Artificial Insemination with Canine Semen Stored at a Low Temperature

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1 FULL PAPER Theriogenology Artificial Insemination with Canine Semen Stored at a Low Temperature Toshihiko TSUTSUI 1), Tomoyuki TEZUKA 1), Yukiko MIKASA 1), Hirofumi SUGISAWA 1), Nobuyuki KIRIHARA 1), Tatsuya HORI 1) and Eiichi KAWAKAMI 1) 1) Department of Reproduction, Nippon Veterinary and Animal Science University, 7 1 Kyonan-cho 1 chome, Musashino-shi, Tokyo , Japan (Received 12 February 2002/Accepted 3 December 2002) ABSTRACT. Cooled canine semen solutions for storage were investigated with three stock solutions: egg yolk-citrate-glycine-glucose solution, egg yolk Tris-fructose citrate solution (EYT-FC), and egg yolk sodium citrate dihydrate solution (EYCD). For the control group, the second fraction of semen was examined. Nine male beagles and 37 female (47 experimental cases) beagles for artificial insem ination (AI) were used. The qualities of semen stored at 4 C deteriorated earlier in the control and EYCD groups. In the other two groups, sperm motility was 60% or higher after storage for 6 days and 20% or higher after storage for 12 days. On a comparison of these two groups, the sperm motility and viability were slightly higher in the EYT-FC group. A high conception rate was obtained by AI using semen stored at a low temperature for a maximum of two days in the control group and four days in the EYT-FC group. KEY WORDS: artificial insemination, canine, cooled semen. J. Vet. Med. Sci. 65(3): , 2003 For artificial insemination (AI) of dogs, fresh semen [3, 12, 18, 20, 21], cooled semen [6 9, 12, 15], and frozen semen [3, 12, 16, 19] have been investigated. We have shown that when using fresh semen, sperm [21] are required for intravaginal insemination and sperm [20] are required for unilateral uterine horn intrauterine insemination. We have also shown that a high conception rate, 90%, could be obtained by insemination of frozen semen supplemented with egg yolk Tris-fructose citrate (EYT-FC) containing Orvus ES Paste (OEP, Nova Chemical Sales, Scituate, Inc., MA, U.S.A.) [19], but intrauterine insemination requires a surgical technique. When the male dog is alive, intravaginal insemination of cooled semen is considered. In studies of cooled canine semen, semen preparations with milk [6, 8, 9, 13, 15] and egg yolk [1, 2, 4, 5, 7, 11 14] as the extender have been investigated. In studies with egg yolk-citrate-glycine-glucose extender (EYC-GG) performed by Foote [4, 5] and egg yolk Tris-glucose extender performed by Iguer-ouada and Verstegen [11], canine sperm could be stored at 4 C for a prolonged period, although these were in vitro studies. Some attempts at fertilization have succeeded when using cooled semen. Gill et al. [6] and Linde-Forsberg [12] reported a high conception rate for cooled semen within 24 hr. Successful fertilization with semen stored for a long period has been reported. Harrop [8] stored semen in sterilized milk for 4 days, and this is the first successful fertilization with cooled semen. Seager and Fletcher [15] also stored semen in sterilized milk for 1 4 days similarly to Harrop [8] and obtained a conception rate of 53.0% (8/15), but the semen volume and number of sperm used for AI and the frequency of collecting semen were not clarified. Therefore, although many in vitro experiments have been performed with cooled semen as mentioned above, semen preparation applicable to AI for several days has not been clarified. We therefore investigated extenders, storage methods, and AI methods with cooled semen in dogs. For the extender, EYC-GG with which Foote [4, 5] succeeded in long-term storage of canine sperm, EYT-FC, which we used for frozen canine semen [19], and egg yolk sodium citrate dihydrate (EYCD), which has long been used for domestic animals, were investigated. MATERIALS AND METHODS Animals: Nine male dogs aged 2 5 years were repeatedly used. For AI, there were 29 female dogs aged 7 months-1.5 years at the initial estrus and two multiparous dogs. Because of repeated use, the number of cases was 47. Since many dogs were used for AI at the initial estrus, the conception results were obtained from 58 dogs mated at the initial estrus in the same colony for comparison as the mating group. Collection of semen and semen quality test: Semen was manually collected in three fractions and the qualities of the second fraction containing sperm were examined as reported previously [21]. Sperm viability was assessed by eosin-nigrosin staining. The presence or absence of acrosomes in the head of sperm was examined by the triplestain technique [17]. Osmotic pressure of semen was measured with a VAPOR PRESSURE OSMOMETER 5100B (WESOR Inc., Logan, Utah, U.S.A.). The second and third fractions of semen were used in the experiment. Low-temperature storage of semen: Semen diluted with the three extenders shown in Table 1, and original semen as the control were stored at a low temperature. The semen fraction was centrifuged at 1,500 rpm for 5 min to remove semen plasma, and the number of sperms was adjusted to /ml with each extender or the third fraction (control). These semen preparations were aliquoted

2 308 T. TSUTSUI ET AL. Table 1. Composition of extender Ingredient EYCD a) EYT-FC b) EYC-GG c) Sodium citrate dihydrate (g) 3.00 Citric acid (g) Tris aminomethane (g) 2.40 Fructose (g) 1.00 Glycine (g) 0.75 Glucose (g) 1.00 Egg-yolk (ml) Didistilled water (ml) Penicillin G potassium (IU) 100, , ,000 Streptomycin sulfate (g) a) Egg yolk sodium citrate dihydrate. b) Egg yolk Tris-fructose citrate. c) Egg yolk-citrate-glycine-glucose. 2 ml into 10-ml glass tubes. Temperature was decreased from 20 C to 4 C during a one-hour period in a programmed low-temperature bath (UH-JF, Chino Ltd., Japan) and the semen preparations were stored at a low temperature in a low-temperature semen processing system (two-window type, Fujihira Ltd., Japan). Sampling for the semen test was carried out in a low-temperature semen processing system. The semen quality test of the control was performed daily for 4 days, and that of semen diluted with extenders was performed every other day until day 12 of storage. Artificial insemination: Semen for AI was prepared by the same dilution method as in the low-temperature storage experiment described above. For AI, sperm stored for one day and and sperm stored for 2 days were inseminated in the vagina in the control group. Among semen preparations diluted with 3 extenders, semen diluted with the most preservative extender was selected for AI. Semen was stored at 4 C for 4 9 days then sperm were inseminated. The number of sperm in semen stored at a low temperature and used for AI was the number of viable sperm before storage. AI was performed during the optimum mating period 3 5 days after ovulation day estimated from the blood progesterone level [10]. Semen was infused in the deep vagina of a female dog restrained in the handstand position as previously reported [18]. Diagnosis of pregnancy: Pregnancy of artificially inseminated dogs was determined with an ultrasonographic diagnostic system (ECHOVISION SSD-500EV, Aloka Co., Japan) 25 days after AI. The number of newborns was counted on the delivery day. Statistical analysis: Data obtained in this study were analyzed by Student s t-test or χ 2 test, and a P value of < 0.05 was regarded as significant. Fig. 1. Changes in motility and viability of canine sperm stored at a low temperature (mean ± SE). a: Significantly different from that of EYT-FC (p<0.01). b: Significantly different from that of EYT-GG (p<0.01). RESULTS 1. Qualities of semen stored at a low temperature Time-course changes in the qualities of semen stored at a low temperature are shown in Figs Mean sperm motility was decreased to 17.3% or lower Fig. 2. Changes in abnormality and acrosome defective rate of canine sperm stored at a low temperature (mean ± SE).

3 AI WITH COOLED CANINE SEMEN 309 Fig. 3. Changes in ph and osmotic pressure of canine sperm stored at a low temperature (mean ± SE). Table 2. Conception results of AI with canine semen stored at 4 C for one day (Control) ( /2 ml) Bitch number Semen quality Dog Sperm Sperm number motility viability Number of pups Conception rate (%) 181 a) / (80.0) 193 a) Mean b) ± SE a) Multiparous bitch. b) Mean ± SE of the number of fetuses for eight animals that delivered. after storage for 2 days and to 0% after storage for 3 days in the control group. The EYCD group showed similar changes to the control group. In the EYT-FC and EYC-GG groups, sperm motility was 60% or higher after storage for 6 days and 20% after storage for 12 days, being significantly higher than that in the other two groups. Motility was higher in the EYT-FC group than in the EYC-GG group (P<0.01). The mean viability of sperm on day 4 was 87.6% in the control group, but it was 85% or even higher on day 12 in the three additive extender groups. Among these groups, viability was slightly higher in the EYT-FC group. The abnormality of sperm rapidly increased in the control group. The mean rate was 19.7% and 26.2% after storage for 2 days and 4 days, respectively. The increase was mainly due to an increase in sperm with a curved middle portion. Among the three additive extender groups, the abnormality of sperm was slightly higher in the EYT-FC group than the other two groups, and the mean rate was 15.9% after storage for 8 days. The rate was about 10% in the other two groups. The abnormal site was located mainly in the tail region in the EYCD and EYC-GG groups, whereas abnormality increased in the middle and tail of sperm in the EYT- FC groups. The rate of sperm lacking acrosomes rapidly increased in the control group and reached a mean rate of 17.8% after storage for 4 days. In the additive extender groups, the defective rate gradually increased compared to that in the control group. The semen ph gradually increased and reached a mean ph of 7.1 after storage for 4 days in the control group. In the additive extender groups, the ph was slightly decreased by storage. Osmotic pressure of semen was about 305 mos/kg in the control group. In the additive extender groups, osmotic pressure ranged widely from to mos/kg immediately after addition of the extender, and osmotic pressure increased as the storage period was prolonged. Among stock semen prepared with the three extenders, the sperm motility was significantly higher in the semen diluted with EYT-FC and EYC-GG than semen diluted with EYCD (P<0.01). On comparison of EYT-FC and EYC-GG, the semen diluted with EYT-FC was slightly superior in sperm motility, viability, and rate of sperm with defective acrosomes. Thus, EYT-FC was selected for the extender for the subsequent AI experiments. 2. Artificial insemination with semen stored at a low temperature 1) Conception results of AI in the control group The conception results obtained by insemination of semen stored for one and 2 days are shown in Tables 2 and 3. The conception result in the copulation group is shown in Table 4. With regard to the qualities of semen used for AI, mean sperm motility and viability were 28.0 ± 3.2% and 88.7 ± 1.3%, respectively, after one-day storage and 13.5 ± 2% and 88.0 ± 1.3%, respectively, after two-day storage. When sperm were inseminated in the vagina after storage for one day, the conception rate was 80.0% (8/10) and the number of pups was 1 6, mean 3.4 ± 0.6 (SE). The conception rate was 82.8%, and the mean number of pups was 6.2 ± 0.3 in the copulation group. There was no significant difference between the control and copulation groups

4 310 T. TSUTSUI ET AL. Table 3. Number of spermatozoa ( 10 8 ) Conception results of AI with canine semen stored at 4 C for 2 days (Control) Bitch number Semen quality Dog Sperm Sperm number motility viability Number b) of pups Conception rate (%) / (30.0) a) a) a) / a) (70.0) Mean ± SE a) Multiparous bitch. b) The mean numbers of pups for three and seven dams inseminated with and sperms were 4.7 ± 1.5 and 5.3 ± 1.1, respectively. Table 4. Conception by copulation at initial estrus (copulation group) Number of Number of pups bitches Total 58 Conception rate: 82.8% (48/58). Number of pups: 6.2 ± 0.3 (Mean ± SE, n=48) Table 5. Stored days Conception in AI with canine semen with EYT-FC stored at 4 C for 4 9 day Bitch number Semen quality Dog Sperm Sperm number motility viability Number of pups Conception rate (%) 202 a) / (83.3) Mean b) ± SE /2 208 a) (50.0) 175 a) a) a) Mean ± SE a) a) Mean ± SE a) /1 (0) a) Multiparous bitch. b) Mean number of fetuses for five animals that delivered. 0/5 (0) 1/3 (33.3)

5 AI WITH COOLED CANINE SEMEN 311 in the conception rate but the number of pups was significantly lower in the control group (P<0.01). When sperm stored for 2 days were inseminated, the conception rate was significantly lower than that obtained with sperm stored for one day (30.0%, 3/10, P<0.01). Nevertheless, when sperm stored for 2 days were inseminated, the conception rate was 70.0% (7/10) and the number of pups was 2 9 mean 5.4 ± 0.4, and these were not significantly different from those in the copulation group. 2) Conception results for artificial insemination with semen diluted with EYT-FC and stored at a low temperature The conception results obtained by AI of semen prepared with EYT-FC and stored at a low temperature are shown in Table 5. The conception rate and number of pups obtained with semen stored for 4 days were 83.3% (5/6) and 1 6 (mean: 3.2), respectively, and the number of newborns was significantly lower than that in the copulation group (P<0.01). The conception rates obtained with semen stored for 5, 6, 7, and 9 days were 50.0% (1/2), 0% (0/5), 33.3% (1/ 3), and 0% (0/1), respectively. The numbers of pups obtained with semen stored for five and seven days were two and one, respectively. DISCUSSION When the canine semen was stored at 4 C, it could be used for AI for 2 days. Preservation with EYT-FC prolongs the storage period to 4 days, which allows AI with cooled semen. But the number of pups was significantly lower in the group inseminated with semen stored for 4 days than in the copulation group. Since the number of sperm was limited to for AI, increasing the number of sperm may increase the conception rate and number of pups. It is necessary to clarify whether semen used for AI can be stored for more than 4 days by using semen diluted with EYT-FC. When the canine semen was stored at 4 C, the mean viability on day 2 was high, 90.7%, but the mean sperm motility was low, 17.3%, but when sperm stored for 2 days were inseminated in the vagina, a conception rate of 70% was obtained. In contrast, although the sperm motility was high, 60.7%, after storage for 6 days, none of the 5 animals were fertilized in the EYT-FC group. There was no problem as to qualities other than sperm motility after storage for six days. In this study we attached great importance to sperm motility in the evaluation of three extenders. Although it cannot be excluded that sperm motility is most important, it may be necessary to investigate what parameters other than sperm motility are necessary. The above findings showed that canine semen supplemented with EYT-FC and stored at 4 C for 4 days can be used for intravaginal insemination. 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6 312 T. TSUTSUI ET AL. ber of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn. Jpn. J. Vet. Sci. 51: Tsutsui, T., Tezuka, T., Shimizu, T., Murao, I., Kawakami, E. and Ogasa, A Artificial insemination with fresh semen in beagle bitches. Jpn. J. Vet. Sci. 50: