. 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...

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 ...

ABSTRACT. Because of weak resistance of canine sperm to freezing, an applicable method of preparing canine frozen semen has not yet been established. We added various concentrations of Orvus ES Paste (OEP) to egg yolk Tris-fructose citrate, and investigated its effectiveness on survival of spermatozoa. Addition of 0.5-1.0% OEP to the extender for freezing canine semen was effective in prolonging post-thaw survival of spermatozoa.-KEY WORDS: canine, frozen semen, Orvus ES Paste.The first successful fertilization using frozen canine semen involved semen pellets, and was achieved by Seager and Platz in 1969 [15]. Then, in 1972, Andersen reported a successful case by the straw method, which is convenient for storage [1]. Thereafter, many researchers have investigated extenders [5,7,8,17,18], glycerol concentrations [2,4,5,10,12,16], freezing methods [2,10,[16][17][18], and thawing methods [4, 5, 7, 10-12, 17, 20] for frozen canine semen, but an applicable method of preparing frozen canine semen has not yet been established. It is recognized that canine sperm have poor freezing resistance, and sperm motility cannot be maintained for a prolonged time after thawing [3,7,13]. This is considered to be due to major injury of the acrosomes during thawing.A surfactant, Orvus ES Paste (OEP, Nova Chemical Sales, Inc., MA, U.S.A.), is added to the extender of boar sperm, because of weak freezing-resistance. OEP is known to protect the sperm acrosomes after thawing, resulting in improvement of sperm motility and conception rate [6]. In 1993, Nöthling and Volkmann [9] succeeded in obtaining conception using canine frozen semen diluted with an extender supplemented with 0.5% OEP. In 1997 and 1999, Rota et al. [13, 14] also showed the effectiveness of addition of 0.5% OEP to the extender for canine frozen semen by observing the sperm motility and acrosomes after thawing.Although Nöthling and Volkmann [9] and Rota et al. [13,14] set the OEP concentration to 0.5%, the criterion for determining this concentration is not clear. Therefore, in this study, using Tris-fructose-citrate extender containing 20% (v/v) egg yolk (EYT-FC), as used by Rota et al. [13,14], we investigated the effect of concentration of OEP supplemented to the extender on post-thaw survival of canine spermatozoa. In previous reports dealing with frozen canine semen [2, 4, 8-11, 13, 14, 16, 17, 20], 0.5 ml volume straws were used. Since intravaginal insemination of frozen semen in dogs requires a large number of surviving spermatozoa, we investigated the possibility of semen preservation in 1.0 ml straws .The male dogs used in this study were 10 beagles from 2 to 6 years of age with copulation ability and fertility. The semen samples, collected by digital manipulation, were divided into three fractions. The volume of semen, the number, motility, and viability of sperm were determined by methods described previously [19]. The motility of spermatozoa was represented by the percent ratio of spermatozoa that actively moved forward using a test plate for semen prope...

ABSTRACT. Using the triple-stain technique, we investigated whether sperm acrosomes in frozen canine semen were protected during freezing and thawing by addition of a surfactant, Orvus ES Paste (OEP), to the extender. Acrosomes were clearly shown to be protected by the addition of OEP to the entender when compared with those in sperm frozen without OEP addition (p<0.05).-KEY WORDS: acrosome, canine, Orvus ES Paste.A high percentage of boar sperm are damaged by freezing. Graham et al. [1] demonstrated that adding a surfactant, Orvus ES Paste (OEP), to the extender protects acrosomes, making frozen boar semen available for practical use. By electron microscopy, Toyama and Itoh [7] showed that acrosomes are maintained in a normal state by adding OEP to the extender used to freeze boar semen. Kato et al.[2] obtained a similar result using sodium laurylsulfate (1.2-1.6 mg/ml, SLS), which is the major ingredient of OEP.Similar to boar sperm, canine sperm has a low resistance to freezing, and no practical procedure for use of frozen semen has been developed yet. In 1993, Nöthling and Volkmann [3] initially added 0.5% OEP to frozen canine semen. However, they did not discuss the effectiveness of adding OEP to frozen canine semen. Later in 1997 and 1999, Rota et al. [4, 5] reported that adding 0.5% OEP to the extender of frozen canine semen increases its effectiveness. We also investigated various concentrations of OEP added to the extender of frozen canine semen, and their effectiveness. The result showed that addition of 0.5-1.0% OEP improves the sperm motility after thawing and maintains the motility for a prolonged time after thawing [8]. The reason for these effects may be that OEP protects acrosomes, as Toyama and Itoh [7] showed in boar sperm.We therefore, added OEP at a final concentration of 0.75% to Tris-fructose-citrate extender containing 20% (v/ v) egg yolk (EYT-FC), which was used by Rota et al. [4,5] as an extender for frozen canine semen, and investigated whether acrosomes were protected using the triple-stain technique (TST) reported by Talbot and Chacon [6].The five dogs used in the experiments were 2-6 year-old beagles with copulation ability and fertility. The semen samples, collected by digital manipulation, were divided into three fractions. Semen volume, and the number, motility, and viability of sperm of the 2nd fraction were determined by methods previously described [9]. After examination, semen was subjected to the first dilution with EYT-FC, and was then divided into two fractions. In the second dilution, one fraction was diluted with the secondary extender containing OEP, and the other fraction was diluted with the secondary extender without OEP addition. Both primary and secondary dilutions were performed at room temperature (20°C). The final sperm concentration was adjusted to 1 × 10 8 /ml, and 0.5 ml straws were used. The final glycerol concentration was adjusted to 7%. Semen was frozen using a conventional freezer with the freezing temperature conditions and method previously described...

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