbergs H, Bovine spermatozoa a suitable cell model for pharmacological studies: effects of some homeopathic drugs on mitochondrial activity and other important parameters of cell function. Research paper Faculty of Veterinary medicine – Bonn University, Germany
4. Dr Ray Ferguson – Monash Veterinary Hospital – Melbourne Australia: pers. com. And reported in “The Melbourne Age” April 23rd 2004
   

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Dr. John F. Newell B.V.Sc	Phone: 02 4375 1001
Tony Wiseman Crt. Camelot.F.S.	Fax: 02 4375 1010