Introduction

The demand for artificial insemination (AI) and semen freezing is increasing. Chilled semen is increasing as an alternative to mating or use for AI with frozen semen (Thomassen & Farstad, 2009). New commercial diluents for fresh semen are being developed that may enable storage of chilled semen for several days, which eliminates transport distance as an obstacle to AI with fresh semen. The shipping of cooled and frozen semen has opened the door to a wide variety of breeding opportunities for dog owners, providing that their breed organization permits AI with shipped semen.

Although the first studies on the use of AI in dogs date from the late XVIIIth century, small animal reproduction has not seen much development since. Only in recent years has the development and routine application of such biotechniques in dog breeding gained attention (Silva et al., 2008).

Semen technology is important for biodiversity preservation, for dog breeders and for basic research. The interest for development and application of such biotechnique in dog breeding is steadily increasing. This paper provides an overview of cooling and freezing canine. The advances in the development of such biotechnique will contribute to improve dog reproduction and for their application in wild canidae.

Semen Collection

Semen collection is easily performed in dogs, but the presence of a female in estrus can improve the quality of the ejaculate. Frozen swabs impregnated with vaginal secretion from estrus females (Silva, 2001) or the synthetic pheromone methyl-ρ-benzoate (Goodwing et al., 1979) can also be used.

Electroejaculation is restricted to collection of dogs that will not allow digital manipulation and has been compared to the employment of artificial vagina, with no differences concerning the quality of the ejaculate (Ohl et al., 1994).

Semen collection from the epididymis is an alternative for retrieval of sperm cells from animals that cannot ejaculate or from those recently dead (Silva et al., 2004). Epididymal sperm can be retrieved from dogs submitted to orchiectomy and dissection of the epididymis and stored at 4°C for up to 8 days (Yu & Leibo, 2002). Sperm cells can also be retrieved from epididymis and kept between 15 and 25°C for up to 20h. Following retrieval, epididymal sperm can be employed directly for AI or frozen (Hewitt et al., 2001; Hori et al., 2004).

Semen Processing

Immediately after collection the semen is analyzed under the microscope. At this time, it can be determined if the collection is worthy of preserving based on the sperm count, volume, and motility. When semen is collected for posterior use, sperm viability can be improved by cooling and adding extenders such as egg yolk Tris (Stornelli et al., 2001) and coconut water in powder (Madeira et al., 2003).

Cooled Semen

Cooled semen can be transported in different types of containers and is viable for one to five days, as long as temperature is kept between 4 and 5°C (England & Ponzio, 1996). Changing the extender promotes viability of canine semen for another 20 days, once the energetic substrate available for the sperm cells is renewed (Iguer-Ouada, 2001). Also, supplementation of the Tris buffer with fructose instead of glucose improves preservation of sperm cell motility for up to 23 days under 5°C (Ponglowhapan et al., 2004).

Cooled semen should be used within 24-72 hours depending on the semen quality. Importers must be able to get the semen through their country's customs quickly. Some countries require import permits and extensive testing of the donor dog before collection. Have the foreign importer provide all documentation necessary before entering into agreements for providing semen for export.

When an individual receives a fresh chilled sample, the package should immediately be opened. Attention should be paid to the impression of coldness. The ice packs should be at least cold, if not still frozen. The tube should contain the extended semen in a liquid state. Unfortunately, occasional mishandling by the shipping company or by the shipper placing the semen package in a non-pressurized compartment of the airplane will cause the sample to arrive frozen. The freezing kills the sperm cell and renders the sample useless.

Frozen Semen

Frozen semen does require even more precise handling than fresh cooled semen.

Canine semen can be frozen and stored indefinitely, without losing fertility when reheated and employed for AI (Concannon & Battista, 1989). At this low temperature all molecular activity is halted and the cells can be preserved for many years without loss of fertility. The thawed spermatozoa has a maximum life span of 12 to 24 hours after insemination into the bitch. The ultra-short life span makes success with frozen semen only possible with precise mapping of the bitch's estrous cycle with definitive detection of the LH release and the exact time of ovulation.

Several extenders can be used for canine semen freezing, such as lactose, Tris, Bes-lactose, Triladyl, Tes/Tris, Biociphos W482, Laiciphos 478, CLONE extender, skimmed milk and a coconut water extender in powder. However, the Tris buffer remains the most commonly used extender by most research groups, with excellent in vitro and in vivo results (Silva, 2005).

Egg yolk is added to the extender usually at 20% (Silva et al., 2003) but is at risk of disease transmission because of its biological origin. Thus, replacement by other synthetic and purified lipids like the hydroxytoluene butylate analogues has been suggested. The use only of the low density lipoproteins in the egg yolk for semen conservation has been proposed (Varella et al., 2004).

Cryoprotectors such as glycerol, dimethylsulfoxide, methanol, ethylene glycol and dimethylformamide have been employed in canine semen freezing procedures, but glycerol still is the most commonly used, in concentrations between 2 and 8%.

Concentrations employed for sperm freezing vary from 10 x 10⁶ (Tsutsui et al, 2000) to 8 x 10⁸ sperm cells/mL (Peña & Linde-Forsberg, 2000). Other researchers employ a fixed dilution based on the volume of sperm to be frozen and varying between one part of semen to one part of extender and one part of semen (Cardoso et al., 2003; Silva et al., 2003) to four parts of extender (England & Ponzio, 1996).

Sperm thawing is performed at 37°C (Silva et al., 1998) to 75°C (Olar et al., 1989) and storage can be in tablets, 5mL aluminum canes (Ivanova-Kicheva et al., 1997) or preferably in straws (Thomas et al., 1993). Frozen/thawed semen can also be maintained at 4°C for up to three days post-thawing (Verstegen et al. 2002).

Final Considerations

The employ of cooled or frozen semen that are available for assisted canine reproduction, it becomes clear that the choice of one or another depends on many factors that shall be considered in each case. The advances in canine semen technology will contribute for enhancement of domestic dog reproduction and extrapolation to wild canidae.

References

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