两种抗冷冻剂对不同品系小鼠精子冷冻的影响

目的建立一套比较理想的小鼠精子冷冻复苏的方法。方法采用R18S3和FERTIUPTM-CPA两种保护剂,对DBA/2、C57BL/6J、KM和B6.129S7-Ldlrtm1Her/J四个品系的小鼠精子进行冷冻,冷冻精子用三种方法复苏,以体外受精率来评价精子冷冻的效果。结果以R18S3作为冷冻保护剂,DBA/2(73.3%,88.4%,55.6%)和KM(64.9%,60.2%,39.6%)品系小鼠冷冻精子复苏后体外受精率差异显著(P<0.05),C57BL/6J(3.0%,10.3%,3.7%)和B6.129S7-Ldlrtm1Her/J(0%,5.0%,0%)结果差异不显著(P>0.05);以FERTIUPTM-CPA作为冷冻保护剂,DBA/2(33.6%,14.1%,91.6%)、C57BL/6J(8.4%,21.0%,4.9%)和B6.129S7-Ldlrtm1Her/J(8.2%,10.0%,28.9%)品系小鼠冷冻精子复苏后体外受精率差异显著(P<0.05),而KM(48.1%,48.0%,48.1%)小鼠冷冻精子复苏后体外受精率差异不显著(P>0.05)。结论对于DBA/2和KM品系小鼠来说,用R18S3或FERTIUPTM-CPA冷冻精子,选择一种恰当的复苏方法,均可以得到较理想的体外受精率,而C57BL/6J和B6.129S7-Ldlrtm1Her/J品系小鼠无论采用哪种冷冻保护剂,选择何种方法复苏精子,得到的体外受精率都较低。     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

JCB：研究发现可促进精子卵子成功受精的关键糖蛋白

在精子和卵子受精之前。精子往往会结合并且穿过卵子外部的透明带结构。透明带（Zona pellucida）是包围在卵子与着床前胚胎外的透明胶质结构,其主要成分为糖蛋白;尽管研究者们为此进行了很多年的研究,但是其背后的生化机制依然并不清楚,近日,刊登在国际杂志The Journal of Cell Biology上的一篇研究论文中。来自美国国立糖尿病、消化和肾脏疾病研究所的研究人员通过研究鉴别出了透明带上帮助精子和卵子成功受精的关键蛋白质。     

系统低温生物学技术在实验小鼠资源保存中的建立与应用

目的建立小鼠胚胎与配子冷冻库,以安全、有效地保存小鼠资源。方法选择不同遗传背景（近交系、远交群、免疫缺陷、疾病模型和基因改变等）的实验小鼠,系统进行了超数排卵、体外受精（IVF）率、胚胎与精子的冷冻复苏效果、卵巢冷冻与移植、辅助体外受精等比较研究。结果①小鼠年龄和遗传背景的不同,其超数排卵的结果也不同（P〈0．01）。三个日龄段中,28日龄最好,其次为112日龄,56日龄最差;不同遗传背景小鼠的超数排卵结果显示,封闭群和大部分近交系小鼠优于转基因小鼠（P〈0．05）,自发性疾病小鼠和基因剔除小鼠的结果最差;②不同品系小鼠的新鲜精子和冻融精子的体外受精率差异有显著性（P〈0．05）,特别是C57BL／6J小鼠冻融精子的IVF率（10．3±4．2％）与新鲜精子（89．8±4.8％）相比,差异极显著（P〈0．01）;③不同品系小鼠的胚胎复苏率,除MRL／mp小鼠的复苏率略低外,其他小鼠品系均有较高的冷冻胚胎复苏率（58．2％～83．9％）,表明,不同遗传背景小鼠之间差异有显著性（P〈0．05）,但均可以达到有效保存小鼠资源的目的。④小鼠的遗传背景、年龄等对小鼠精子的冷冻效果都有影响,采用改良的FERTIUP冷冻保护剂和细胞质内单精注射（ICSI）技术可有效提高以C57BL／6J为背景的基因改变小鼠的精子冷冻复苏率。⑤卵巢冷冻保存可以改善雌性小鼠的繁殖困难或不孕。结论小鼠资源的安全保存,除了长期连续繁殖保种外,最好的或最保险的方法是低温保存。通过将胚胎、配子、卵巢等长期保存在液氮（-196℃）中避免遗传性状的改变,并在将来复苏后获得正常的小鼠后代,以用于生物学和医学等研究。     

小鼠卵子在不同条件下的体外受精

本实验比较了小鼠精、卵细胞在不同生理状态下体外的受精能力。结果表明,体内受精率明显地高于体外(p<0.05),自发排出的卵子比超数排出的卵子受精率高(p<0.05),体外获能的附睾精子比体内获能的子宫精子受精率高(p<0.01)。唯独用超数排出的卵子和体外获能的附辜精子体外受精时,其受精率和体内相似。     

人及哺乳动物受精与糖蛋白的关系

越来越多的研究表明精卵识别与精子质膜及卵透明带中所含糖蛋白有直接关系。在精卵识别中存在着精子受体和卵透明带（zona pellucida,ZP）配体相互作用的糖类识别机制及精子质膜与卵子质膜的糖蛋白识别。本文主要从结构功能上对与受精相关的精卵表面糖蛋白作一介绍。卵子表面受精相关的糖蛋白主要是ZP1、ZP2、ZP3。与卵子表面糖蛋白相比,精子表面参与精卵识别的糖蛋白种类较多,在SP56、SP95、PH－20、FA－1、甘露糖结合蛋白、顶体素、fertilin蛋白等。     

α-D-甘露糖残基在猪透明带中的分布及其在受精中的作用

为证明α-D-甘露糖残基在猪透明带中的分布及其在受精中的作用,用异硫氰酸荧光素络合小扁豆凝集素(fluorescein isothiocyanate-labelled Lens culinaris,FITC-LCA,一种D-甘露糖特异结合植物凝集素)标记去除卵丘细胞的体外成熟猪卵母细胞,用于观察LCA的标记情况及体外受精,并用甘露聚糖进行体外受精的竞争抑制实验及检测对精子顶体反应的影响.结果显示,LCA均匀标记于整个透明带且呈强荧光反应;LCA标记能够显著减少透明带表面精子结合数量并几乎完全阻断卵母细胞的受精能力;甘露聚糖能够显著降低透明带精子结合数量和卵母细胞的受精率;甘露聚糖在精子培养的不同时间段上都能明显增加顶体反应的精子数量.这些结果表明,猪透明带糖蛋白上的α-D-甘露糖残基是精子受体的重要组成部分,它能够诱导精子发生顶体反应,促进精子侵入透明带.     

α-D-甘露糖残基在猪透明带中的分布及其

为证明α-D-甘露糖残基在猪透明带中的分布及其在受精中的作用, 用异硫氰酸荧光素络合小扁豆凝集素(fluorescein isothiocyanate-labelled Lens culinaris, FITC-LCA,一种D-甘露糖特异结合植物凝集素)标记去除卵丘细胞的体外成熟猪卵母细胞, 用于观察LCA的标记情况及体外受精, 并用甘露聚糖进行体外受精的竞争抑制实验及检测对精子顶体反应的影响. 结果显示, LCA均匀标记于整个透明带且呈强荧光反应; LCA标记能够显著减少透明带表面精子结合数量并几乎完全阻断卵母细胞的受精能力; 甘露聚糖能够显著降低透明带精子结合数量和卵母细胞的受精率; 甘露聚糖在精子培养的不同时间段上都能明显增加顶体反应的精子数量. 这些结果表明, 猪透明带糖蛋白上的α-D-甘露糖残基是精子受体的重要组成部分, 它能够诱导精子发生顶体反应, 促进精子侵入透明带.     

两种冷冻保护剂和冷冻程序对兔精子冷冻效果的比较

目的比较不同冷冻保护剂和冷冻程序对兔精子冷冻保护的影响,以期提高兔精子冷冻保存的效果和效率。方法用三步降温法（程序Ⅰ）和两步降温法（程序Ⅱ）两种冷冻程序与终浓度分别为2%,3%,4%,5%的甘油和乙酰胺两种冷冻保护剂配合进行精液冷冻保存,统计精子复苏率。结果使用程序Ⅱ添加3%乙酰胺的冷冻保护剂实验组的精子复苏率较高,同其它组比较差异有显著性意义（P〈0.05）;程序Ⅱ比程序Ⅰ节省约70%的时间,同种浓度冷冻保护剂的不同冷冻程序组之间精子复苏率差异无显著性意义（P〉0.05）。结论程序Ⅱ与3%乙酰胺配合可以取得良好的冷冻保存效果;用程序Ⅱ进行兔精液冷冻保存可以大幅缩短操作时间。     

两种玻璃化胚胎冷冻方法对不同品系小鼠胚胎冷冻效果比较

目的探讨EFS和DAP两种玻璃化冷冻方法对不同品系小鼠胚胎冷冻的效果。方法6个品系小鼠（KM、ICR、BALB／c、C57BL／6J、OB／OB、LAP／~TAOF59）的2-cell胚胎分别用EFS和DAP两种玻璃化冷冻方法进行冷冻和复苏,比较两种冷冻方法的胚胎复苏率和着床率。结果6个品系小鼠冷冻胚胎EFS方法的平均复苏率为69．97％（47．9％～83．6％）,DAP方法的平均复苏率47．23％（26．3％-76．7％）,EFS方法明显优于DAP方法。其中KM、ICR和BALB／c小鼠EFS方法的冷冻复苏率显著高于DAP方法（P〈0．01）;冻融胚胎移植后EFS方法的平均着床率27．23％（1．75％一45．0％）,DAP方法的平均着床率31．43％（7．0％一46．3％）。除KM、ICR小鼠外,其他4个品系小鼠的着床率DAP方法高于EFS方法。结论KM和ICR远交群小鼠胚胎适合用EFS方法冷冻保存;C57BL／6J、OB／OB、LAP／aTAOF59三个品系小鼠DAP方法优于EFS方法,但差异不大;BALB／c小鼠两种玻璃化冷冻方法的冻融胚胎着床率均较低,需进一步研究。     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.     

Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.     

Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods

This study is to investigate the change of morphology of the meiotic spindle and the extent of zona hardening relating to the morphological survival and developmental competence of thawed oocytes. Four- to 8-week-old female mice (C57BL/6) primed with an intraperitoneal injection of pregnant mare's serum gonadotropin and human chorionic gonadotropin. Cryopreserved oocytes using two protocols: vitrificaton using ethylene glycol (EG) and slow freezing using propanediol (PROH). The freezing oocytes were thawed and were fertilized and subsequently cultured in vitro. Spindle/chromosome imagery, dissolution of zona pellucida, and post-thawing survival and development were comparable between two groups. The vitrification cryopreservation method proved to be better than the slow-freezing protocol when comparing the frequency of normal-shaped spindle development post-thawing. The difference in the time required for the dissolution of the zona pellucida under treatment of pronase that was determined to exist between the two cryopreservation methods was statistically significant (P<0.005). The survival rate of post-thawed mature oocytes was significantly greater for the vitrification group than it was for the slow-freezing cryopreservation group (P=0.005). The vitrification cryopreservation of mature murine oocytes would appear to be more satisfactory than the slow controlled-rate freezing method as regards the post-thawing oocyte survival and also the incidence of the normal spindle apparatus in the ooplasm.     

Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

三种不同冷冻保护剂对小鼠附睾冷冻效果的比较

目的探讨三种不同冷冻保护剂对C57BL/6J小鼠附睾冷冻的效果。方法性成熟并交配过的6~8周龄C57BL/6J雄鼠附睾,分别用3种不同冷冻保护剂二甲基亚砜(DMSO)、丙二醇(PROH)、R18S3进行玻璃化冷冻、复苏和精子采集,观察比较三个实验组的复苏精子形态、存活率以及生殖能力。结果三组冷冻复苏分离后的精子形态完整,具有镰刀头状结构;荧光染色后PROH组精子存活率为(88.17±3.43)%,明显高于DMSO组:(61.17±10.65)%和R18S3组(16.83±6.49)%(P0.05);经辅助体外受精后均具有受精能力。获得的胚胎移植后可得到正常子代小鼠(DMSO∶PROH∶R18S3=13∶8∶17)。结论 3种冷冻保护剂均适用于C57BL/6J小鼠附睾冷冻,但PROH冷冻效果较好。     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.