Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

Tada, Norio; Sato, Masahiro; Yamanoi, Junko; Mizorogi, T.; Kasai, Kazuhiro; Ogawa, Shyoso

doi:10.1530/jrf.0.0890511

Cited by 187 publications

(90 citation statements)

References 9 publications

(13 reference statements)

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“…Low concentrations of sugar are usually incorporated into semen freezing diluents as a sperm metabolic source, but high sugar concentrations in the freezing diluent have improved cryoprotection of mouse epididymal spermatozoa (Tada et al, 1990). Up to a 7-fold increase in glucose concentrations in Tris-based diluents had no effect on postthawing motility or fertility of pellet frozenthawed ram spermatozoa (Molinia et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Molinia

Evans²,

Maxwell³

1994

Reprod. Nutr. Dev.

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Summary ― Diluents based on the zwitterion buffers Tes, Hepes and Pipes titrated to pH 7.0 with NaOH or Tris were compared with Tris-citrate diluents by assessment of post-thawing motility and acrosome integrity of frozen ram spermatozoa. Varying buffer osmolalities between 300 and 360 mosmol had no effect but there was a decrease (P < 0.001 ) in motility between 360 and 420 mosmol. There was a quadratic effect (P < 0.001 ) on motility of increasing glucose concentration in the diluent (30, 85, 140, 195 and 250 mM) with a maximum at 85 mM. At this sugar concentration, motility was higher (P < 0.001 ) for diluents containing glucose and fructose than lactose, sucrose or trehalose. Inclusion of egg yolk (optimum at 13.5% v/v; P < 0.05), centrifugation of the diluents (P < 0.01 use of low dilution rates (3 or 6-fold; P < 0.05) and freezing in pellets rather than minitubes or straws (P < 0.001 ) improved both the motility and acrosome integrity of spermatozoa in the zwitterion diluents. It is concluded that zwitterion buffers are superior to Tris-citrate in ram semen freezing diluents. zwitterion buffer / motility / acrosome integrity / frozen ram spermatozoa Résumé ― Effet de l'addition de tampons amphotères à des dilueurs sur la congélabilité des spermatozoïdes de bélier. Nous avons comparé l'efficacité de dilueurs à base de tampons amphotères TES, HEPES et PIPES ajustés à pH 7,0 avec NaoH ou tris, à celle de milieux à base de tris-citrate, en évaluant la motilité des spermatozoïdes de bélier et l'intégrité de leur acrosome après congélation. Entre 300 et 360 milliosmoles, l'osmolarité des tampons n'a aucun effet. Au-delà, et jusqu'à 420 milliosmoles, on observe une baisse significative (P < 0, 001) de la motilité. On obtient un effet biphasique sur la motilité en augmentant la concentration de glucose dans le dilueur (30, 85, 140, 195, 250 mM) avec un maximum pour 85 mM. À cette concentration, la motilité est plus élevée avec le milieu contenant du glucose et du fructose qu'avec ceux contenant du lactose, du sucrose ou du tréhalose (P < 0,001). L'addition de jaune d'œuf (optimum à 13,5% vlv; P < 0,05), la centrifugation des dilueurs (P < 0,01), l'utilisation d'un taux de dilution peu élevé (3 ou 6 fois ; P < 0,05) et la congélation en pastilles plutôt qu'en minitubes ou en minipaillettes (P < 0,001) améliorent la motilité et l'intégrité de l'acrosome des spermatozoïdes congelés dans des milieux contenant un tampon amphotère. Cette étude montre donc que les tampons amphotères sont plus efficaces que le tampon tris-citrate pour les dilueurs utilisés pour la congélation du sperme de bélier.bélier / mobilité l acrosome / sperme congelé / dilueur / tampon * Current address:

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Section: Discussionmentioning

confidence: 99%

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Molinia

Evans²,

Maxwell³

1994

Reprod. Nutr. Dev.

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“…[10] 35-36 -** -** Okuyama et al [9] 42 -** -** Takeshima et al [12] 34-36 -45 Nakagata & Takeshima [15] 71-95 -51 Tada et al [16] 85 57 -ICR (CD-1) Storey et al [25] 33-58 -** -** <hybrid> B6CF1 (C57BL/6 X BALB/c) Yokoyama et al [11] 19-37 -17-75 B6C3F1 (C57BL/6N X C3H/He) Nakagata & Takeshima [15] 59 -56 B6D2F1 (C57BL/6J X DBA/2J) Sztein et al [20] 89-93 37 38 Songsasen and Leibo [21] 64-73 -** -** Songsasen and Leibo [22] 61 -42 Songsasen et al [23] 26-39 -24-47 M.m.musculus Nakagata et al [18] 48-63 -24-51 *PZD oocytes were inseminated with frozen-thawed spermatozoa with low motility. **Fertilized oocytes were not transferred.…”

Section: -Review-mentioning

confidence: 99%

“…The first reports of successful cryopreservation of mouse spermatozoa were published in 1990 by 3 independent groups of Japanese investigators [9][10][11]. We were also subsequently successful in the cryopreservation of mouse spermatozoa by means of an improved method [12,13].…”

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confidence: 99%

Mouse Spermatozoa Ctyopreservation.

Nakagata

2000

J.Mamm.Ova.Res.

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“…This has been one of the major technical challenges associated with sperm cryopreservation since the first success about 20 years ago [12,13]. The sperm membrane, especially around the acrosome, is very sensitive to freezing-thawing procedures, and injury to the membrane can decrease the fertilization rate following IVF [14].…”

mentioning

confidence: 99%

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Hasegawa

Yonezawa

Ohta

et al. 2012

Journal of Reproduction and Development

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Abstract. The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 µl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation. Key words: C57BL/6, Glutathione, Mouse, Spermatozoa (J. Reprod. Dev. 58: [156][157][158][159][160][161] 2012) L arge-scale mutagenesis/knockout and phenotyping programs using mouse strains have been launched to provide the research community with a long-lasting resource for the study of mammalian gene function [1,2]. In addition to these large organized programs, individual laboratories generate mice with gene modifications of specific interest using conventional transgenic and knockout methods [3,4]. These uses have increased the demand for the efficient and safe preservation of invaluable mouse strains. Since the first success by Whittingham in 1972 [5], embryo cryopreservation has been the main strategy for preserving mouse genetic resources because of its high reproducibility and technical ease.From the practical viewpoint, however, preservation of mouse strains as embryo stocks has several technical disadvantages because of the steps required before the production of embryos, including superovulation of females, in vitro fertilization (IVF), and in vitro embryo culture. All of these steps should be well controlled for the best results. In addition, the number of oocytes collected from a single female is limi...

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Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

Cited by 187 publications

References 9 publications

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Mouse Spermatozoa Ctyopreservation.

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

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