Semen cryopreservation in dogs and foxes

SCIENCE ELSEVIER

Animal ReproductionScience 42 ( 1996)25 l-260

Semen cryopreservation in dogs and foxes W. Farstad Department of Reproduction and Forensic Medicine, Norwegian College of Veterinary Medicine, P.O. Box 8146, Dep. N-0033, Oslo, Norway

Abstract Low temperature preservation of canid semen has been a subject of increasing interest among dog breeders, fur breeders and scientists. Research has focused on the use of different buffers, cryoprotectants, egg yolk additions, equilibration and cooling rates, semen packaging techniques, and freezing-thawing protocol. Some laboratories have seen a beneficial effect of post-thaw dilution, especially with vaginal semen deposition. Few reports have given fertility results on a larger scale as a basis for recommended protocol. However, it is established that artificially inseminated frozen s&men can yield good fertility results (75-80% whelping rate) in dogs and in blue fox-silver fox crossbreeding, whereas pure breeding with frozen blue fox semen yields fertility results far below those from natural service. More basic research on membrane function during exposure to freezing regimes and media is called for in this species. Keywords:

Dog; Fox; Spermatozoa;Cryopreservation;Freezing

1. Introduction Since the first birth of live offspring from frozen dog semen @eager, 19691, dog breeders worldwide have put increasing pressure on the scientific world to be part of the progress in reproductive biotechnology. As a result of this, sperm banks at universities and kennel clubs, as well as private companies offering breeding services, have been established. Artificial insemination with frozen semen in the blue fox was first reported in 1972-1973 (Aamdal et al., 1972; Fougner et al., 1973). The fox industry must be able to adapt to the demands of a market governed by fashion. During the early 198Os, when the fox industry was booming and the use of fresh semen was increasing rapidly, interest was generated among breeders in preserving genetic material from rare mutants and otherwise valuable animals. The rapid availability of genes through semen cryopreservation reduces the need to keep live male breeding stock with pelt characteristics currently 0378-4320/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01483-2

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out of fashion. Another incentive for freezing fox semen was introduced when cross breeding between silver fox males and blue fox females was made possible with the use of artificial insemination with fresh semen, since these species do not usually mate with each other. Since their breeding season has only a short period of overlap, it was considered beneficial to use frozen semen to produce the valuable bluefrost pelts on a large scale (Fougner, 1989; Farstad et al., 1992a).

2. Low temperature preservation of canid spermatozoa Damage to spermatozoa by cooling from physiological temperatures to the freezing point is called cold shock (Watson, 1981). The ability of spermatozoa to withstand chilling and freezing differs between species. Spermatozoa from horse, cat, dog and humans are relatively insensitive to cold shock, whereas spermatozoa from cattle, sheep and goat show medium sensitivity, and boar sperm are extremely sensitive (Watson and Plummer, 1985; Bwanga, 1991). Silver fox sperm seem to be similar to dog sperm as far as post-thaw fertility is concerned, whereas blue fox sperm seem to be highly sensitive with impaired fertility when they are processed using the buffers, cooling and freezing procedures used by our laboratory for dog (Farstad, 1984) and silver fox semen (Hofmo, 1988; Farstad et al., 1992~). Physical and chemical alterations in the cell membrane caused by cooling may be irreversible and differ from the damage caused by freezing and thawing; cold shock and freezing damage may therefore be considered separate phenomena (Paulenz, 1993). Irreversible alterations in the sperm membrane (i.e. disturbances in the protein lipid bilayer structure), such as decreased membrane fluidity, increased membrane permeability, acrosome damage, dehydration, enzyme and phospholipid liberation, reduced metabolic activity and diminished consumption of ATP, are all consequences of cooling and freezing which may partly or totally compromise fertility (for review, see Hammerstedt et al., 1990). The most evident result of these changes is loss of sperm motility (Paulenz, 1993). However, studies on frozen-thawed silver fox and blue fox spermatozoa have shown that reduced fertility is not revealed by reduced motility or even by the degree of acrosome integrity (Farstad et al., 1992~). Also, in the dog no clear relationship has been found between post-thaw motility and fertility (Kosiniak et al., 1992; Thomas et al., 1993).

3. Semen processing Successful preservation of spermatozoa by cooling, freezing and thawing is dependent on a series of steps aimed at reducing damage to the cell and securing adequate longevity in vitro and in vivo, i.e. optimal dilution, addition of extenders, type of buffer, cooling protectant and cryoprotectant addition, cooling rate and equilibration time, seeding, freezing and thawing rate, and possibly, also the removal of cryoprotectant after

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thawing. The latter may be dependent on in vitro post-thaw storage time prior to insemination and on the method of semen deposition (i.e. vaginal or intrauterine).

4. Addition of extenders Extenders dilute the semen and remains of prostatic fluid and provide favourable osmotic conditions, as well as provide energy to spermatozoa during the storage period between cooling/equilibration and freezing. Empirical adaptation of extenders used for cooling and freezing of bull spermatozoa initiated the use of skimmed milk (Martin, 19631, glucose-phosphate (Lofstedt, 19561, citrate (Harrop, 1962), chloride-phosphate (Wales and White, 1963), lactose @eager, 19691, Tris (trimethylhydroxyaminomethane) (Gill et al., 1970) and Tris-fructose-citrate (Foote, 1964; Andersen, 1972; Andersen, 1975) buffers for dilution in early experiments with short-term (liquid) or long-term (frozen) preservation of dog spermatozoa. Most of the recent research groups have used the original Tris-fructose buffer without modifications, or have continued to develop the Tris buffer, for instance by substituting monosaccharide fructose with glucose, which is nutritional and osmotic, or by the non-penetrating disaccharides sucrose and lactose, which act as extracellular cryoprotectants in addition to providing a favourable osmotic effect (Farstad, 1984; Olar, 1984; Linde-Forsberg and For&erg, 1989; Ivanova-Kicheva et al., 1992; England, 1992; Thomas et al., 1993; Wilson, 1993; Ivanova-Kicheva et al., 1995; Niithling et al., 1995). The commercially available Triladyl (Minitiib, Tiefenbach, Germany) also contains Tris as the major buffer (Niithling et al., 1995). England (1992) preferred a mixture of Tris and TES (n-Tris(hydroxymethyl)methyl-2-amino etbane-sulphonic acid, N-(2-((hydroxy- 1,l -bis(hydroxymethyl)ethyl))amino) ethanesulphonic acid), but found the regular Tris buffer to work better for some dogs (G.C.W. England, personal communication, 1993). Addition of the detergent sodium dodecyl sulphate to Tris-citrate extender was found to improve the longevity of frozen-thawed spermatozoa (Thomas et al., 1992). Also, a variety of commercial companies have developed their own buffers (Laichipos 478, L’Aigle, IMV, France, Silva et al., 1995; C.L.O.N.E, Pennsylvania, USA; ICG, Pennsylvania, USA). The exact composition of many of the commercially available extenders is unknown. In recent publications, Tris-citrate buffer has been shown to be superior to other buffers for either short-term storage of chilled semen (Rota et al., 1995) or for freezing of semen in straws (Thomas et al., 1993). In foxes, only the Triscitrate-fructose buffer has been employed for cryopreservation (Hofmo, 1988; Farstad et al., 1992a; Farstad et al., 1992b), whereas EDTA extenders without egg yolk are still exclusively used for dilution and short-term preservation of fresh semen (Fougner, 1989). 4.1. Cooling protectants and cryoprotectants The addition of cooling protectants such as egg-yolk and cryoprotectants such as glycerol or DMSO (dimethylsulphoxide), the sugars lactose or sucrose, macromolecular polymers or detergents such as polyvinyl pyrrolidone, Orvus ES-Paste (Olar, 1984;

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Zom, 1987) or Equex STM-paste (Niithling et al., 1995) is necessary to protect spermatozoa during cooling and freezing/thawing. Egg yolk is widely used as an addition to Tris-based and skimmed milk extenders. Egg yolk contains phosphatidylcholine (lecithine) which is believed to protect the sperm membrane by restoring the phospholipids lost during cold shock (Watson and Plummer, 1985; Hammerstedt et al., 1990). In boar sperm phosphatidylserine is claimed to be more efficient as a cryoprotector than lecithine or other low density lipids (Bwanga, 1991). In most reports on freezing of dog spermatozoa whole egg yolk has been used as the major cooling protectant. It is not known whether components other than lecithine in egg yolk may be beneficial for dog sperm, and it seems sensible at this point in time to continue to use whole egg yolk. Egg yolk does, however, present a possibility for disease transmission, and in our laboratory we use egg yolk from specific-pathogen-free hens. Research on substitution with other purified and synthetic lipids is called for with the increasing movement of frozen semen between countries. Interesting candidates for such substitution are butylated hydroxytoluene (BHT) analogs which have been shown to protect sperm plasma membrane from cold shock injury (Graham and Hammerstedt, 1992). Egg yolk contents in Tris extenders used for freezing dog and fox spermatozoa vary from 10 to 20% v/v (Andersen, 1975; Farstad, 1984 (dog); England, 1992; Farstad et al., 1992a; Farstad et al., 1992b (fox); Wilson, 1993). England (1992) found that 10% egg yolk (v/v) gave better post-thaw quality than 20%. Since egg yolk also has a buffering capacity, the amount of egg yolk may depend on the buffering capacity of other ingredients in the extender. Glycerol is the most widely used cryoprotectant for freezing of sperm from domestic animals. It has been shown, however, that glycerol has toxic effects on spermatozoa (McLaughlin et al., 19921, involving all zones of the membrane (Hammerstedt et al., 1990). Also, it alters the osmolarity of the extender. The optimum osmolarity for sperm media seems to be within the range of normal plasma, i.e. 300 mOsm 1-l. The osmolarity of a solution containing 6% (v/v) glycerol in Tris buffer was measured to be approximately 1300 mOsm 1-l (W. Farstad, personal observations), and this has been confirmed by Hammerstedt et al. (1990). The glycerol concentration in different extenders used for freezing of dog and fox semen varies from 2 to 10% v/v, the concentration possibly depending on the dilution rate of the final extended semen, since some authors quote the concentration in the extender before the addition of semen (Andersen, 1972; Farstad, 1984; Hofmo, 1988 (fox)), whereas others give the final concentration after dilution (GettlC, 1982; England, 1992). However, 2-4% (v/v) in the final dilution seems sufficient to protect sperm cells during rapid freezing with the minimum risk of profound toxic effects (England, 1992; Hammerstedt et al., 1990). Another point to be addressed concerning the addition of cryoprotectant, is whether to add the substance at 37°C room temperature (22-25°C) or after cooling (5°C) (Hammerstedt et al., 1990). For boar semen, addition of cryoprotectant is more efficient at 5°C than at room temperature (Almlid and Johnson, 1988; Hofmo and Almlid, 1990). In our laboratory we have always added glycerol to the semen in one step at room temperature both for fox and dog semen. Others have used two-step dilution with glycerol added in the second step, also at room temperature (OettlC, 1982). Thomas et al.

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(1993) added one-third of the final volume of the freezing extender just prior to freezing, but did not offer any explanation for this protocol. The effect of adding glycerol after cooling compared with at ambient temperature should be looked into more systematically. 4.2. Equilibration/adaptation

time and cooling rate

Yubi (1984) suggested that dog semen should be allowed to equilibrate at 7°C and adapt to this temperature for 2-3 h before freezing. In our laboratory, 2-3 h of equilibration and cooling time has been used for dog semen since 1972 (Andersen, 1972). For silver fox semen Hofmo (1988) used 2 h equilibration time, and this procedure was used to process semen in large-scale field trials of Al in foxes (Farstad et al., 1992a; Farstad et al., 1992b). In 1993, equilibration and cooling times from 45 min to 3 h were tested in fertility trials with frozen-thawed silver fox semen; 2 h equilibration time (including cooling to +5”C) yielded significantly higher conception rates than shorter or longer times (W. Farstad, unpublished data, 1993). Currently, we use 2 h for dog semen also. Olar (1984) could not detect differences in post-thaw motility between semen samples cooled and equilibrated for 1, 2 or 3 h. For dog semen, England (1992) found the optimal equilibration to be 4 h with his methodology. Penetration of the cryoprotectant glycerol is reported to be only 30 s for boar spermatozoa (Almlid and Johnson, 19881, and the need for relatively long equilibration times (hours) may not be dependent on cryprotectant penetration alone. A weakness in research on equilibration time has been that equilibration time is not usually separated from cooling time. 4.3. Semen packaging Dog semen has been successfully frozen either in pellets @eager, 1969; Battista et al., 1988; Thomas et al., 1993), 0.25 ml PVC straws (Niithling et al., 1995), 0.5 ml PVC straws (Andersen, 1972; Farstad, 1984, Olar, 1984) or 2.5 ml straws (Thomas et al., 1993). Fox semen has been frozen in 0.5 ml straws (Aamdal et al., 1972; Hofmo, 1988; Farstad et al., 1992a; Farstad et al., 1992b3). Both Battista et al. (1988) and Thomas et al. (1993) reported that semen frozen in 0.5 ml straws with Tris extender gave similar or better post-thaw motility than semen frozen in pellets. Thomas et al. (1993) also found higher motility post-thaw for 0.5 than 2.5 ml straws. One problem to consider, however, is that freezing curves may be essentially different for pellet and straw freezing, and using the same thawing rate when comparing the two methods of packaging may complicate the interpretation of results. Freezing in straws has an advantage over pellets in that it facilitates identification of semen donor and reduces the possibility of contamination which becomes increasingly important with the movement of frozen semen from one country to another. 4.4. Cooling and freezing rates and freezing method

Cooling at a moderate rate (2-5°C min- ’1 from 4-5°C to below the freezing point, i.e. from - 7 to - 15 or - 20°C) and freezing at a rapid rate from - 20 to - 50 or

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-70°C has been successful both for dog (England, 1992) and silver fox (Hofmo, 1988) semen, but not for blue fox semen (Farstad et al., 1992c). Also, rapid freezing by placing straws on a rack already placed above liquid N, with a constant flow of vaporised nitrogen has yielded good results for canine semen (Andersen, 1972; Farstad, 1984; Olar, 1984; Wilson, 1993; Fontbonne and Badinand, 1993). However, Dobrinski et al. (1993) claimed that a slower overall cooling and freezing rate of - 5°C min- ’ from + 3 to - 157°C gave better motility values than an intermediate rate of - 8°C min- ’ from + 3 to - 164°C or fast rate of - 18°C min- ’ from + 3 to - 191°C for all extenders tested when thawed at 37°C for 2 min. The different freezing rates were achieved by placing straws different distances from the liquid N, and for different periods of time. Freezing by lowering straws onto a rack placed above the surface of liquid N, is a static method which allows little control over the freezing procedure (different containers, metal or plastic racks), and it is advised that controlled dynamic freezing (variable flow of N vapour) in freezing machines be used for commercial purposes. When freezing the large number of straws used in the field trials for silver fox semen, semen was frozen in a Planer 10 freezer to reduce batch-to-batch variation due to the freezing process (Farstad et al., 1992a; Farstad et al., 1992b). ‘Ihe regime used to freeze fox semen has also been applied to dog semen in our laboratory when large numbers of straws are frozen. 4.5. Thawing rate It is well established that optimum cell survival requires that the rate chosen for freezing is paired with an appropriate rate of thawing (Mazur, 1984; Hammerstedt et al., 1990). Generally it is believed that fast freezing requires fast thawing to reverse the osmotic balance, rehydrate and restore the lipid protein configuration of the membrane in a fashion similar to the events induced during freezing. A variety of freezing regimes and thawing protocols has been reported for dog semen, even when the same packaging method and extender is used. For 0.5 ml PVC straws, thawing regimes in a water bath at 70°C for 6.5 s (Andersen, 1972), 70°C for 8 s (Farstad, 1984; Hofmo, 1988 (fox)), 50°C for 30 s (Silva et al., 19951, 30°C for 30 s (GettlC, 1982), 37°C for 2 min and 55°C for 5 s (Dobrinski et al., 1993) have been reported. These are moderate to fast rates of thawing compared with thawing at 4°C which Hofmo (1988) found was inferior to fast thawing of silver fox semen. Usually, pelleted semen is thawed in a thawing solution (usually saline or sodium citrate) at 37°C (Linde-Forsberg, 19951, but thawing in plastic bags has also been reported (Thomas et al., 1993). It is advisable to use the thawing rate recommended by the person or laboratory providing the frozen semen, since thawing rates are closely connected with the freezing protocol. 4.6. Post-thaw dilution In bovine artificial intrauterine insemination with frozen semen, and intrauterine insemination of dogs and foxes carried out in Norway with domestically frozen semen, the removal of cryoprotectant from the semen occurred in the female genital tract. Some commercial companies have instructed inseminating veterinarians to dilute the semen

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after thawing prior to insemination with special post-thaw extenders (C.L.O.N.E. and ICG, USA), although one recent instruction in the ICG manual is not to use post-thaw dilution when performing intrauterine insemination. Vaginal insemination with post-thaw dilution of 3-5 ml homologous or heterologous prostatic fluid has produced good results (Niithling et al., 1995). It is not known if the beneficial effect of redilution before insemination is only that of removal of cryoprotectam or if it has other effects as well. The addition of prostatic fluid points to some interesting possibilities because prostatic fluid does not maintain dog sperm motility as well as some extenders both at ambient (NWling and Volkmann, 1993) and refrigerator temperature (Rota et al., 1995). It has been shown that there are male-to-male variations in the composition of seminal plasma, such as in the content of sterol exchange proteins (Hammerstedt et al., 1990). The possibility of the seminal plasma acting as a source of proteins which may interact with the sperm membrane by adsorption to the glycocalix region of the surface cannot be excluded. Such recoating of the membrane may be favourable when vaginal insemination is performed, since the vagina is not considered an appropriate environment for spermatozoa as opposed to the uterus. Research on protein additions to the thawed semen, with the goal of improving fertility results with vaginal inseminations is therefore recommended. 4.7. Evaluation offrozen-thawed

semen

The ultimate test of success of semen processing techniques for frozen-thawed semen is the evaluation of fertility, i.e. whelping rate and litter size in canids. The problems associated with fertility tests are, however, that they usually require a substantial number of animals and also depend on a variety of factors other than semen quality per se, such as insemination dose and volume, number of inseminations, site of semen deposition, timing of insemination and individual female factors. Using intrauterine deposition of semen (Andersen, 19751, one or two inseminations 24 h apart and doses of 50-200 million spermatozoa per insemination, our fertility results, pooled from 1994 until October 1995 for all inseminations with imported and domestic frozen-thawed dog semen, were: a 74% whelping rate (79/107) and a mean litter size of 5.5 pups (range 1- 17). No selection of data with regard to number of inseminations (one or two), timing of insemination, insemination dose (i.e. total number of spermatozoa in the inseminate), freezing protocol or semen quality post-thaw was done. In fox field trials in 1989-1990, crossing 617 blue fox females with silver fox males by artificial insemination (two inseminations 24 h apart, intrauterine semen deposition of 37.5-150 million frozen-thawed spermatozoa per insemination), an overall whelping rate of 81% and mean litter size of seven cubs was obtained (Far&d et al., 1992a). In 1991 (single intrauterine insemination of 150 million spermatozoa), 456 of 608 vixens (75%) whelped with a mean litter size of six cubs. A significant effect of day of insemination was recorded in this trial (Farstad et al., 1992b). Insemination of blue fox vixens with frozen blue fox semen has not yielded similar success with whelping rates of 33-48% and decreased litter sizes (Farstad et al., 1992~). These results show that for the dog and the silver fox, the fertility of frozen-thawed semen is not only secured, but the results are close to those obtained by natural service even when material is not

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selected. For the blue fox, in which large male-to-male differences were observed in the freezability of semen, similar to that in the boar, freezing protocol, type of buffer, cryoprotectant type and concentration, as well as sperm membrane composition, must be investigated further before results corresponding to those of the dog and silver fox can be expected. Alternative investigations to fertility trials, i.e. assessing semen quality by conventional methods, such as motility estimates (speed of progression and per cent motile cells) and acrosome integrity do not seem to reveal the differences observed with regard to fertility between the two fox species (Farstad et al., 1992c). Post-thaw acrosomal damage is extensive for both species (Zalewski and Andersen Berg, 1983; Hofmo and Andersen Berg, 1989). The recently introduced hypo-osmotic swelling test (HOS), in which the ability of canine spermatozoa to swell in hypo-osmotic solutions has been assessed, offers interesting possibilities for testing membrane function (Rodriguez-Gil et al., 1994). Similar tests may be applicable for blue fox spermatozoa as well, and could indicate the ability of these sperm to behave as osmometers in hypo-, iso- or even hyperosmotic media (such as that of glycerol containing media). Although the test has not been evaluated for the ability to predict fertility in dogs, some experiments carried out with human spermatozoa have indicated positive correlation between the HOS test and fertility (Rodriguez-Gil et al., 1994). Also, if successful in vitro fertilisation systems could be established, sperm function could be tested without using expensive field trial regimes (Farstad et al., 1993).

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