Comparative Studies of Canine Semen Freezing Protocols

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1 Bulletin UASVM, Veterinary Medicine 67(2)/2010 ISSN ; Electronic ISSN Comparative Studies of Canine Semen Freezing Protocols Manuela STĂNESCU (PASCAL), Alin Ion BÎRŢOIU Faculty of Veterinary Medicine Bucharest, 105 Splaiul Independenţei, , Bucharest, Romania; Abstract. The freezing of sperm is a method of conservation of spermatozoa, theoretically for an unlimited time. The freezing methods are an important tool for preservation of genetic diversity of the species and in assisted reproduction. The principle for cryopreservation of semen is reducing temperature of sperm determines a reduction of the metabolic activity of spermatozoa that permits their storage. A number of freezing methods have been described in the literature in the last 30 years. Some methods are registered marks and require buing a francise to be used. Proprietary agencies include Canine Cryobank, CLONE, International Canine Semen Bank (ICSB) and Synbiotics. Nonproprietary systems with published protocols include the Norwegian Tris extender, the Uppsala-Equex system 1 and 2 and its modifications, the CERREC extender, the CERCA extender, the extender developed by England C.G.W. and Lofstedt R. (Concannon and Battista 1989; England 1993; Farstad 1996; Linde- Forsberg et al. 1995; Peña and Linde-Forsberg 2000 a, b) and other extenders and cryopreservation methods. Key words: sperm, dog, cryopreservation In most cases the sperm is collected by digital manipulation and the second fraction of the ejaculate (the sperm-rich fraction) is used for cryopreservation. The main steps of freezing are: centrifugation, dilution, termic equilibration, packing, freezing and thawing. Different canine sperm cryopreservation techniques include as the first step the centrifugation of sperm. The goal of centrifugation is removing prostatic fluid excess wich was proved to have a negative effect over the motility and the vitality of frozen-thawed spermatozoa (England and Allen, 1992; Rota et al., 1995; Sirivaidyapong et al. 2000) and to standardize the extension of the semen to a controlled final sperm concentration and glycerol concentration etc. (Linde-Forsberg, 2005). Shuttleworth R. (2002) demonstrated that the addition of homologous prostatic fluid to the thawed sperm before intrauterine insemination has a beneficial effect on fertility by increasing the number of fertilized oocytes and embryos that survive the implantation. The most used centrifugation regimen in the cryopreservation protocoles is centrifugation at g for 5 10 minutes (Igna, V., 2007). Johnson D. (2000) demonstrated that collection into warmed extender media lessened the cold and ph shock to the spermatozoa reported in previous studies, as shown by the improved semen parameters (life span of the spermatozoas, motility). The dilution of the sperm has to roles: protecting the spermatozoa from low temperatures negative effects and obtaining a previously established final concentration. In most systems dog semen is frozen at a final concentration of million spermatozoa per 0.5 ml straw, and 2-4 straws are used per insemination. When different spermatozoa concentrations were tested using a Tris-based extender, the best post-thaw 209

2 results were obtained at a final sperm concentration of 200x10 6 spermatozoa/ml (Peña and Linde-Forsberg 2000a). The extender should be isotonic with the sperm, have a ph of 6.9 to 7.1, to provide nutritional support for sperm (glucose, fructose), it should contain crioprotective agents (glycerol) and have antibacterial activity (penicillin, streptomycin) (Igna, V., 2007). Currently there are many forms of extenders, different in structure and proportion of ingredients. The dilution of the semen can be done in one or two steps. When the two-step dilution is used, the first step takes place at room temperature and the second step is done after chilling/equilibration and just before freezing (Linde -Forsberg et al. 1999, Peña and Linde- Forsberg 2000 a,b). No significant difference was found in post thaw viability or longevity of canine spermatozoa whether glycerol was added all at once or in two steps with increasing concentrations (Smith, 1984, Fontbonne and Badinand 1993, Peña and Linde -Forsberg 2000b). In the case of Equex STM paste (a product of Nova Chemical Sales Inc. Containing dodenil sodium sulphate) recent studies indicate that it is better to add it just before freezing, therefore in its case the dilution should take place in two steps (Peña and Linde -Forsberg 2000b). The preparation of the sperm for cryopreservation includes its packaging and division that allows storing the sperm in the form of easily identifiable insemination doses: flakes, pellets, tubes. Currently, the most common form of packaging of frozen semen in the dog are the straws, since they present several advantages over other methods of packaging: low volume (increase s storage capacity), uniform freezing, the possibility of marking important data for identification (dog's name, date of collection, etc.) (Igna V., 2007). Before being frozen, diluted semen is cooled to 4 C and maintained at this temperature for a variable time, depending on the protocol used (1 4 hours). This process is called balancing and allows sperm to develop a higher resistance to thermal shock associated with freezing. Although canine spermatozoa are considered relatively resistant to cold shock, it has been shown that in this species a comparatively slow cooling is preferable (White, 1993; England, 1993). Freezing can be done by using a programmable freezer or by placing the straws at different distances over liquid nitrogen. The last stage of cryopreservation is the plunge in liquid nitrogen (-196 o C) (Igna, V., 2007). For thawing, straws are immersed in a waterbath at the temperature and for the time recommended by the person/agency who did the freezing because an interaction has been found between freezing and thawing rates. In the case of dog semen, thawing straws at higher rates has been shown to improve spermatozoal viability. Thawing 0.5 ml straws in a waterbath at 70 C for 8 sec resulted in a higher post-thaw survival than thawing at 37 C for 15 to 60 sec (Olar, 1984; Concannon and Battista, 1989; Rota et al., 1998; Peña and Linde- Forsberg, 2000b). 1. Uppsala freezing technique (Linde-Forsberg, C., 2005): The second sperm rich fraction of the ejaculate is collected. After assessing morphology and motility and counting the total number of spermatozoa the ejaculate is centrifuged at 700 x g for 6 min. The supernatant is removed (and if it still contains spermatozoa it can be centrifuged again) and the pellett diluted at room temperature in Uppsala Equex 2 or Uppsala extender 1 (Tris, fructose, glycerol, egg yolk, Equex) to a concentration of 400 x 10 6 spermatozoa/ml and allowed to equilibrate for min at +4 C. An equal volume of Uppsala Equex 2/Extender 2 is also cooled to +4 C and added slowly dropwise after the equilibration period. The sample is mixed carefully and 210

3 immediately filled in 0.5 ml straws, resulting in a final concentration of 200 x 10 6 spermatozoa/ml. The straws are frozen in liquid nitrogen-vapor tank containing ml of LN2. The freezing is performed in three steps, with the goblets at the top of the canes and with the top of the canes 7, 13 and 20 cm (for 2, 2 and 1 min) below the opening of the tank, respectively, whereupon the canister is placed in the tank. Not more than 4 straws should be placed in each goblet, and not more than 4 goblets (i.e. a total of 16 straws) be frozen in each batch. The straws can also be frozen using a styrofoam box containing liquid nitrogen with the straws lying horizontally on a rack placed 4 cm above the surface of the liquid nitrogen for 10 min whereupon the straws are immersed into the liquid nitrogen. The semen is usually frozen so that the final number of spermatozoa per straw is around 100 x10 6. Depending on the semen quality, usually 2-3 straws are used for each AI. In dogs of the smaller breeds which produce fewer spermatozoa per ejaculate it may be desirable to freeze the semen less concentrated to obtain more straws. The straws are best thawed in a water bath at +70 C for 8 sec. Any water remaining on the outside of the straw is carefully wiped off before opening the straw. Each straw is emptied into 1 ml of the Uppsala Equex 2/thaw medium at +37 C and left at this temperature for approximately 5 min before assessing semen quality and performing the insemination. Linde-Frosberg (2002a, b) reported significantly higher whelping rates and larger litter sizes when semen is deposited in the uterus rather than in the vagina in the bitch, not only when frozen-thawed semen in used, but this also applies for fresh as well as for chilled semen. The post-thaw total motility was 68.48% at thawing time (T 0 ) and 64.62% after 30 minutes (T 30 ), the post-thaw progressive motility was 57.00% at T 0 and 54.88% at T 30, 55.4% live sperm with intact acrosome, 27.0% live sperm with damaged or reacted acrosome and 71.9% whelping rate. Martins-Bessa and col. (2006) found no advantages in using ethylene glycol instead of glycerol in the Uppsala Equex II and Norwegian extenders, while Rota. A. and col. (2006) demonstrated that ethylene glycol significantly improves dog semen quality at thawing: all the motility parameters that were considered are in agreement and all resulted higher with ethylene glycol than with glycerol, at least at thawing. Neagu and col. (2010) suggest that the Uppsala extender can be improved with the addition of the antioxidant butylated hydroxytoluene (BHT) since the percentage of sperm membrane (71.6%) was significantly higher in semen samples frozen in the presence of BHT. Hermansson and Linde-Forsberg (2006) found that dog spermatozoa can be frozen after 1 or 2 days of cold storage with Uppsala Equex II without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. 2. The norwegian extender and freezing procedure (Andersen, 1972): A well fractionated ejaculate is collected by digital manipulation. (If the ejaculate is not obtained well fractionated it should be diluted about 1 : 5 with the extender and then centrifuged at about 1000 g for 5 min and subsequently resuspended to the original ratio with the extender). The sperm rich fraction is diluted 1 : 5 at 35 C with the Tris-glycerol-fructose extender. Immediately before dilution 20% (v/v) egg yolk is added to the extender. The diluted semen is placed in a water bath at 35 C and cooled in the refrigerator for 2 hours. (The final temperature is then about 4 C). The material is filled in medium straws and frozen in nitrogen vapour for about 10 min., 4 cm above the surface and then transferred to the liquid nitrogen container for storage. The final sperm concentration should be approximately 100 x 211

4 10 6 spermatozoa per ml. The semen is thawed in a water bath at 70 C for 8 sec. The semen is deposited intrauterine (the norwegian catheter methode). In 2006, Thomassen and col. reported 73.1% overall whelping rate and 5.1±0.1 litter size. The whelping rate was higher after intrauterine insemination (75.0%) than after intravaginal insemination (10.0%). Insemination at the optimal time resulted in a higher whelping rate (78%) and larger litter size (5.8±0.2) than inseminations performed late or too late (55.7% and 4.5±0.5). Szasz F. and col. (2000) added Triladyl to the norwegian extender. The pregnancy rate was 57%, but the litter size was low (2.8). The results are lower than the ones obtained using the original method. 3. CERREC cryopreservation technique (Centre d Etude et de Recherche en Reproduction et Elevage Canins Lyon) (Briffaut, 2007): The sperm collected by digital manipulation is evaluated (spermogram) and only the ejaculates of good quality (mobility over 70-80%, anomaly 20 30%) are processed. Semen is centrifuged and the supernatant is removed. CERREC uses a Tris-based extender, with glucose, glycerol and egg yolk that is prepared extemporaneously. The volume of CERREC extender added depends on the desired final concentration of spermatozoa. Balance mixture for 2 hours at 4. Then, sperm is packed in 0.25 or 0.5 ml straws, previously identified with specific data on dog and freezing. The straws remain 10 minutes in nitrogen vapor at -70 o C and then are plunged into liquid nitrogen at -196 o C. The semen is thawed in a boiling water bath: 8 seconds at 70 o C or 10 seconds at 37 o C. According to the 2009 study conducted by Barthelemy, fertility was 62.4% with frozen semen by this method, while in 2003 Rostagnat reported 66.7% gestation and a prolificacy of 5.3 ± 2.7 litters/calving. Fertility is higher for intrauterine insemination (66.5%) compared to the intravaginal insemination (63.3%), while litter size did not differ significantly for the two methods of insemination. For a single insemination fertility was 60.3%, 72.5% for double insemination and 78.3% for triple insemination. 4. The CERCA freezing protocole (Centre d Etude en Reproduction canine Assistee Alfort) (Benechet, N., 2007): The rich sperm fraction of the ejaculate is centrifuged at g for 5-10 minutes. Remove the supernatant. The CERCA extender type I (Tris, fructose, glycerol, and egg yolk) is mixed (drop by drop) at ambient temperature (22 o C) with the rich sperm fraction of the ejaculate. The amount of extender used is 1.5 ml to 100 million spermatozoa. Equilibrate for 1 hour at 4 C. After equilibration, a second dilution is made with CERCA extender type II (same as type I plus Equex). Diluted semen is packed in straws and frozen 10 minutes in nitrogen vapor at -70 o C, then plunged into liquid nitrogen bath at -196 o C. At hours after freezing check residual motility. Only the ejaculates that have at least 40% motility are stored. The straws are thawed in a water bath at 70 o C for 8 seconds or 45 seconds at 37 o C. Reported characteristics for the thawed ejaculate are: 85% spermatozoa without abnormalities, 57% live spermatozoa, 60% motility at thawing and 57% after 1 hour, 68% spermatozoa with membrane integrity at the hypoosmotic swelling test. After a study period of 8 years, Rostagnat (2003) reported a post-thawing motility of 54.5% (± 11.7%). Benechet (2007) reported a pregnancy rate of 50.5%, Esparel (2010) 57.1%, while Mimouni and Dumon (2005) reported a calving rate of 30 40% after intravaginal insemination and 60 65% after intrauterine insemination. 212

5 DISCUSSIONS AND CONCLUSIONS There are no significant differences in the stages of sperm collection and centrifugation of the four analyzed protocols. The components of the extenders differ significantly and also the way of their addition: the norwegian method uses dilution in a single step, while Uppsala, CERCA and CERREC protocols use the two-stage dilution. In the Uppsala method, the final concentration of glycerol is 5% plus 0.5% SDS and the thawed straws are emptied in Uppsala Equex II extender. For the other methods, no extender has to be added after thawing. Leblanc (2004) compared the Uppsala and CERCA methods of cryopreservation. His study showed that the Uppsala protocol achieves better results in terms of motility (68.48%), resistance to heat and acrosomal integrity (55.4%). The fertility percentage obtained by CERCA with frozen semen is 50.5%, a lower performance then the one of the other international centers. In Sweden, in 1999, Linde- Forsberg et al. published a success rate of 71.9% for the Uppsala technique significant difference from the results of CERCA (χ2 = 15.9). In Norway, Thomassen et al. published in 2006: 73.1% success rate with an average of 5.7 litter size for the period using the norwegian (Andersen) method significant difference from the results of CERCA (χ2 = 21.6) (Esparel, 2010). For all the methods of freezing there are significant differences in favor of the intrauterine insemination compared to the intravaginal insemination. Fontbonne and Badinand (1993) compared the intravaginal insemination of semen frozen by the norwegian method (exception: straws were thawed 45 seconds at 37 o C) with its intrauterine insemination. They reported 52.6% pregnancy rate after three intravaginal inseminations with 4.2 litter size and 73.6% pregnancy after three intrauterine inseminations with 5.5 litter size. This study demonstrates a higher prolificacy for intrauterine insemination than for the intravaginal insemination. Linde-Forsberg (2002a, b) reported 35-37% success rate for intravaginal insemination with semen frozen by Uppsala method and 52-55% for the intrauterine insemination and Thomassen et al. (2006) 10% fertility for intravaginal insemination and 75% for the intrauterine insemination with semen frozen by the norwegian method. REFERENCES 1. Andersen, K. (1972). Fertility of frozen dog semen. Acta Veterinaria Scandinavia, 13: Barthelemy, C. (2009). Statistical study of the inseminations carried out at CERREC since 1995 (in french). PhD these, Lyon, France. 3. Benechet, N. (2007). Artificial insemination with frozen semen in the dog: analysis of records of dogs followed in the Center for Reproduction of Carnivorous at the National Veterinary School of Alfort (CERCA) from january 2001 to december Phd thesis, Alfort, France. 4. Briffaut, A.S. (2007). Freezing of canine sperm: determination of the optimal combination or four different factors (in french). PhD thesis, Lyon, France. 5. Concannon, P.W., Battista, M. (1989). Canine semen freezing and artificial inseminations. In: Kirk RW (ed). Current Veterinary Therapy X: Small Animal Practice. Philadelphia, WB Saunders, p England, G.C.W., Allen, W.E. (1992). Factors affecting the viability of canine spermatozoa. II. Effects of seminal plasma and blood. Theriogenology 37, p England, G.C.W. (1993). Cryopreservation of dog semen: a review. J. Reprod. Fert. 47: (suppl). 8. England, G.C.W., Lofstedt, R. (2000). Canine reproduction. Seminar Atlantic Vet. College. 213

6 9. Esparel, A., A-G. (2010). Contribution to the study of reproduction in the dog: an analysis of the records of the bitches followed in the Center for the study of reproduction in the carnivorous at Alfort since 2005 to 2008 (in French). PhD thesis, Alfort, France. 10. Farstad, W. (1996). Semen cryopreservation in dogs and foxes. Animal Reproduction Science, 42: Fontbonne, A., Badinand, F. (1993a). Canine artificial insemination with frozen semen: comparison of intravaginal and intrauterine deposition of semen. J. Reprod. Fert. Suppl., 47: Fontbonne, A., Badinand, F., (1993b). Studies on freezing dog spermatozoa: effect of glycerol on motility after thawing. J. Reprod. Fert. (suppl.) 47: Hermansson, U. (2006). Studies of canine and feline sperm viability under different storage procedures. With special reference to chilling, freezing and use of zona pellucida binding assays. PhD thesis, Uppsala, Sweden. 14. Igna, V. (2007). Sperm preservation, p In: Canine reproduction, obstetrics and gynecology (in Romanian). Ed. Brumar, Timişoara; Romania. 15. Johnson, D.L. (2000). Improving semen parameters through modification of semen collection/extension. Master degree thesis, Texas Tech University, USA. 16. Leblanc, B. (2004). Improvements in techniques of cryopreservation of dog sperm for use at CERCA (in French). PhD thesis, Creteil, France. 17. Linde-Forsberg, C. (1995). Artificial insemination with fresh, chilled-extended and frozen-thawed semen in the dog. Sem. Vet. Med. Surg. (Small animal) 10: Linde-Forsberg, C., Ström Holst, B., Govette, G. (1999). Comparison of fertility data from vaginal vs uterine insemination of frozen-thawed dog semen: a retrospective study. Theriogenology 52: Linde-Forsberg C. (2002a). Fertility data from 2041 controlled artificial inseminations in dogs. Proceeding of the 4th Int. Symp. Canine Feline Reproduction, Oslo, Norway. 20. Linde-Forsberg C. (2002b). What can be learnt from 2500 Ai s in the dog? 98-th World Small Animal Veterinary Association Congress, Granada, Spain. 21. Linde-Forsberg, C. (2005). Artifici al insemination. Proc. ESAVS-EVSSAR-ENVN Congress, Nantes, France. 22. Martins-Bessa, A., Rocha, A., Mayenco-Aguirre, A. (2006). Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders. Theriogenology 66: Mazur, P. (1984). Freezing live cells: mechanisms and implications. Am. J. Physiol. 247: Mimouni, P, Dumon, C. (2005). Vademecum of reproduction pathology in the dog (in french). Ed.Med com, Paris, p Neagu, V.R., Macias Garcia, B., Salazar Sandoval, C., Morillo Rodriguez, A., Ortega Ferrusola, C., Gonzalez Fernandez, L., Tapia, J.A., Pena, F.J. (2010). Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves post thaw sperm membrane integrity. Theriogenolgy 73: Olar, T.T. (1984). Cryopreservation of dog spermatozoa. PhD thesis. Fort Collins, Colorado State University, USA. 27. Peña, A.I., Linde-Forsberg, C. (2000a). Effects of spermatozoal concentration and post -thaw dilution rate on survival after thawing of dog spermatozoa. Theriogenology, 54: Peña, A.I., Linde-Forsberg, C. (2000b). Effects of Equex, one- or two- steps dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa. Theriogenology, 53: Peña, A.I., Lòpez-Lugilde, L., Barrio, M., Becerra, J.J., Quintela, L.A., Herradón, P.G. (2002). Studies on the intracellular CA2+ concentration of frozen-thawed dog spermatozoa: Influence of Equex from different sources, two thawing diluents and post-thaw incubation in capacitating conditions. Proc. 3rd EVSSAR Congress, Liège,

7 30. Rostagnat, L. (2003). Contribution to the study of artificial insemination in the dog: analysis of records kept at the Center for Study and Research of Reproduction and Growth of Carnivorous (in french). Alfort, France. 31. Rota, A., Ström, B., Linde-Forsberg, C. (1995). Effects of seminal plasma and three different extenders on canine semen stored at 4 C. Theriogenology, 44, p Rota, A., Linde-Forsberg, C., Vannozzi, I., Romagnoli, S., Rodriguez-Martinez, H. (1998). Cryosurvival of dog spermatozoa at different glycerol concentrations and freezing/thawing rates. Reproduction in Domestic Animals 33, Rota, A., Milani,C., Cabianca, G., Martini, M. (2006). Comparison between glycerol and ethylene glycol for dog semen cryopreservation. Theriogenology 65: Shuttleworth, R. (2002). Fertility of frozen-thawed dog sperm with the addition of homologous prostatic fluid or protein-free sperm TALP prior to intravaginal insemination of bitches. Degree thesis, Pretoria, South Africa. 35. Sirivaidyapong, S., Ursem, P., Bevers, M.M., Colenbrander, B. (2001). Effect of prostatic fluid on motility, viability and acrosome integrity of chilled and frozen-thawed dog spermatozoa. Journal of Reproduction and Fertility, Suppl 57, Smith, F.O. (1984). Cryopreservation of canine semen, technique and performance. PhD Thesis. University of Minnesota, USA. 37. Szasz, F., Gabor, G., Solti, L. (2000). Comparative study of different met hods for dog semen cryiopreservation and testing under clinical conditions. Acta Veterinaria Hungarica 48(3): Thomassen, R., Sanson, G., Krogenaes, A., Fougner, J.A., Andersen Berg, K., Farstad, W. (2006). Artificial insemination with frozen semen in dogs: A retrospective study of 10 years using a nonsurgical approach. Theriogenology 66 (6-7): White, I.G. (1993). Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod. Fert. Dev. 5: