Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

Abstract

Abstract. Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca 2+ ), phosphate (PO4 3-) and lactate. In all media containing no Ca 2+ , including medium lacking Ca 2+ , lacking Ca 2+ and PO4 3-, lacking Ca 2+ and lactate and lacking Ca 2+ , PO4 3-and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca 2+ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca 2+ .In conclusion, preincubation of thawed sperm in medium containing no Ca 2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca 2+ is practical for use in cryopreserved C57BL/6J sperm. Key words: C57BL/6, Calcium, Cryopreservation, In vitro fertilization, Sperm (J. Reprod. Dev. 55: [386][387][388][389][390][391][392] 2009) ryopreservation of mouse sperm is useful because it is simple, rapid and inexpensive. A large number of sperm can be frozen immediately after collection from a male. Females are required only when the frozen sperm are thawed for fertilization. Successful cryopreservation of mouse sperm was reported in 1990 using raffinose with glycerol [1], ME2SO [2] or skim milk [3]. Since then, cryoprotectant solutions and freezing methods for mouse sperm have been investigated [4][5][6][7][8][9][10][11]. At present, the 18% raffinose plus 3% skim milk method [12] is relied upon in most laboratories. However, with this technique, the fertility of cryopreserved sperm is far less than that of fresh sperm, especially with inbred mice [13][14][15][16]. In particular, fertility decreases markedly in C57BL/6J, a principal inbred strain used for transgenesis studies. Frozen C57BL/6J sperm exhibit acrosome damage with loss of contents caused by the freeze-thaw procedure [17], and almost no sperm can penetrate the zona pellucida. When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23]. Successful IVF with fresh sperm depends on the media used. Various IVF media have been developed, and the effects of media on capacitation and IVF have been investigated. However, the effects of media on frozen mouse sperm have not been investigated.In this s...

Optimized Cryopreservation of Mouse Sperm Based on Fertilization Rate

“…Keeping the sperm relatively concentrated may prove advantageous if motile sperm need to be selected and used in the IVF procedure. Selection of motile sperm may prove useful for sperm from mouse strains found to have poor post-thaw fertilization rates [1,3]. The results of the current study suggest that it may be prudent to extend the sperm from a single mouse in no more than 200-300 μl of cryoprotectant.…”

“…However, similar high fertilization rates have been demonstrated with C57BL/6J sperm frozen in 18% raffinose [3]. To achieve this, thawed sperm were preincubated in calcium-free medium and then motile sperm were selected for the IVF.…”

“…Clearly, the significantly lower fertilization rate achieved by the sperm in the wells was caused not by the presence of raffinose, but by factors generated in the fertilization medium by the sperm, as discussed elsewhere [5]. No difference in fertilization rate was observed using sperm thawed in a water bath set at 37 C or 53 C. Suzuki-Migishima et al [3] also found that C57BL/6J sperm best fertilized eggs after thawing in a 54 C water bath. In contrast, Ostermeier et al [2] noted that sperm thawed in a 54 C water bath fertilized significantly fewer eggs than sperm thawed in a 37 C water bath.…”

“…In those terms, raffinose pentahydrate dissolved in water is the most successful mouse sperm cryoprotectant [1,[3][4][5][6][7]. Commonly, skim milk powder is included [1,2,4,6] to stabilize the pH [8] and to reduce damage to the plasma membrane sustained during the freeze-thaw process [1].…”

“…57: 92-98, 2011) ryopreservation of mouse sperm has become an acceptable and cost-effective procedure for archiving the genomes of genetically engineered mice. Although procedures for in vitro fertilization (IVF) with cryopreserved sperm have, to some extent, been optimized [1][2][3][4][5], there is little objective information demonstrating optimization of a cryoprotectant and cryopreservation procedure for mouse sperm. Hence, that was the purpose of this study.…”