Intrauterine and Intravaginal Insemination with Frozen Canine Semen Using an Extender Consisting of Orvus ES Paste-Supplemented Egg Yolk Tris-Fructose Citrate

Abstract

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 ...

Effects of Final Dilution Rate, Sperm Concentration and Times for Cooling and Glycerol Equilibration on Post-Thaw Characteristics of Canine Spermatozoa

“…Canine semen was diluted with an extender without glycerol followed by a second dilution with glycerol, both at 20°C, and then frozen in liquid nitrogen [28][29][30]. The method corresponds to cooling for 0 hr and equilibration for 1 hr in this study (Table 3), and the percentage of acrosomeintact spermatozoa after thawing was increased by the addition of Orvus ES paste [29] whereas in this study, the prolonged cooling time increased acrosomal integrity (Table 3).…”

“…In fact, offspring were obtained by insemination with canine semen frozen after cooling followed by no particular period of glycerol equilibration; indeed, the time corresponded to that required for loading the semen into straws (about 15-30 min) [27]. The concentration of glycerol used here was 8% whereas other studies employed less [28][29][30]. Since at this concentration, the post-thaw sperm characteristics were generally acceptable, no further investigations were carried out.…”

“…On the other hand, if semen were collected from wild animals in the field, manipulation of the ejaculate would have to be carried out under unfavorable conditions. Thus, modifications would be needed for the application of techniques developed for dogs to wild animals.Successful artificial insemination with frozen canine semen has been well documented [18,22,23,25,27] since the first conception was reported by Seager [21], and recently, the freezing protocol for canine semen has been improved [1,3,20,[28][29][30]. If semen is diluted to a constant final sperm concentration, the dilution rates may differ.…”

The effect of homologous prostatic fluid on motility and morphology of dog epididymal spermatozoa extended and frozen in Biladyl with Equex STM paste or Andromed

“…As early as 1993, Nöthling and Volkmann [7] showed that ejaculated dog sperm frozen in Triladyl ® (Minitüb GmbH, Tiefenbach, Germany) with the surfactant Equex STM paste (Nova Chemical Sales, Scituate, MA, USA) and to which homologous prostatic fluid was added after thawing resulted in better fertility of vaginally-inseminated bitches than bitches inseminated with similar semen but to which no fluid was added after thawing. In support of the findings of Nöthling and Volkmann with respect to the group that received no prostatic fluid [7], Tsutsui et al [8] reported very poor fertility in bitches that were inseminated intravaginally with high numbers ((1.5-10) × 10 8 ) of progressively motile spermatozoa that were frozen in a similar extender (including a surfactant) to the one used by Nöthling and Volkmann [7], but without prostatic fluid. The beneficial effect of prostatic fluid that Nöthling and Volkmann [7] observed could not be achieved by replacing prostatic fluid with albuminfree TALP, suggesting that prostatic fluid promotes the fertility of vaginally inseminated bitches via a mechanism other than merely changing the physical characteristics of the inseminate [9].…”

“…Equex STM, which Nöthling et al [7] and [9] added to the Triladyl, prolongs the maintenance of motility [15], [16], [17] and [18], membrane integrity [15] and [19] and acrosomal integrity [17], [20] and [19], calcium uptake [18] and fertility after intrauterine insemination [8] of frozen-thawed dog spermatozoa. Biladyl ® (Minitüb, GmbH, openUP introducing infectious agents into semen via the extender.…”