Canine sperm requirements and legislation for international trading.

Catharina Linde Forsberg, DVM, PhD, Dipl ECAR

Division of Reproduction, P.O. Box 7054, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden. CaniRep HB; Fjällbo 110, 755 97 Uppsala, Sweden.

 

Introduction

Breeding of dogs is today an international hobby and the international shipments of dog semen are rapidly increasing. 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 just one or 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 the rules that apply for importation of chilled semen may be different from those for frozen 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 regulations may change at any time. Before undertaking to export dog semen the bitch owner or importer should, therefore, always be advised to contact the Ministry of Agriculture (or the corresponding authority) and the Kennel Club in the importing country to get accurate information about the latest set of rules and regulations. These contacts should be made well in advance of the planned semen export, so that all the necessary health certificates and blood tests can be made in accordance with the requirements. (For a list of addresses etc. see Linde Forsberg, 2010). Within the European Union is now for the most part a free exchange of dog semen. Italy is an exception.

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 four categories:

Category 1.

No regulations. In general, the lack of regulations means that the importation of dog semen is not subject to any restrictions. In some countries, however, for instance Italy, it means that it is not officially allowed.

Category 2.

A request for an import permit, with no further requirements.

Category 3.

A request for 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.

Category 4.

A request for an import permit and a health certificate and for 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, or 3-6 weeks after) the semen collection.

Kennel Club requirements:

Some Kennel Clubs have no regulations which, again, may mean that everything is allowed, or that registration of litters by imported semen is not officially allowed. However, the lack of regulations in general 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.

?he 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.

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.

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, including a statement that the dog has a normal testicular status, and that the identity of the dog was properly confirmed.

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 pelletted semen, should be used for each AI.

It should always be the official, registered name of the dog that is used for all official documents, including the blood test report, not its pet name.

Always enclose a set of copies of the certificates, either attached on the outside of the shipment, or inside it in case the air waybill with the original certificates is lost under way, or removed by mistake, for instance by the freight company or the 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 placed 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. The number of the seal should be stated in the veterinary certificate.

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 registered name (which may be abbreviated),

the dog’s KC registration number or microchip number,

? the date of semen collection (obligatory when blood tests and veterinary certificates are required), and

? where the semen was collected/processed.

Example:

Afghan hd B.S. Sandstorm SE-06689-09 1-6-2010 SLU

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

Chilled extended dog semen

Good quality and properly handled chilled extended dog semen may maintain its fertilizing capacity for several days up to one week or more if kept at +5°C.

Extenders for chilled dog semen:

A number of chilled semen extenders are available, most of which are proprietary, such as the CLONE Chilled Kit, Synbiotics Fresh Express and CaniPro. The non-proprietary and scientifically well documented CaniRep Chilled Semen Extender is now also commercially available (www.canirep.com). Extenders for chilled dog semen should always contain some antibiotics, to prevent bacterial growth and the risk for spread of infections.

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 2-4mL extender (of the same temperature as the semen) added to the sperm pellet and this is then well mixed. Using larger volumes for AI will only lead to loss of spermatozoa due to backflow of semen from the uterus or vagina. Use sterile plastic tubes with a tight screw cap for the semen sample, preferably the small 5 mL cryovials, but the 12 ml Nunc tubes are also acceptable, although they contain more air and empty space. 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 remain in the refrigerator to avoid temperature fluctuations, which are detrimental to sperm longevity. Mark the semen vial with the dog’s breed, KC-registered name, registration or microchip number, and date and place of collection (see above).

Shipment of chilled dog semen:

The chilled semen is preferably sent in a neopore box with ice packs (www.minitübe.de) which chills the sample at a proper rate during transport and then keeps the temperature low for 48 hours. 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 to below 0?C to ensure that the semen does not freeze. Therefore, the tube containing the semen must be protected from direct contact with cold-packs or ice cubes, for instance by putting it in an outer tube casing, or by wrapping it in paper or cotton wool. The neopore or styrofoam box and the thermos flask weigh little and has a low value and need not be returned, which keeps the shipping costs low.

Results using chilled extended dog semen:

Whelping rates from 374 artificial inseminations using chilled extended semen were reported to be 45.1% 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)(Linde-Forsberg, 2000). The results of this study emphasize the importance of intrauterine semen deposition not only for frozen-thawed semen (Linde-Forsberg et al., 1999), but also when using chilled extended and fresh dog semen.

Freezing of chilled, extended dog semen.

It is possible to successfully freeze dog semen that has been collected and then chilled for 2-3 days before being frozen (Verstegen et al., 2005; Hermansson & Linde Forsberg, 2006). It is thus a viable option to have a dog collected nearer to home and ship the chilled semen to a semen bank for freezing.

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. Especially the racing Greyhound associations are, however, by tradition often using the pellet system.

Extenders for freezing of dog semen:

There are a number of agencies which freeze dog semen, using proprietary extenders, such as for instance Camelot Farms, Canine Cryobank, CaniPro, CLONE, International Canine Semen Bank and Synbiotics. Others may use the non-proprietaty Uppsala Equex extender (Rota et al., 1997), and its modification the CaniRep Uppsala-Equex II extender (Linde Forsberg, 2002).

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 it for from 1 to 4 hours and freezing it for instance either in liquid nitrogen vapour on a rack 4cm above the LN2 surface in a styrofoam box (Andersen, 1975; Peña and Linde-Forsberg, 2000a,b) or in three steps directly into a liquid nitrogen tank (Linde-Forsberg et al., 1999, Peña and Linde-Forsberg, 2000a,b). Most semen banks freeze dog semen at a final concentration of 50-100 million spermatozoa per 0.5 mL straw, and use 2-4 straws per AI. Some use a thaw medium, usually 0.5 – 1 mL for each straw or 1-2 mL per vial of pellets. Thawing of the semen is done in a waterbath either at 37?C for 15-60 sec or at 70?C for 8 sec. To obtain the best result with frozen-thawed semen it is important to adhere to the thawing instructions provided by the semen freezing agency, as the thawing method is dependent on the freezing method.

Shipment of frozen dog semen:

To ship frozen semen a liquid nitrogen container is required, which keeps the temperature at –197ºC. Today, all semen banks use dry-shippers, which absorb the liquid nitrogen into a porous material in their walls. These will not spill and therefore need not to be shipped as dangerous goods, which is more expensive. They are, however, fragile and may break by rough handling. The tank is usually kept in a plastic outer casing for protection. These outer casings come in different shapes. The mushroom-shaped casing seem to offer the best protection, because this shape prevents the tank from being transported lying on the side, and from having something stored on top of it. Some semen banks request that the dry-shipper is insured against damage 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 (dry-shipper) fully absorbed in a porous material and intended for transport, at low temperature, of non-dangerous products are not subject to these Regulations....” The new single-use Dry-Shipper 3L (from ST Technologies, USA) with a holding time of 4 days , and a weight of only 4.5kg, considerably reduces the freight costs, by up to 3/4. A larger version with a longer holding time is said to come on the market soon. Dry-shippers may be accepted by some air companies as passenger baggage. (I.A.T.A. Dangerous Goods Regulations; Subsection 2.3; Table2.3.A), if the design of the dry shipper 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. Whenever this is practically possible a numbered seal should be applied on the package. This is required by some countries, and it prevents unauthorized persons from opening the inner package containing the semen. The number on the seal should be stated in the veterinary certificate.

Check the liquid nitrogen tank

Before shipment it should always be made a routine to check that the liquid nitrogen tank 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 Dry-Shipper 3L from ST Technologies. It must be weighed).

? Weigh the tank both before and after filling to check that it contains the proper amount of liquid nitrogen. The most commonly used dry-shippers take from 1 to 4kg of LN2, weigh between 9 and 14kg full, and have a holding time of from 7-20 days. Always check the manufacturer´s information. The single-use Dry-Shipper 3L weighs 4.5kg 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 despatch.

? 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 certificate should accompany it for the return freight, 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 (Linde-Forsberg et al., 1999) and 71% from 312 frozen-thawed semen AIs (Thomassen et al., 2001) have been reported when the semen was deposited in the uterus using the Scandinavian catheter (Linde-Forsberg, 2001), and 87.5% using a rigid endoscope in 161 Greyhound bitches (Pretzer et al., 2006). In a 10-year field study including 286 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 many different veterinarians the whelping rate by vaginal AI was 34.6% compared to 52.0% by intrauterine AI (Linde-Forsberg, 2000).

How much semen should be sent?

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 is informed about the situation and has given 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 only one AI can be done. Vaginal AI may require up to 10 times more spermatozoa to obtain the same results as by intrauterine AI both with fresh and frozen-thawed semen (Tsutsui et al., 1989; Linde-Forsberg et al., 1999). Chilled semen can be stored for up to 1-2 weeks, if originally of good quality and kept in an unbroken cold chain, and enough semen sent in one shipment for repeated AIs. 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 semen collection and freezing, it may also be reasonable to send all the resulting semen doses, unless other agreements have been made.

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 procedures. 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 will 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. 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, other taxes, or a customs fee. Consider insuring the tank against damages during the transport.

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 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 dogs. When it comes to frozen semen, however, this is unlikely, because of the special qualifications needed for the handling of the semen and the generally poor results obtained with vaginal AI.

Cooperation between the semen banks and the inseminating veterinarians.

Semen banks must 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 chosing 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 and frozen-thawed semen AIs. The optimal time for AI is considered to be 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 (Linde Forsberg et al., 2008). Methods for AI in bitches include vaginal deposition of the semen, transcervical intrauterine deposition (TCI) using the Norwegian/Scandinavian catheter or with the aid of a rigid endoscope, and intrauterine (IU) insemination by laparoscopy or full abdominal surgery. In for instance The Netherlands, Norway and Sweden the use of surgical AI is not considered ethically acceptable, and therefore is illegal, whereas in the UK its use is very restricted.

References:

For a list of references and veterinary forms see: Linde-Forsberg, C. (2010). Regulations and recommendations for international shipment of chilled and frozen canine semen. In: Recent Advances in Small Animal Reproduction, A1209.0501. Eds. P.W. Concannon, G.C.W. England & J. Verstegen www.ivis.org. International Veterinary Information Service, Ithaca.