Effects of Orvus ES Paste on Canine Spermatozoal Longevity after Freezing and Thawing

Abstract

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...

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

“…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.…”

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

“…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 report by Mahi and Yanagimachi [7] that capacitation of canine sperm required seven hours in in vitro experiments, it appears difficult to achieve conception by artificial insemination (AI) using such frozen semen.…”