. Recently, canine frozen semen has been attracting attention for breeding purposes, and methods of judging ovulation and optimum timing for insemination have become important. As methods of predicting the canine ovulation, vaginal smear, plasma sex hormone levels and ultrasonographic diagnosis system (US) have been investigated in combination, but a standard technique has not yet been established. Therefore, we investigated a method of predicting canine ovulation in dogs by US, and by measuring plasma LH and progesterone (P) levels three times a day. Ovulation could be observed by detecting irregularly shaped ovarian follicles by US in six of 11 dogs (54.5%). In these dogs, the time between the LH peak and ovulation was 24-48 hr, 38.0 hr on average. The P level on the ovulation day was 1.88-2.81 ng/ml, 2.34 ng/ml on average. A value of 1.88 ng/ml was detected in one dog, but the other five dogs showed P levels of 2 ng/ml or higher. The P level on the day before ovulation was 0.8-1.56 ng/ml, 1.12 ng/ml on average. Assuming that ovulation occurred two days after the LH peak in the 11 experimental dogs, the P level was 2.12-4.06 ng/ml, 2.78 ng/ml on average. The period of a high LH level, not less than 10 ng/ml, continued for 12 hr around the LH peak. Based on these findings, to predict ovulation using US and LH level, it would be necessary for the tests to be performed several times a day. In contrast, it was shown that the day on which a plasma P level of 2 ng/ml or higher was detected by the test performed once a day corresponded to the ovulation day.-KEY WORDS: canine, LH, ovulation, progesterone, ultrasonography.J. Vet. Med. Sci. 62(3): 243-248, 2000 determining the ovulation is difficult, and a technique to predict accurately ovulation in dogs has not yet been established. In this study, therefore, we investigated a method of predicting the ovulation in dogs by thrice daily observation by US and thrice daily measurement of blood LH and P levels. Since the mating period in dogs is 3-5 days after ovulation [17,18,20], a method of predicting the ovulation during the mating period was investigated. MATERIALS AND METHODSExperimental animals: Eleven in-bred beagle dogs aged 2 to 5 years maintained at Nippon Veterinary and Animal Science University were used in the experiments. These dogs were known to be reproductively normal from previous estrous cycles and consisted of nine multiparous dogs and two nulliparous dogs. The dogs were maintained in cages measuring 160 × 75 × 65 cm and were fed commercial dog foods once a day. The dogs were given drinking water three times a day, in the morning, afternoon and evening.Observation of estrus: The female dogs were observed every day for pudendal enlargement and the presence or absence of vulval bleeding.Blood collection for hormone measurements: Blood samples for measurements of plasma LH and P levels were collected once a day (19:00) between the first day of vulval bleeding and the first day on which ovarian follicles could be observed by US, then collected t...

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ABSTRACT. Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 × 10 7 sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 × 10 7 sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats. KEY WORDS: artificial insemination, epididymal sperm, feline.J. Vet. Med. Sci. 65(3): 397-399, 2003 It has been demonstrated in many species that epididymal sperm have fertility in in vitro fertilization and artificial insemination (AI) [4,8,[11][12][13][14][15]25], and fertilization with frozen epididymal sperm was confirmed in pigs [11] and dogs [9].Freeze-storage of epididymal sperm is an important technique for gamete preservation of species, particularly for preservation of animals becoming extinct. As wild animals in the cat family are becoming extinct, we selected cats as an animal model.We have performed studies of reproductive techniques such as in vitro fertilization [5,6], embryo transfer [6,24], and AI [18,[20][21][22][23] in cats.With regard to feline epididymal sperm, studies of the qualities [1,2], state of fertility acquisition [2,3,12], and freeze-storage method [4,8,17] are progressing, and fertility of fresh epididymal sperm [12] and frozen semen [4,8] has been shown, but no in vivo study with frozen epididymal sperm has been performed. With frozen feline ejaculated semen, Platz et al.[16] and we [23] have succeeded in obtaining newborns by intravaginal and intrauterine insemination, respectively. Therefore, we froze epididymal sperm by the same method as for freezing ejaculated sperm and investigated the possibility of inducing conception by intrauterine insemination. It has been shown that frozen semen supplemented with Orvus ES Paste (OEP, Nova Chemical Sales Scituate, Inc., MA, U.S.A.) has superior properties after thawing semen without OEP in dogs [9] and pigs [7]. In this way, the usefulness of OEP was also investigated in this study.Animals: Ten male crossbred cats aged 1.0-3.3 years (mean: 1.9 ± 0.3, SE) were used. For AI, 11 female cats aged 2-4 years were used. The cats were kept in groups in an animal room measuring 4 × 7 m with temperature controlled to 23 ± 2°C. The animals were given commercial dry cat food (Hill's Feline Maintenance Dry, U.S.A.).Transmigration of sperm from epididymis: Surgically excised testis and epididymis were weighed. The epididymis was cut at the corpus into two equal parts. Each part was minced in 1 ml of egg yolk Tris-fructose citrate solution (EYT-FC) [23] to transmigrate the sperm. The solution was filtered through a stainless steel mesh (80 µm) and sperm were recovered. The time from excision of the epididymis to transmigration of the sperm was within 1 hr.Sperm quality test and freezing: The recovered sperm were examined by the gen...

ABSTRACT. Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 ± 1.6 (SE) %; sperm viability, 89.1 ± 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 × 10 7 , mean 61.5 ± 10.0 × 10 7 . Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 ± 2.5% and 53.1 ± 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 × 10 8 , 3 × 10 8 , or 4 × 10 8 sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 × 10 8 sperm was fertilized (1/16, 6.3%). When 1 × 10 8 sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate. KEY WORDS: canine, epididymal sperm, frozen, intratubal insemination, intrauterine insemination.J. Vet. Med. Sci. 66(1): [37][38][39][40][41] 2004 Sperm reach maturation during migration from the caput to the cauda of the epididymis and are retained in the cauda epididymis until ejaculation. It has been demonstrated in many species that sperm in the cauda epididymis exhibited fertility in in vitro fertilization and artificial insemination [1,4,[7][8][9]14]. For animals that die unexpectedly, transmigration of epididymal sperm, and its freeze-storage are important techniques for gamete preservation. These techniques are considered important for preventing species from becoming extinct. Conception obtained by artificial insemination of frozen epididymal semen has been reported in one dog [7], pigs [8], goats [1], and cats [14]. Marks et al. [7] successfully obtained delivery of one pup after intrauterine insemination despite poor semen quality after thawing. There is only one study about the quality and resistance to freezing of canine epididymal sperm, reported by Hewitt et al. [5]. Their study showed that the number of sperm that reached maturation was lower in the epididymis than in frozen ejaculated sperm, but there was no difference in oocytepenetrating ability [5]. In the method of epididymal sperm recovery, Marks et al. [7] reported that the epididymis was perfused from the seminal duct to the epididymis in boxer dogs and Hewitt et al. [5] used the mincing method. Sirivaidyapong [11] com...

ABSTRACT. Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 × 10 8 spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 × 10 8 spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 × 10 8 spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 ± 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 × 10 8 or 40 × 10 8 spermatozoa, but two of three bitches that received insemination of 20 × 10 8 spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.-KEY WORDS: artificial insemination, canine, frozen semen, Orvus ES Paste.Conception using frozen canine semen was initially achieved by Seager and Platz using pellet form semen in 1969 [14]. Following this, conceptions by the straw method were reported by Andersen in 1972 [1]. Thereafter, conceptions using frozen semen have been obtained at a small number of research institutions, using egg yolk Trisfructose citrate (EYT-FC) [1-4, 11-13, 17, 18, 24], Triladyl [8,10], laiciphos [15,16], biciphos [16], and CLONE [17] as extenders. The composition of some of these extenders has not been revealed. With regard to the insemination site, both intravaginal [2,4,[8][9][10][11][12][13]15] and intrauterine [3,4,6,12,13,15,16,24] insemination (IVI, IUI) have been performed. Moreover, the numbers of sperm and modes of insemination vary among researchers. A high conception rate has been documented in many of these reports. However, it is generally recognized that currently frozen canine semen is not practical for clinical use, contradicting the research reports.We have previously reported [20] that the quality of frozen semen prepared using EYT-FC, which is used as an extender of frozen canine semen, is reasonable, however, the sperm motility rapidly decreased in the early phase after thawing, and was reduced to 0% after three hours. Referring to the ...