Regulations and Recommendations for International Shipment of Chilled and Frozen Canine Semen

1. Introduction

The interest in international shipment of dog semen is increasing at an accelerating rate as dog breeding is becoming a more and more international hobby, and with the increasing awareness of the need to widen the gene pool to keep the recognized breeds healthy and sound. Dog semen can be shipped as extended and chilled, or frozen. What is most convenient will depend on a number of factors, such as shipping distance, and whether the shipped semen is intended for artificial insemination (AI) of one or of several bitches, but also the rules and regulations of the country in question. In several countries there are different rules for semen from different countries and some also have rules that regulate the exportation of semen. The rules for importation of chilled semen may be different from those which apply to frozen semen. Sometimes the regulations in the importing country preclude the possibility of using chilled semen. The national Kennel Clubs may request prior application for permission to use imported semen, and they usually request proper identification of the semen donor by microchip or ID-tattoo, and/or a DNA test.

It should be kept in mind that both national legislation and Kennel Club (KC) regulations may change at any time. Before undertaking to export dog semen, the bitch owner or the importer of the semen should, therefore, always be advised to contact the Ministry of Agriculture (or the corresponding authority) and the Kennel Club in the importing country to receive information about the latest set of rules and regulations. These contacts should be made well in advance of the planned semen collection and shipment, so that all the necessary health certificates and blood tests can be made in accordance with the requirements. A list of addresses can be found in Appendix 1. (This Appendix 1. was originally compiled in 2001, and although considerably revised the rules may have changed in many countries again. Always check the latest versions).

2. Ministry of Agriculture Requirements

When it comes to the national legislations pertaining to the importation of dog semen, the different countries basically fall into one of the following five categories:

Category 1
No restrictions.

Category 2
A veterinary health certificate issued at the time of semen collection, also stating the microchip number or ID-tattoo marking of the semen donor. (This requirement applies today for most of the countries within the EU).

Category 3
An import permit is required.

Category 4
An import permit and a veterinary health certificate for the semen donor, stating the microchip number or ID-tattoo, and issued at the time of semen collection, or at a stipulated time interval before, and/or after, the semen collection is required.

Category 5
An import permit and a veterinary health certificate for the semen donor issued at the time of semen collection, or at a stipulated time interval before, and/or after the semen collection, and a serological test for leptospirosis (usually l. canicola and l. ichterhaemorrhagica) and/or for Brucella canis. [Note that the blood samples in some cases should be taken prior to (usually within 15-45 days before), in other cases at the time of, and sometimes after (usually 20-30 days, 30-45 days, or 3-6 weeks after) the semen collection].

Some additional requirements that may apply:

• That the semen donor must not have been outside the exporting country for a period of from 2 to 12 months prior to the semen collection
• That the semen donor has been tested for leishmaniasis between 30-45 days after the semen collection (currently required for semen import to Australia)
• That the donor dog has not been used for natural breeding from the time of the blood test until semen collection
• That the semen donor has, or has not, been vaccinated with specified vaccines (e.g. rabies) for a varying time prior to the semen collection
• That the inseminated bitch must not be bred by another dog during the same cycle and that in case she should abort the Ministry must be immediately notified and the fetuses examined
• That the import permit in original must accompany the shipment, or be presented at customs on arrival
• That the liquid nitrogen must be new or unused
• That the liquid nitrogen container must be new or unused; or has been properly disinfected.
• That eggs used for the semen extenders must be from a flock of hens free from Newcastle disease

3. Kennel Club Requirements

• Some Kennel Clubs have no regulations and this usually means that the use of imported dog semen is freely allowed, but it can also mean that registration of litters by imported semen is not officially allowed. This, therefore, should be checked well in advance. However, the lack of regulations generally means that there are no restrictions, or that there are no controls made of whether the bitch has been abroad to be mated, or the semen has been imported and the bitch artificially inseminated.
• Most Kennel Clubs request that the semen donor should be registered by an officially recognized Kennel Club.
• The Kennel Clubs also usually request that the semen donor is permanently identified by an ID-tattoo or a microchip, and that the veterinarian in charge of the semen collection certifies that the dog’s identity has been checked.
• Most Kennel Clubs request that the semen donor has a normal testicular status.
• Some Kennel Clubs request prior application for permission to use imported semen.
• The American Kennel Club requests that a DNA sample is submitted. It is taken with the aid of a special cheek-swab supplied by the AKC (see Appendix 1).
• Some Kennel Clubs have an eradication scheme for various hereditary diseases (for instance Hip Dysplasia, Progressive Retinal Atrophy, and Progressive Nephropathy) for some breeds, and request that the semen donor has been tested free from any such disease.
• Other requirements may be that the dogs have shown that they can mate normally or have proved that they are fertile.

4. General Recommendations for Minimum Documentation to Accompany International Shipments of Dog Semen

In order to comply with the majority of the various national rules and regulations, and as a courtesy to the colleague who will perform the AI, it is recommended that each international shipment of dog semen is accompanied by the following documents, and that the following procedures are adhered to, also when this is not formally required:

• A veterinary health certificate, also stating the microchip number or ID-tattoo marking of the donor dog, and with a statement that the dog has a normal testicular status. (A template for such a certificate is given in Appendix 2).
• A semen quality assessment form, and in the case of frozen semen also thawing instructions, together with a recommendation of how many straws, or vials in the case of pelleted semen, should be used for each AI. (A template for such a certificate is given in Appendix 3).
• The official, Kennel Club registered name of the dog should always be used for all official documents, including the blood test report, never its pet name.
• Always enclose a set of copies of the certificates, either attached on the outside of the shipping case, or on its inside, as a precaution if the airway bill with the original certificates is lost under way, or removed by mistake, for instance by customs in a transit country.
• Straws or pellet vials should be packaged so that they are easy to move from the shipping dewar to the storage tank, i.e. they should be in goblets and canes, or in plastic sheaths, which are properly marked (for marking, see below).
• If the semen is frozen in pellets, do not forget to enclose the plastic bags used for the thawing (Whirl-pak, Nasco, USA) as these are not readily available in all countries.
• Whenever it is possible, always apply a seal to the neopore box, styrofoam box, thermos flask, or LN2 dewar, even when this is not an official requirement. This prevents unauthorized persons from opening it and, thus, minimizes the risks of damage. Preferably put the seal directly on the box, flask, or dewar and not on the outer case. The LN2 dewars usually are equipped with a separate ring for seals. If a numbered seal is used the number should be stated in the veterinary certificate.

5. How to Mark the Semen Straws or Vials

To comply with the requirements from most countries the semen-containing straws or vials should always be marked with the following information:

• The breed (which may be abbreviated),
• The dog’s KC registered name (which may be abbreviated),
• The dog’s KC registration number or microchip number, (for Australia the microchip number is required)
• The date of semen collection (obligatory when import permit, blood tests and veterinary certificates are required), and
• Where the semen was collected/processed.

Example:

The identification markings of the straws/vials should also appear in the certificates.

6. Chilled Extended Dog Semen

Dog semen is commonly shipped after extension and chilling to 5°C (41.0°F). Chilled extended dog semen can retain its motility and membrane integrity for up to 1-2 weeks or more [12,14], although it still remains to be determined in more detail for how long it also can retain its fertilizing capacity.

6.1. Extenders for Chilled Dog Semen

A number of chilled semen extenders are available on the market, the majority being proprietary, such as the CLONE Chilled Kit, the Synbiotics (Zoetis) Fresh Express, Minitübe’s 5 or 10 days CaniPro etc. A non-proprietary and scientifically well documented Chilled Semen Extender (Appendix 4) is now also commercially available (www.canirep.com). It comes in 4 mL cryovials that are stored frozen and ready-to-use after thawing. One such vial is enough for the chilling of 1-2 ejaculates. Extenders for dog semen should always contain some antibiotics, to prevent bacterial growth and the risk for spread of disease. In winter, or when the chilled semen is shipped on one of the long-haul flights and there might be a risk that the temperature drops to below the freezing point, an extender containing 3% glycerol, e.g. CaniRep Uppsala Equex II, extender 1 or the CLONE first freezing extender, will protect the semen sample from freeze damage.

6.2. Chilled Semen Preparation

The second, sperm-rich fraction of the ejaculate is collected and checked for total number of spermatozoa, sperm morphology and motility, and then centrifuged at 300-700G for 5-6 min. The supernatant seminal plasma (which is detrimental for spermatozoa during storage) is discarded and 2-5 ml extender added (of the same temperature as the semen) to the sperm pellet and this is well mixed. Using larger volumes for AI will only lead to loss of spermatozoa due to backflow of semen from the uterus or vagina. The semen is then chilled to around 5°C (41°F) over 30-45 min, or put in a neopore of styrofoam box with 1-2 ice-packs, to chill during transport. If a thermos flask is used for the shipment it should also be chilled before putting the semen sample into it. Check a re-warmed drop of semen for vitality after chilling, before putting the sample in the box or flask. Semen of originally good quality preserved in this fashion and stored chilled has been used successfully after more than 1 week, and can likely be used for even longer if kept in an unbroken cold chain. Mark the semen vial with the dog’s breed, registered name, registration number or microchip number, and date and place of collection (see above). Whenever this is practically possible a numbered seal should be applied on the neopore- or styrofoam box, or the flask. This is required by some countries, and as mentioned above it prevents unauthorized persons from opening the inner package containing the semen. If the seal is numbered, the number should be stated in the veterinary certificate.

6.3. Shipment of Chilled Dog Semen

Use sterile plastic tubes for the semen, preferably the small 5 mL cryovials, or the bigger 12 ml Nunc tubes with a tight screw cap, which do not leak or break during the transport. If the semen is to be used for more than one AI the sample should be split into 2 separate tubes, so that the portion to be used for the 2nd insemination can be kept in the refrigerator to avoid temperature fluctuations which are detrimental to sperm longevity. The chilled semen is preferably sent in a neopore box with ice packs (www.minitube.com) which chills the sample at a proper rate and then keeps the temperature low for 48 hours. If the shipment takes longer than 48 hrs, for instance over a week-end, an Equitainer which has a holding time of 72 hours, can be used. A styrofoam box or an ordinary thermos flask can also be used, but have a shorter holding time. It is imperative that the temperature does not drop below 0°C (32.0°F) to ensure that the semen does not freeze. Therefore, when not using the purpose-made neopore boxes, the tube containing the semen must be protected from direct contact with ice cubes or cold-packs, for instance by putting it in an outer tube casing, or by wrapping it in cotton wool. The neopore or styrofoam box and the thermos flask weigh little and are of little value and thus are not returned, which keeps the shipping costs low. The Equitainer, on the other hand, represents a value and is therefore usually returned.

Figure 1. The Minitübe neopore cool box for chilled dog semen has a holding time of 48 hours.

Figure 2. The Equitainer can be used also for chilled dog semen and has a holding time of 72 hours.

Figure 3. The single-use disposable dry-shipper has a weight of 4.5 to 5.0 kg when full and a holding time of 4 days.

Figure 4. The ordinary dry-shippers weigh around 15 kg when full and have a holding time of 2-3 weeks. A mushroom shaped protective outer casing is preferable to a square box as the former will not be placed on its side, and nothing will be put on top of it during shipping.

Results Using Chilled Extended Dog Semen

Whelping rates from 577 artificial inseminations using chilled extended semen were reported to be 45.7% when the semen was deposited in the cranial vagina and 65.6% by transcervical intrauterine semen deposition. The average litter size was 5.8 ± 3.0 pups by vaginal AI and 6.4 ± 3.2 by intrauterine AI. (P<0.001) [3,Linde Forsberg and Andersson, in preparation)]. The results of these studies emphasize the importance of intrauterine semen deposition not only for frozen-thawed semen [8]., but also when using chilled extended and fresh dog semen.

7. Frozen Dog Semen

Frozen dog semen can be stored practically indefinitely, probably for 1-2000 years or more. Dog semen can be frozen in 0.5 or 0.25 ml French straws or in pellets. The 0.5 ml straws are in Europe preferred by most semen freezing agencies and practitioners performing the AIs. An added advantage is that their identity cannot be manipulated after the freezing, whereas the content in a vial with pellets easily can be changed.

7.1. Extenders for Freezing of Dog Semen

There are a number of agencies which freeze dog semen, using proprietary extenders, such as Camelot Farms, Canine Cryobank, IMV Canifreeze, Minitübe CaniPro, CLONE, International Canine Semen Bank, and Zoetis. Others may use the non-proprietary CaniRep Uppsala-Equex II extender (Appendix 5) [5].

7.2. Frozen Semen Preparation

The semen is collected and centrifuged as described above for chilled semen preparation. It can be further processed in many different ways, using various extenders and one or two-step dilutions (the first at room temperature and the second after chilling and just before freezing), equilibrating for from 1 to 4 hours and freezing for instance either in liquid nitrogen vapour on a rack 4 cm above the LN2surface in a styrofoam box [1,10,11] or in three steps directly into a liquid nitrogen tank [8,10,11]. Most agencies freeze dog semen at a final concentration of 50-100 million spermatozoa per 0.5 ml straw, and use 2-4 straws per AI. With the pellet freezing system, one breeding unit is usually put in each pellet containing cryovial. Some use a thaw medium, usually 0.5–1 ml for each straw or 1-2 ml per pellet vial. The semen is thawed in a water bath either at 37°C (98.6°F) for 15-60 sec or at 70°C (98.6°F) for 8 sec. It is important to adhere to the thawing instructions provided by the semen freezing agency, as the thawing method is related to the freezing method.

7.3. Shipment of Frozen Dog Semen

To ship frozen semen a liquid nitrogen container which keeps the temperature at around -197°C (-322.6°F) is required. For this purpose semen banks today use the so-called dry-shippers which are constructed so that the liquid nitrogen is absorbed into a porous material in their walls. These dewars, therefore, will not spill and thus are not classified as dangerous goods, which is more restricted and expensive to ship. They are, however, fragile and may be broken by rough handling. The dry-shippers, therefore, during transportation are protected by an outer plastic casing. These outer casings come as square or mushroom-shaped boxes. The mushroom-shaped casing seem to offer the best protection, because in contrast to the square ones their shape prevents them from being transported lying on the side, and from having something stored on top of them. Some semen processing agencies request that the dewar is insured against damaging during freight. The shipping code for the shipper’s declaration for liquid nitrogen is: UN1977, which tells that it is Restricted Goods. The regulations pertaining specifically to the dry-shippers are found in the IATA Packing Instruction 202, Note, which reads: "Insulated packagings containing refrigerated liquid nitrogen fully absorbed in a porous material, and intended for transport, at low temperature, of non-dangerous products are not subject to these Regulations….".

When the ordinary dry-shippers are full and in their outer protective casing they usually weigh between 7 and 15 kg, and they need to be shipped back, why the freight costs will be quite high. On the other hand several doses of semen, or semen from several dogs can be sent in one shipment. The freight cost for the single-use DryShipper 3.0 (www.strepro.com; In Europe: www.huvesearch.com) with a holding time of 4 days and a charged weight of only 4.5-5.0 kg is considerably lower, and there is no return which reduces the freight cost by up to 75% compared to that of the ordinary dry-shippers. The DryShipper 3.0 is compliant with the IATA Special Provision A152 and it has also been accepted as passenger baggage by several air companies: (I.A.T.A. Dangerous Goods Regulations; Subsection 2.3), because the design of the DryShipper is such that it does not allow the build-up of pressure within the container and would not permit the release of any refrigerated liquid nitrogen irrespective of the orientation of the insulated packaging.

7.4. Check the Liquid Nitrogen Tank

Before shipment it should always be made a routine to check that the liquid nitrogen dewar is not broken. Therefore:

• Always fill the tank with liquid nitrogen at least 24 hours before the shipment is due. The dry-shippers have to be filled over a period of time, and left to saturate between fillings. Some liquid nitrogen that is seen to remain at the bottom of the tank is the sign that it is full (this does not apply to the single-use DryShipper 3.0).
• Weigh the tank both before and after filling to control that it contains the proper amount of liquid nitrogen. The most commonly used dry-shippers take from 1 to 4 kg of LN2, weigh between 9 and 15 kg fully charged and with their outer casings, and have a holding time of from 7 to 21 days. Always check the manufacturer´s information. The single-use DryShipper 3.0, weighs only 4.5-5.0 kg when full, and has a holding time of 4 days.
• Leave the filled tank for 24h over-night and check its weight again before it is shipped. If needed it should be topped up with some more LN2 before dispatch.
• Also when receiving a tank, it is a good routine to check its weight on arrival in case something has gone wrong with the shipment.

The ordinary dry-shippers are expensive and are usually rented from the semen banks and should be returned as soon as possible. To avoid problems at customs when the rented tank is returned, a customs clearance instruction and a certificate for its return should accompany it, for instance:

"The LN2 tank and shipping case are both the sole property of ….. and on loan to …… and will be returned to … as duty free Return Goods."

Results using Frozen-thawed Dog Semen

Whelping rates of 84.4% from 327 frozen-thawed semen AIs [8] and 71% from 312 frozen-thawed semen AIs [15] have been reported when the semen was deposited in the uterus using the Scandinavian catheter [4]. In a 20-year field study including 889 frozen-thawed semen AIs with semen of variable quality that had been processed by a large number of different agencies world-wide and AIs performed in bitches with varying fertility by 114 different veterinarians the whelping rate by vaginal AI was 30.1% compared to 49.5% by intrauterine AI (Linde Forsberg and Andersson, in preparation). Whelping rates by intrauterine AI in the dog are significantly better than those obtained by vaginal AI not only for frozen-thawed semen (+51%) but also for chilled (+44%) as well as for fresh semen (+30%). Litter size using intra-uterine AI of frozen-thawed semen is also significantly larger than by vaginal AI (Linde Forsberg and Andersson, in preparation). Litter size has been estimated to be 25-30% smaller in bitches receiving frozen semen compared to fresh and chilled [6-7].

8. How Much Semen Should be Sent?

How much semen should be sent mainly depends on what has been agreed. In Europe it is still generally recommended to use at least 150-200 million motile, morphologically normal spermatozoa per AI, and to inseminate the bitch twice. In, for instance, the USA 100 million normal spermatozoa and just one AI is considered adequate.

If the ejaculate contains abnormal spermatozoa or the post-thaw motility is low the total number of spermatozoa per breeding unit should be increased accordingly in an attempt to compensate for the poor semen quality. Semen of unsatisfactory quality, however, should not be shipped unless the bitch owner or importer is informed about the situation and has given his/her consent.

If the AI is to be done by surgery, which is still the most common method in many countries, fewer spermatozoa may be required per AI and usually only one AI is done. Some colleagues combine TCI and surgical AI to be able to do 2 AI:s in a bitch. Using vaginal AI up to 10 times more spermatozoa are required to obtain the same results as by intrauterine AI both with fresh and frozen-thawed semen [8,16]. Chilled semen can be stored for up to 1-2 weeks, if originally of good quality and when kept in an unbroken cold chain. Enough semen for repeated AIs can therefore be sent in one shipment.

Considering the high costs involved in the shipping of frozen semen it might be a good idea to send enough semen for a repeat breeding at the next oestrus in case the bitch doesn’t get pregnant at the first attempt. If the importer has paid for the freezing, it may also be reasonable to send all the resulting semen doses, unless other agreements have been made.

9. Who Should Organise the Shipment?

Most canine semen banks co-operate with a freight company. It can be quite time consuming to organize the shipments, as they often involve customs clearance and other related procedures, such as the need for a proforma invoice and a decision about the value of the shipment for VAT/sales tax and/or insurance. Others chose to leave it to the dog or bitch owner to organize the shipments themselves. In case of countries with more complex import requirements expert knowledge is, however, required. For the shippers’ declarations they need to know that the code for liquid nitrogen is UN1977, and for the dry-shippers the IATA Packing Instruction 202:Note, which clarifies that this kind of tank can be shipped as non-dangerous goods. Also: Not restricted, approved by IATA Special Provision A152.

In the I.A.T.A. Dangerous Goods Regulations there are also provisions for dangerous goods carried by passengers or crew (Subsection 2.3; Table 2.3.A). It states: "Insulated packagings containing refrigerated liquid nitrogen (dry shipper), fully absorbed in a porous material and intended for transport, at low temperature, of non-dangerous products are not subjected to these Regulations provided the design of the insulated packaging would not allow the build-up of pressure within the container and would not permit the release of any refrigerated liquid nitrogen irrespective of the orientation of the insulated packaging."

There should also be an accompanying statement for customs that the tank is on loan and that it will be returned as an empty packaging, if that is the case, to avoid VAT/sales tax, or a custom's fee.

10. Who may Perform A.I.?

Both national legislation and the Kennel Clubs may have regulations regarding the right to perform AI in dogs, and where dog semen may be stored. In several countries this is an exclusively veterinary matter, and in some countries the veterinarians are expected to have passed a special course for AI in dogs. In other countries the breeders themselves may inseminate their own bitches. When it comes to frozen semen, however, this is uncommon, because of the special qualifications needed for the handling of the semen and the generally poor results obtained with vaginal AI.

11. Cooperation between the Semen Processing Agencies and the Inseminating Veterinarians

Semen processing agencies should provide the practitioners with adequate information about the quality of the semen they send and, in the case of frozen semen, information about how the semen should be thawed, because the method of thawing is dependent on how the freezing was done, and the methods vary between freezing agencies.

The responsibility of choosing the right time during the bitch’s oestrous cycle to perform the AI, on the other hand, lies with the inseminating veterinarian. The bitch’s peripheral plasma level of progesterone should always be checked both for fresh, chilled or frozen-thawed semen AI:s. The optimal time for AI is considered to be 4 to 7 days after the LH-surge, i.e. 2-5 days after the estimated time of ovulation. The level of progesterone is then usually between 10 and 20 ng/ml (30 and 60 nmol/L). However, progesterone values should be interpreted bearing in mind that plasma levels of progesterone fluctuate during the day by up to 30-40%, in a not diurnal fashion [9]. It is recommended to take a vaginal smear at the time of progesterone testing as this will help to determine whether the bitch is still in prooestrus, in proper oestrus, or already in metoestrus/dioestrus. Especially the transition from oestrus to metoestrus cannot be done only based on the level of progesterone.

The AI-technique is also extremely important to obtain good pregnancy results when using dog semen. Significantly better results are obtained with intrauterine compared to vaginal deposition of fresh, as well as chilled extended or frozen-thawed canine semen [3,Linde Forsberg and Andersson, in preparation]. Intrauterine AI in the dog can be done with the Scandinavian/Norwegian catheter [4] or with the aid of a rigid endoscope and a dog urinary catheter [17]. Surgical AI and AI by laparoscopy is also used. Whether these latter methods are ethically acceptable is under debate. In several countries in Europe surgical AI is illegal, or very restricted.

An important part of the cooperation between the semen banks and the practitioners, and one that seldom functions to satisfaction, is the reporting back of the results that are obtained. Although time-consuming, this follow-up obviously is of great value for both parties, and critical for any objective evaluation of a semen processing system, an insemination facility, or of a specific procedure or modification.

Appendixes

Appendix 1 - A Quick Guide to Regulations for International Shipment of Chilled and Frozen Canine Semen - Download PDF
Appendix 2 - Veterinary Health Certificate for Export of Dog Semen - Download PDF
Appendix 3 - Certificate of Collection of Dog Semen - Download PDF
Appendix 4 - Tris-egg Yolk Extender for Chilled Dog Semen - Download PDF
Appendix 5 - CaniRep Uppsala Equex II - Download PDF
Appendix 6 - The CaniRep Uppsala Freezing Procedure - Download PDF

Appendix 1 - Download PDF

A QUICK GUIDE TO REGULATIONS FOR INTERNATIONAL SHIPMENT OF CHILLED AND FROZEN CANINE SEMEN

In parallel with the growing interest in international shipment of canine semen there is a demand for a source of quick reference to the varying rules and regulations for import of dog semen that apply in different countries. The present Guide was first published in 2001. When now updating it I realized that we are today living in a totally different world, and that most of the information can easily be found on the internet. Instead of listing postal addresses, phone and fax numbers I, therefore, give the web addresses to the Ministries of Agriculture and the Kennel Clubs in a number of countries. As will be evident this list is far from being complete, and despite my efforts it may in part also be incorrect, still I hope that it can be of some use. It should be remembered that the rules and regulations may be changed at any time. Therefore, always check for the latest official versions.

The author would greatly appreciate input from readers worldwide with updates and corrections to this list, and with information from countries not yet included in the list (Contact Dr. Linde Forsberg).

Some Useful Links

Information on almost 90 Kennel Clubs world-wide can be found on the website of the Fédération Cynologique Internationale, (FCI): http://www.fci.be/membres.aspx. Others may be found by searching their name on the internet.

European Union list of Ministries of Agriculture of the member countries: http://ec.europa.eu/agriculture/links-to-ministries.

EU: For import of dog semen from other countries within the EU it is usually only required a veterinary health certificate issued at the time of semen collection, also stating the microchip number or ID-tattoo marking of the dog, and that the dog has normal testicular status.

Australia

Australian Quarantine and Inspection Service (AQIS/DAFF)
http://www.daff.gov.au/biosecurity/import/live-animals, http://apps.daff.gov.au/icon32
Australian National Kennel Council (ANKC) P.O. Box 1005, St. Marys, New South Wales 2760, Australia.
Phone: +61-2 98 34 40 40; Fax: +61-2 98 34 60 38; email:[email protected];
http://www.ankc.org.au/Contact-Us.aspxm

Austria

Federal Ministry for Health (Bundesministerium für Gesundheit), Veterinärvervaltung, www.bmg.gv.at
Austrian Kennel Club (Österreichischer Kynologenverband, ÖKV), email: [email protected]; http://www.oekv.at

Belgium

The Belgian Ministry of Agriculture (Ministère de l´agriculture), Service de l´Inspection Vétérinaire. See above under EU.
The Belgian Kennel Club (Union Royale Cynologique Saint Hubert), http://www.srsh.be, email: [email protected]

Canada

Canadian Food Inspection Agency (CFIA), http://www.inspection.gc.ca/eng/
Canadian Kennel Club (CKC), http://www.ckc.ca

Czech Republic

Czech Republic State Veterinarian (Státni Veterinární Správa Ceské Republiky),
http://eagri.cz/public/web/mze
The Czech Kennel Club (Ceskomoravská Myslivecká Unie), email:[email protected]; http://www.cmku.cz

Denmark

The Danish Veterinary and Food Administration, (Ministeriet for Fodevarer, Landbrug og Fiskeri), http://www.fvm.dk, email:[email protected]
The Danish Kennel Club (Dansk Kennel Klub), http://www.dansk_kennel_klub.dk

Finland

The Finnish Ministry of Agriculture and Forestry (Jord- och skogsbruksministeriet), http://www.mmm.fi
The Finnish Kennel Club (Suomen Kennelliitto-Finska Kennelklubben r.y.), http://www.kennelliitto.fi

France

The French Ministry of Agriculture, http://www.agriculture.gouv.fr
The French Kennel Club (Société Centrale Canine de France), http://www.scc.asso.fr

Germany

The German Ministry of Agriculture (Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft), email: [email protected]
The German Kennel Club (Verband für das Deutsche Hundewesen, VDH), http://www.vdh.de

Hungary

The Hungarian Board of Agriculture (Földmûvelésügyi és Vidékfejlesztési Minisztérium), email: [email protected]; for website see under EU above.
The Hungarian Kennel Club (Magyar Ebtenyésztok Országos Egyesülete, MEOE), http://www.kennelclub.hu

Iceland

Ministry of Agriculture, http://www.landbunadarraduneyti.is; email: [email protected]
The Icelandic Kennel Club, http://www.hrfi.is/, email: [email protected]

Ireland

The Department of Agriculture & Food, http://www.agriculture.gov.ie, email: [email protected]
The Irish Kennel Club, http://www.ikc.ie/index.php, email: [email protected]

Italy

The Italian Ministry of Agriculture, email:[email protected]; http://www.politicheagricole.it
The Italian Kennel Club (Ente Nazionale della Cinofilia Italiana, ENCI), email:[email protected];; http://www.enci.it

Japan

Japan ministry of Agriculture, http://www.maff.go.jp/e/
Japan Kennel Club, http://www.jkc.or.jp

The Netherlands

The Dutch Ministry of Agriculture, Nature Management and Fisheries, http://www.minlnv.nl
The Dutch Kennel Club (Rad van Beheer op Kynologish Gebied in Nederland), http://www.raadvanbeheer.nl/

New Zealand

The Ministry of Agriculture and Forestry, P.O. Box 2526, Wellington, New Zealand. http://www.biosecurity.govt.nz/imports/animals/standards/general-info-germplasm.htm
The New Zealand Kennel Club, email:[email protected], http://www.nzk.org.nz

Norway

The Norwegian Ministry of Animal Health (Statens Dyrehelsetilsyn), email: [email protected], http://www.mattilsynet.no
The Norwegian Kennel Club (Norsk Kennel Klub, NKK), email: [email protected], http://www.nkk.no

Spain

The Spanish Ministry of Agriculture, http://www.mapya.es/indices/pags/info/index.htm
The Spanish Kennel Club (Real Sociedad Central de Fomento de Razas Caninas en España), email: [email protected], http://www.rsce.es

Republic of South Africa

National Department of Agriculture, Forestry and Fisheries, email: [email protected], http://www.daff.gov.za
Kennel Union of Southern Africa, email: [email protected], http://www.kusa.co.za

Russia

Russian Federation Ministry of Agriculture, http://government.ru/en/department/59/
Russian Kennel Club: Russian Kynological Federation, email: [email protected], email (international department): [email protected]

Singapore

Agri-Food & Veterinary Authority of Singapore, Phone: +65-6227 0670, Fax: +65 6227 6305
The Singapore Kennel Club, http://www.skc.org.sg

Sweden

Swedish Board of Agriculture, http://www.sjv.se
Swedish Kennel Club, http://www.skk.se

Switzerland

Federal Veterinary Office (Bundesamt für Veterinärwesen), http://www.bvet.admin.ch
Swiss Kennel Club (Schweizerischen Kynologischen Gesellschaft), http://www.skg.ch

United Kingdom/Great Britain

Department for Environment, Food and Rural Affairs (DEFRA), 1A Page Street, London, SW1P 4PQ, United Kingdom, http://www.defra.gov.uk, email:[email protected],
The Kennel Club, Clarges Street, London, http://www.the-kennel-club.org.uk

United States of America

United States Drug Administration (USDA), http://www.usda.gov
American Kennel Club (AKC), http://www.akc.org

Appendix 2 - Download PDF

Download The Veterinary Health Certificate for Export of Dog Semen for printing.

Appendix 3 - Download PDF

The semen can be identified as follows [breed, name, KC reg. no./microship no, date and place of collection]:……………………………………………………………………………………….

………………………………………………………………………………………………………………

Colour of straws/plug:……………………………………………………………………………………

Type of extender/cryoprotective:………………………………………………………………………..

Recommended thawing procedure:……………………………………………………………………..

………………………………………………………………………………………………………………

………………………………………………………………………………………………………………

Recommended number of straws or vials per artificial insemination:………………………………

Total number of spermatozoa:…………………………………  Motility:………………………………%

Number of spermatozoa/straw or vial:………………………..  Abnormal sperm……………………..%

At the time of semen collection the semen donor was clinically examined and was found free from signs of disease. He has two normal testicles, fully descended in the scrotum.

Signature of veterinarian:………………………………………………………………………………..

Printed name:…………………………………………………………………………………………….

Address:…………………………………………………………………………………………………..

Phone:……………………………………… Email:………………………………………………………

Click here to download The Certificate of Collection of Dog Semen. for printing

Appendix 4 - Download PDF

This extender can be ordered from CaniRep HB (www.canirep.com).

Appendix 5 - Download PDF

CaniRep Uppsala Equex II

Appendix 6 - Download PDF

The CaniRep Uppsala Equex II-Method for Cryopreservation of Dog Semen

Freezing Semen

1) The sperm rich 2nd fraction of the ejaculate is collected.

2) Total number of sperm, sperm motility and morphology is assessed.

3) The ejaculate is centrifuged at 600-700 x g for 3-5 min depending on sperm density (higher RPM and/or longer time for more sperm dense samples) and the supernatant is removed. Avoid that the resulting sperm pellet is too densely packed.

To calculate the g-force of a centrifuge the following formula can be used:

4) The sperm pellet is diluted at room temperature with CaniRep Uppsala Equex II/ extender no. 1 to a concentration of 400 x 106 spz per mL and mixed well with a pipette.

5) The extended semen sample is cooled for 75 – 90 min from room temperature to 3-5°C (37-41°F) (Fig.1). The same volume of CaniRep Uppsala Equex II/ extender no. 2 is cooled in a syringe together with the extended semen (if necessary, longer cooling times of the semen extended with the first freezing extender by several hours is not detrimental).

Figure 1. The semen sample extended with CaniRep Uppsala Equex II/extender no. 1 is cooled from room temperature to 3-5°C (37-41°F) for 75-90 min, together with the syringe with the CaniRep Uppsala Equex II /extender no. 2, the marked straws, the filling equipment, the canes, a forceps and 2-3 paper towels to wipe off fluid from the outside of the straws before freezing.

6) The 0.5 mL straws are marked with: breed, name of the dog, Kennel Club registration no. or microchip no., date, and the name of the clinic (a requirement for import in many countries). The straws and the syringe with the adaptor are cooled together with the canes and goblets that are used for freezing, the paper towels, the forceps and the extended semen and CaniRep Uppsala Equex II/extender no. 2.

Calculate the number of straws needed based on a final concentration of 200 x 106 spermatozoa (spz) per mL, i.e. 100 x 106 spz per straw. (Some of the fluid will be absorbed by the filter at the top end of the straw, which functions to seal the straw at that end. Therefore, add 0.1 to 0.3 mL more of each of the extenders 1 and 2 than what is calculated by the total number of spz).

7) After 75 – 90 min of cooling, the same volume of CaniRep Uppsala Equex II/extender no. 2 is added slowly as previously done with extender no. 1, dropwise at 3-5°C (37-41°F). Final concentration is then 200 x 106 spz per mL. Mix the sample well by turning the tube with extended semen upside down a couple of times. Leave for a couple of minutes. Take care to maintain the temperature at 3-5°C (37-41°F) during this procedure, and the following step.

8) Again, mix the sample well and then fill the straws. Using a 5mL syringe with a special connector (Fig. 2) or a piece of plastic tubing with a dimension that fits the diameter of the straws. Because the volume of the extended semen will expand during the freezing the straws should not be completely filled, leaving 5-10 mm of air at the open end. Avoid dipping the straws deeply into the semen. To avoid that the straws freeze together in bundles carefully wipe off any fluid on the outside of the straws by quickly rolling them between the paper towels in the cooler, without warming them with the hand. The straws do not need to be sealed at their open end. If one prefers to seal the straws, place the air bubble in the middle of the straw and seal by either dipping the open end in sealing powder or by pressing a sealing ball in the open end. The frozen straws should be stored with the filter end pointing down.

Figure 2. A 5mL syringe with a connector for filling of 0.5mL straws.

9) The straws are frozen either in a styrofoam box with a lid lying on a rack 4 cm above the LN2 level for 10 min and then plunged into LN2 (Fig. 3). They are left for some time in the LN2 to stabilize and are then packaged and moved to a storing dewar.

Figure 3. Styrofoam box with an inner steel box for the LN2 and a rack for the straws. The rack with the straws should be placed 4 cm above the level of LN2 and the lid be closed for 10 min.

10) Freezing can also be done directly into an Apollo SX-18 or a TW-10 XT or a similar size LN2-tank (Minnesota Valley Engineering, Inc., New Prague, MN, or Taylor Wharton, Theodore, AL, USA) with a level of LN2 of 15-18 cm measured from the hub at the bottom of the tank (Fig. 4). (Other tanks can be used after adaption of the markings on the handle of the canister to the same level above the LN2 as described for the Apollo SX-18 and TW-10 XT). The freezing is performed by placing up to 4 straws into each goblet, with the goblet at the upper end of each cane (Fig. 4). Not more than 4 such canes are put into one canister for each freezing.

Figure 4. The shaft of the canister has markings for each of the three levels during freezing. Each cane has a goblet in the upper position. Four straws can go into each goblet. Not more than four canes should go into the canister during freezing.

To freeze the straws, the canister is lifted up and a forceps placed at the lowest marking on its handle (7 cm below the opening of the tank). The canister is placed at this first level in the tank. The canes containing the straws are then moved straight from the cool bench into the canister. The canister is kept at this position for 2 min and then lowered to the second marking (13 cm below the opening of the tank) for another 2 min and then lowered to the third marking (20 cm below the opening of the tank) for 1 min. The canister is thereafter fully lowered and put in its place in the tank. The three levels should be marked on the handle of the canister (Fig. 4 and 5). The straws are left for at least a couple of hours in the tank to stabilize, before re-packaging and moving them to a storage tank.

Figure 5. The freezing is done in three steps, according to the markings on the shaft of the canister. The canister is held at the first and second levels for 2 min each, and at the third level for one min. The canister is then lowered into its place in the tank. In the picture the canes with the straws are in the canister to illustrate their placing, but when freezing the canister should be lowered to its first position before the canes are put in it.

11) Freezing can also be performed using a programmable freezer, according to the instructions for freezing of dog semen from the manufacturer of the equipment.

12) Freezing Using the Pellet System

The CaniRep Uppsala Equex II-extenders can also be used for pellet freezing. The procedure is the same as for freezing in straws except that the cooled extended semen is frozen in droplets on a block of dry ice.

Thawing of Semen when it is Frozen in Straws

1. The contents of each straw should immediately be diluted after thawing with 0.5 - 1 mL of Uppsala Equex II/thawing medium (TM) at 37°C (98.6°F). Therefore, first thaw the required volume of thaw medium and warm this to 37°C (98.6°F).

2. The best result is obtained by thawing the semen in a water bath at 70°C (158°F) for 8 - 9 sec. If this is not practically possible, the semen can be thawed at 37°C (98.6°F) for 30 – 60 sec. Hold the straw with a forceps during the thawing with its open (or sealed) end above the water, but without squeezing it.

3. Be careful to wipe off any water on the outside of the straw before emptying its contents into the thaw medium.

4. Leave the thawed sample at 37°C (98.6°F) for a few minutes before assessing its motility etc, and performing the AI.

Some Practical Hints when Thawing Straws at 70&Deg;C (158&Deg;F) for 8-9 sec

It is actually safer to learn to count the seconds in your head, than to start and stop a timer for each straw to be thawed. In order to get the right time and rhythm, first practice counting the seconds in front of a timer a couple of times. For instance, say (putting stress on the first word): One second, two seconds, three seconds, etc. until nine seconds. When you lift the straw from the water bath, you should check its temperature with your hand. If it feels neither cold nor warm to the touch your timing was correct. If the first thawed straw should feel warm to your hand then thaw the next straw for one second less, and if it feels cold thaw the next straw for one second more.

Thawing of Semen Frozen as Pellets

1. Thaw the contents of the vial with pellets in a water bath at 37°C (98.6°F) using 1 – 2 mL of Uppsala Equex II/Thaw Medium in a Whirl Pak thaw bag. 2. Leave the thawed sample at 37°C (98.6°F) for a few minutes before assessing its motility etc, and performing the AI.