Effects of Liquid Nitrogen Vapor Sensitization Conditions on the Quality of Frozenthawed Dog Spermatozoa

FULL PAPER Theriogenology Effects of Liquid Nitrogen Vapor Sensitization Conditions on the Quality of Frozenthawed Dog Spermatozoa Tatsuya HORI 1), Sanae ODAKA 1), Hiromichi OBA 1), Tatsuji MIZUTANI 1), Eiichi KAWAKAMI 1) and Toshihiko TSUTSUI 1) 1) Department of Reproduction,Nippon Veterinary and Life Science University, 1 7 1 Kyonan-cho, Musashino-shi, Tokyo 180 8602, Japan (Received 7 February 2006/Accepted 8 June 2006) ABSTRACT. The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN 2 ) vapor and the height from LN 2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN 2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN 2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: 4 to 22 C/min) and 10 cm/15 min groups (cooling rate: 6 to 10 C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: 6 to 50.9 C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN 2 to reduce the temperature at a slow cooling rate of about 10 C/min, followed by plunging into LN 2 after sensitization for 10 15 min, provides good semen qualities after thawing. KEY WORDS: canine, cryopreservation, liquid nitrogen vapor, plunging method, simple freezer method. J. Vet. Med. Sci. 68(10): 1055 1061, 2006 Studies of frozen canine semen have not progressed because canine sperm is readily impaired by low-temperature sensitization compared to the sperm of other mammals [4]. It has recently been clarified that the addition of surfactants, such as Orvus ES paste (OEP: Nova Chemical Sales Inc., Scituate, MA, U.S.A.) [19 21] and Equex STM paste [12, 14, 16], to frozen semen extender containing egg yolk protects the acrosome cap after thawing, which prolongs the lifespan of sperm after thawing and increases the conception rate after artificial insemination. However, the preparation method of frozen canine semen varies among researchers, and factors that may affect the qualities of frozen semen after thawing, such as the diluent composition [2, 6, 13, 16, 17], dilution rate [9], cryoprotectant concentration [2], packaging (straw thickness) [8], freezing method (cooling rate) [1, 8, 10, 11, 15, 16, 18, 23], and thawing method (warming rate) [8, 10, 23], have been investigated. For freezing, we previously used a conventional simpletype quick LNG freezer (FA-1652, Fujihira Industry Co., Ltd., Tokyo, Japan) and obtained a high conception rate (90%) by intrauterine insemination using a thawed semen preparation [19 21]. This instrument, developed for the preparation of frozen bovine semen, is capable of preparing a large amount of frozen semen in a single operation. However, the opportunity to prepare such large amounts of frozen canine semen is rare. In another simple method, called the plunging method, semen is exposed to liquid nitrogen (LN 2 ) vapor and then plunged into LN 2, and many researchers investigating frozen canine semen have used this method [3, 8, 10, 12, 15, 16]. However, the height from LN 2 and the sensitization time in the plunging method vary among researchers, and optimum freezing conditions have not yet been established. Although a previous study investigated the distance from the LN 2 surface [3, 8], there has been no report investigating the relationship between the distance from the LN 2 surface and the period of sensitization in LN 2 vapor. The objective of this study was to investigate the effects of sensitization time and the height from LN 2 vapor in the plunging method and to compare the canine semen qualities after thawing with those of frozen semen prepared by the simple freezer method previously reported by us [19 21]. MATERIALS AND METHODS Animals: The male dogs used in this study, 7 beagles from 2 to 10 years of age (mean ± SE: 5.2 ± 1.4), had normal semen quality (sperm motility and viability: > 80%), copulation ability, and fertility. The experimental dogs were kept 2 per cage in 160 75 65 cm cages. Commercial dog food (Hill s Canine Maintenance, Hill s-colgate Ltd., Tokyo, Japan) was given once daily and drinking water was given three times daily. This study was conducted in conformity with the animal study guidelines of Nippon Veterinary and Life Science University. Semen collection and sperm quality test: The semen samples, collected by digital manipulation, were divided into three fractions. Semen volume, the number of spermatozoa, and sperm motility, viability, and abnormality of the 2nd (sperm-rich) fraction were determined by methods previously described [7]. The sperm concentration was deter-

1056 T. HORI ET AL. mined by hematocytometer counts, sperm motility was examined as the percentage of progressively motile sperm with a semen quality examination plate and a warm-plate at 37 C, and the percentages of viable and morphologically abnormal sperm were assessed by eosin-nigrosin staining. Experiment 1: Relationship between the height from the LN 2 surface and the sensitization period in the plunging method Frozen semen was prepared after the semen quality test as previously described [19]. The semen was centrifuged (1,500 rpm, 5 min), and the seminal plasma was immediately removed. The precipitate was diluted with 20% egg yolk-tris/fructose-citrate extender (EYT-FC) [22] to adjust the sperm concentration to 200 10 6 /ml, followed by a 2nd dilution by dripping into it the same volume of EYT-FC containing 14% glycerol and 1.5% OEP for 10 min. The 1st and 2nd dilutions were performed at room temperature. The final concentrations of sperm, glycerol, and OEP were 100 10 6 /ml, 7%, and 0.75%, respectively. The dilution was subjected to 1st cooling for 1 hr at 4 C in a programmed freezer (UH-JF, Chino Corporation, Tokyo, Japan), and the duration of this was designated as the glycerol equilibration time. The semen was then loaded into a 0.5-ml straw in thermostat at 4 C. In freezing, LN 2 was poured into a styrene foam box (20 27 12 cm, 1.8-cm thick) to a level of 2 cm, and the straws were maintained horizontally at 5, 7, and 10-cm heights from the LN 2 surface for 5, 10, and 15 min for sensitization with LN 2 vapor. The straws were then plunged into the LN 2 (plunging method). The cooling velocity of the frozen semen prepared by the plunging method was measured using temperature recorder (EB22005, Chino Corporation, Tokyo, Japan). Semen straws were simultaneously thawed in warm water at 37 C for 45 sec more than 1 week later. After semen motility, viability and abnormality of the thawed semen were evaluated, the semen was kept at 20 C and the timecourse of sperm motility and viability of all semen preparations after 1, 2, 4, and 6 hr was compared. This experiment was performed once for 6 dogs, 6 times in total. Experiment 2: Comparison of freezing between the plunging method and the simple freezer method The best semen qualities of frozen-thawed semen prepared by the plunging method in Experiment 1 were compared with those of frozen-thawed semen prepared by the simple freezer method (using a simple-type quick LNG freezer) as described previously [19]. The procedures from semen collection to loading into straws and the freezing procedure for the plunging method were performed as in Experiment 1. The straws were placed vertically to keep the upper part of the straw 40-cm above the LN 2 surface, and the straw position was lowered by 20 cm over the course of 1 min, kept in place for 15 sec, lowered by 5 cm over the course of 1 min, kept in place for 15 sec, lowered by 5 cm over the course of 1 min, lowered by 2 cm over the course of 30 sec, and kept at 8 cm above the LN 2 for 5 min. The straws were then lowered by 6 cm over the course of 1 min and placed into the LN 2. These processes took a total of 10 min. The thawing method and semen quality test were performed as in Experiment 1. The cooling velocity of the semen prepared by the simple freezer method was also measured using temperature recorder.the experiment was performed 2 5 times for 4 dogs, 17 times in total. Statistical analysis: The semen quality data after thawing was analyzed using one-way ANOVA, and the significance of differences was analyzed using the paired Student s t- test. A significance level of less than 5% was regarded as significant. RESULTS Experiment 1: Relationship between the height from the LN 2 surface and the sensitization period in the plunging method Sperm motility after thawing of the semen prepared by the plunging method and its time-course changes while being kept at 20 C after thawing are shown in Fig. 1. Among the 9 treatment groups, the highest sperm motility immediately after thawing was noted in the 7 cm/10 min group, showing significant differences from those of the 5 cm/5 min, 5 cm/15 min, 7 cm/5 min, and 10 cm/5 min groups (p<0.05). At 6 hr after thawing, the highest value was noted in the 10 cm/15 min group, showing significant differences from those of the 7 groups excluding the 7 cm/ 10 min group (p<0.05, p<0.01). No significant difference was noted for 6 hr after thawing between the 7 cm/10 min and 10 cm/15 min groups. In relation to the height from the LN 2 surface, the lowest value was noted in the 5-min sensitization group at all heights, and no significant differences were noted in the sperm motility for 6 hr after thawing among the three 5-min groups. Sperm viability after thawing the semen prepared by the plunging method and its time-course changes while being kept at 20 C after thawing are shown in Fig. 2. Among the 9 treatment groups, the highest sperm viability after thawing was noted in the 7 cm/10 min group, showing significant differences from those of the 5 cm/5 min and 10 cm/5 min groups (p<0.05 and p<0.01, respectively). At 6 hr after thawing, the highest value was noted in the 10 cm/10 min group, but the differences were not significant when excluding the differences from those of the 5 cm/5 min and 5 cm/ 15 min groups (p<0.05 and p<0.05, respectively). In relation to the height from the LN 2 surface, the lowest value was noted in the 5-min sensitization group at all heights, and no significant differences were noted in sperm motility for 6 hr after thawing among these three 5-min groups. The incidence of morphologically abnormal spermatozoa after freezing-thawing by the plunging method is shown in Table 1. Sperm abnormalities after thawing in all groups were significantly higher than that of fresh semen (2.4 ± 0.4%, p<0.05, p<0.01). The lowest value was noted in the 10 cm/10 min group, and this was significantly different from those of the 5 cm/5 min and 7 cm/5 min groups

EFFECT OF SENSITIZATION TO LN2 ON FROZEN CANINE SEMEN 1057 Fig. 1. Time-course changes in sperm motility after freezing-thawing of canine semen prepared by the plunging method (mean ± SE). a: Significantly different from that of the 5 cm/5 min group at p<0.05(*) and p<0.01(**). b: Significantly different from that of the 5 cm/10 min group at p<0.05 (*). c: Significantly different from that of the 5 cm/15 min group at p<0.05(*) and p<0.01(**). d: Significantly different from that of the 7 cm/5 min group at p<0.05 (*) and p<0.01(**). e: Significantly different from that of the 5 cm/10 min group at p<0.05 (*). f: Significantly different from that of the 10 cm/5 min group at p<0.05 (*). g: Significantly different from that of the 5 cm/10 min group at p<0.05 (*). (p<0.05). Most abnormalities were noted in the tail region. Temperature changes in the frozen semen straws prepared by the plunging method are shown in Fig. 3. The cooling velocities at 5, 7, and 10 cm from the LN 2 surface over 5 min and temperature at 5 min were 6 to 24 C/min and 70.0 C, 4 to 24 C/min and 59.0 C, and 6 to 10 C/min and 37.5 C, respectively, including slow freezing. This was followed by freezing at a range of 0 to 6 C/ min at all heights thereafter. The temperature decreased to 90.5 C, 71.7 C, and 48.0 C, respectively, after 10 min, and 96.0 C, 75.0 C, and 54.0 C, respectively, after 15 min. Experiment 2: Comparison of freezing between the plunging method and the simple freezer method Sperm motility was high immediately after thawing in the 7 cm/10 min and 10 cm/15 min groups prepared by the plunging method in Experiment 1, but no significant difference was noted between these 2 groups. Since the shorter freezing time yielded better results, the 7 cm/10 min group with a high sperm motility immediately after thawing was compared with frozen semen prepared by the simple freezer method. Time-course changes in sperm motility and viability after freezing-thawing of canine semen prepared by the plunging and simple freezer methods are shown in Fig. 4. Sperm motility was higher in semen prepared by the plunging method at all time points from immediately after to 6 hr after thawing (p<0.01). Sperm viability was also higher in the semen prepared by the plunging method, and the difference was significant immediately after and 2 hr after thawing (p<0.05). Temperature changes in the frozen semen straws prepared by the simple freezer method are shown in Fig. 3. The cooling velocity ( 6 to 50.9 C/min) with the simple freezer method was higher than that of the plunging method for about 6 min, indicating more rapid freezing, and the temperature slowly decreased thereafter. The temperature was approximately 190 C when the straws were placed into LN 2.

1058 T. HORI ET AL. Fig. 2. Time-course changes in sperm viability after freezing-thawing of canine semen prepared by the plunging method (mean ± SE).a: Significantly different from that of the 5 cm/5 min group at p<0.01(**). b: Significantly different from that of the 5 cm/15 min group at p<0.01(**). c: Significantly different from that of the 10 cm/5 min group at p<0.05(*) and p<0.01(**). Table 1. The incidence of morphologically abnormal spermatozoa after freezing-thawing by the plunging method (mean ± SE) Height from Sensitization Sperm abnormality (%) LN 2 surface time (min) (cm) Head Mid-piece Tail Total 5 0.0 ± 0.0 0.6 ± 0.2 6.0 ± 1.0 6.6 ± 1.0 5 10 0.1 ± 0.1 0.8 ± 0.3 6.6 ± 2.3 7.5 ± 2.5 15 0.2 ± 0.1 0.8 ± 0.2 5.6 ± 0.9 6.5 ± 0.9 5 0.1 ± 0.1 0.5 ± 0.1 7.2 ± 1.3 7.7 ± 1.4 7 10 0.1 ± 0.1 0.3 ± 0.1 4.2 ± 1.1 b) 4.6 ± 1.1 b) 15 0.2 ± 0.1 0.7 ± 0.2 5.8 ± 1.2 6.6 ± 1.4 5 0.0 ± 0.0 0.8 ± 0.2 5.0 ± 0.7 5.8 ± 0.7 10 10 0.3 ± 0.1 0.6 ± 0.1 3.0 ± 0.6 a,b) 3.8 ± 0.6 a,b) 15 0.1 ± 0.1 0.7 ± 0.2 4.9 ± 1.4 5.7 ± 1.3 a) Significantly different from that of the 5 cm/5 min group at p<0.05. b) Significantly different from that of the 7 cm/5 min group at p<0.05. DISCUSSION Sperm motility was significantly higher in straws kept at 7- and 10-cm above the LN 2 surface than in straws kept at 5 cm. Regarding temperature changes, the cooling rate decreased as the straw position became higher. This finding suggested that rapid cooling impaired the semen qualities after thawing. In a study that similarly investigated the cooling rate reported by Dobrinski et al. [3], cooling from 3 to 157 C at a low velocity ( 5.1 C/min, 20-cm above

EFFECT OF SENSITIZATION TO LN2 ON FROZEN CANINE SEMEN 1059 Fig. 3. Temperature changes in the frozen semen straws prepared by the plunging and simple freezer methods. Fig. 4. Time-course changes in sperm motility and viability after freezingthawing of canine semen prepared by the plunging and simple freezer methods (mean ± SE). a: Significantly different from the simple freezer method at p<0.05(*) and p<0.01(**). LN 2 ), intermediate velocity ( 8 C/min, 12 cm), and high velocity ( 18.8 C/min, 4 cm) was investigated, and the percentage of progressively motile sperm for the slow cooling velocity was significantly higher than those of two other cooling velocities. The low cooling velocity in their report was slower than that in our study, but their finding was consistent with ours; slow freezing was more effective. Rota et al. [15] compared a freezing method in which semen was sensitized at 4-cm above the LN 2 surface (rapid freezing) and a biological freezing method using a freezer (slow freezing) was used in which semen was cooled at 0.5 C/ min from 5 C to 10 C and 8 C/min from 10 C to 60 C, followed by plunging into LN 2. They found that slow freezing significantly improved total post-thaw motility and the number of intact membrane sperm, which is consistent with our findings. However, Nöthling and Shuttleworth [8] reported that no significant differences were noted on comparison between fast ( 20 to 22 C/min, 3.5-cm above LN 2 )

1060 T. HORI ET AL. and slow (about 10 C/min, 8 cm) freezing. They also investigated the influences of 2 thawing methods (37 C for 2 min and 70 C for 8 sec) and found that slow freezing provided good results only when the preparation was thawed at 70 C. Yu et al. [23] compared the cooling rate for the preparation of frozen canine semen, using sperm from the caudal epididymis, not ejaculated sperm and found that the optimum cooling rate was 3 to 11 C/min (thawed at 37 C for 30 sec), but the cooling rate was affected by the thawing velocity. In our study, the thawing condition was specified at 37 C for 45 sec, and the relationship between the cooling rate and thawing velocity was not investigated. Farstad [5] reported that rapidly frozen preparations should be rapidly thawed and slowly frozen preparations should be slowly thawed. In the future, it will be necessary to clarify this relationship. Regarding the sensitization period, the semen qualities after thawing were poor in the 5 min groups regardless of the height from LN 2. Sperm motility may have been impaired because the preparations were put into LN 2 too early. Thus, it is necessary to sensitize for a certain period after decreasing to these temperatures. On comparison of the plunging method with the simple freezer method previously reported by us [19 21], the semen qualities after thawing were higher in the preparations frozen by the plunging method. Although a conception test of semen frozen by the plunging method was not performed, since the conception rate of semen prepared by the simple freezer method was 90% [21], intrauterine insemination of semen frozen by the plunging method may achieve a comparable conception rate. It will be necessary to determine the conception rate using semen frozen by the plunging method in the future. On observation of the temperature changes during the procedures of the 2 methods, the temperature rapidly decreased to about 190 C at 6 to 50.9 C/min using the simple freezer, including that the preparation was rapidly frozen compared to freezing by the plunging method while keeping the preparation at 7 cm above the LN 2 surface. Since the results of Experiment 1 and reports from other researchers [3, 7, 14] show that slow freezing is appropriate for canine semen, a low cooling rate of about 10 to 20 C is recommended as the optimum freezing condition for canine semen. In conclusion, this study clarified that horizontal placement of straws above LN 2 to reduce the temperature to between 50 and 70 C at a slow cooling rate of about 10 C/min, followed by plunging into LN 2 after sensitization for 10 15 min, provides good semen qualities after thawing. We believe this freezing method may improve the conception rate for insemination of canine frozen-thawed semen. REFERENCES 1. Alamo, D., Batista, M., Gonzalez, F., Rodriguez, N., Cruz, G., Cabrera, F. and Gracia, A. 2005. Cryopreservation of semen in the dog: use of ultra-freezers of 152 C as a viable alternative to liquid nitrogen. Theriogenology 63: 72 82. 2. Cardoso, R.C.S., Silva, A.R., Uchoa, D.C. and Silva, L.D.M. 2003. Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations. Theriogenology 59: 743 751. 3. Dobrinski, I., Lulai, C., Barth, A.D. and Post, K. 1993. Effects of four different extenders and three different freezing rates on post-thaw viability of dog semen. J. Reprod. Fertil. (Suppl.) 47: 291 296. 4. England, G.C.W. 1993. Cryopreservation of dog semen: a review. J. Reprod. Fertil. (Suppl.) 47: 243 255. 5. Farstad, W. 1996. Semen cryopreservation in dogs and foxes. Anim. Reprod. Sci. 42: 251 260. 6. Ivanova-Kicheva, M.G., Subev, M.S., Bobadov, N.D., Dacheva, D.P. and Rouseva, I.A. 1995. Effect of thawing regimens on the morphofunctional state of canine spermatozoa. Theriogenology 44: 563 569. 7. Kawakami, E., Tsutsui, T., Yamada, Y. and Yamauchi, M. 1984. Cryptorchidism in the dog: occurrence of cryptorchidism and semen quality in the cryptorchid dog. Jpn. J. Vet. Sci. 46: 303 308. 8. Nöthling, J.O. and Shuttleworth, R. 2005. The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen. Theriogenology 63: 1469 1480. 9. Okano, T., Murase, T., Asano, M. and Tsubota, T. 2004. Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa. J. Vet. Med. Sci. 66: 1359 1364. 10. Peña, A.I., Johannisson, A. and Linde-Forsberg, C. 2001. Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation. J. Reprod. Fertil. (Suppl.) 57: 371 376. 11. Peña, A. and Linde-Forsberg, C. 2000. Effects of Equex, oneor two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa. Theriogenology 54: 859 875. 12. Peña, A.I., Lugilde, L.L., Barrio, M., Herradon, P.G. and Quintela, L.A. 2003. Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca 2+ concentration of dog spermatozoa. Theriogenology 59: 1725 1739. 13. Rota, A., Frishling, A., Vannozzi, I., Camillo, F. and Romagnoli, S. 2001. Effect of the inclusion of skimmed milk in freezing extenders on the viability of canine spermatozoa after thawing. J. Reprod. Fertil. (Suppl.) 57: 377 381. 14. Rota, A., Iguer-Ouada, M., Verstegen, J. and Linde-Forsberg, C. 1999. Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste. Theriogenology 51: 1045 1058. 15. Rota, A., Rota, A., Martini, M., Milani, C. and Romagnoli, S. 2005. Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapors) freezing. Reprod. Nutr. Dev. 45: 29 37. 16 Rota, A., Ström, B., Linde-Forsberg, C. and Rodriguez-Martinez, H. 1997. Effects of Equex STM paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38 C. Theriogenology 47: 1093 1101. 17. Silva, A.R., Cardoso, R.C.S., Uchoa, D.C. and Silva, L.D.M. 2002. Effect of tris-buffer, egg yolk and glycerol on canine semen freezing. Vet. J. 164: 244 246. 18. Thirumala, S., Ferrer, M.S., Al-Jarrah, A., Eilts, B.E., Paccamonti, D.L. and Devireddy, R.V. 2003. Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents.

EFFECT OF SENSITIZATION TO LN2 ON FROZEN CANINE SEMEN 1061 Cryobiology 47: 109 124. 19. Tsutsui, T., Hase, M., Hori,T., Ito, T. and Kawakami, E. 2000. Effects of orvus ES paste on canine spermatozoal longevity after freezing and thawing. J. Vet. Med. Sci. 62: 533 535. 20. Tsutsui, T., Hase, M., Hori, T., Komoriya, K., Shimizu, N., Nagakubo, K. and Kawakami, E. 2000. Effect of addition of Orvus ES paste to frozen canine semen extender on sperm acrosomes. J. Vet. Med. Sci. 62: 537 538. 21. Tsutsui, T., Hase, M., Tanaka, A., Fujimura, N., Hori, T. and Kawakami, E. 2000. Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate. J. Vet. Med. Sci. 62: 603 606. 22. Tsutsui, T., Tezuka, T., Mikasa, Y., Sugisawa, H., Kirihara, N., Hori, T. and Kawakami, E. 2003. Artificial insemination with canine semen stored at a low temperature. J. Vet. Med. Sci. 65: 307 312. 23. Yu, I., Songsasen, N., Godke, R.A. and Leibo, S.P. 2002. Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates. Cryobiology 44: 62 78.