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a The Jackson Laboratory, Bar Harbor, Maine 04609
ABSTRACT
Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.
fertilization, IVF/ART, reproductive behavior
INTRODUCTION
Sperm cryopreservation is a well-established technique for most mammals; however, successful cryopreservation of rodent sperm remains a scientific challenge. The small size and the characteristic shape of rodent sperm are difficult obstacles to systematic cryobiology studies [1]. Ten years ago, the potential utility of mouse sperm cryopreservation increased dramatically with the advent of transgenic technology. None of the sperm-freezing protocols available at that time worked for rodent spermatozoa, and some of the early protocols published specifically for mouse sperm were reported as being unrepeatable [2–4]. Although optimization remains to be done, freezing protocols are today more reliable—particularly the skim milk/raffinose method [5–8], with which many laboratories around the world are routinely banking mouse sperm [9–11]. Nevertheless, the technique seems to be restricted to mice with hybrid or mixed background and to be considerably less effective for inbred mouse strains. This is particularly importance in many transgenic and knockout strains that are backcrossed onto inbred backgrounds such as C57BL/6J.
Mouse models serve as a kind of biological reagent, and genetic background is an important criterion when selecting a strain for a particular experiment. Hybrid mice were commonly used for mouse sperm cryopreservation studies, because their high fertility rates (putatively because of hybrid vigor) make them good models for general reproductive studies.
Inbred mouse strains, like hybrids, have defined reproductive parameters that are so distinctive that they are considered to be a characteristic of the strain [12]. Inbred strains also differ in their capacity for embryo development in culture [13–15] and in survival after embryo cryopreservation [16–18]. Similar variations can be attributed to the sperm, because different strains have different in vitro fertilization (IVF) rates [19–31], differences in abnormal morphology rate [32–34], and differences in their ability to survive cryopreservation [35–39]. Most of these studies used previously frozen inbred sperm to inseminate hybrid eggs [36–38]. Ideally, to reconstitute a mouse strain of defined genetic background from cryopreserved sperm, the thawed sperm should be able to fertilize oocytes of the same genetic background. However, as mentioned, sperm from some inbred strains cannot fertilize in vitro or those gametes from strains of hybrid backgrounds.
Successful sperm cryopreservation is ultimately defined by the recovery of live young from frozen-thawed spermatozoa. This requires that the spermatozoa sustain fertility through the cryopreservation process, and that the subsequent IVF, embryo culture, and embryo transfer be successfully performed. The intention of this study was to determine whether any step involved in generating a live mouse from cryopreserved sperm or the donor strain genetic background is the limiting factor in successful mouse sperm cryopreservation.
MATERIALS AND METHODS
Animals
Inbred male and female C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ mice as well as hybrid male and female B6D2F1/J (C57BL/6J x DBA/2J) and CB6F1/J (BALB/cBy x C57BL/6By) mice were obtained from the Jax Research Systems colonies of the Jackson Laboratory. Mice were maintained under routine husbandry procedures according to standards set forth in the Guide for the Care and Use of Laboratory Animals [40]. This includes a photoperiod of 14L:10D. Food (National Institutes of Health rat & mouse/auto 6F, Lab Diet; PMI Feeds, St. Louis, MO) and water were provided ad libitum. Males were housed singly or at maximum of two per cage for at least 5 days before sperm was collected.
Cryoprotectant Solution
The cryoprotectant solution (CPA), containing 18% raffinose (D[+] raffinose pentahydrate [Sigma, St. Louis, MO]) and 3% skim milk (Bacto, Difco; Becton Dickinson, Franklin Lakes, NJ) in culture-grade water (Millipore, Bedford, MA) was prepared according to the method described by Takeshima and Nakagata [41]. After warming (
60°C) for total dissolution of the sugar, the CPA was centrifuged at 10 000 x g for 10 min. The supernatant was filtered through a 0.45-µm filter, and the solution was stored at 4°C for no more than 5 days before use.
Sperm Collection
The sperm were collected from the vasa deferentia and caudae epididymides of 3- to 5-mo-old male mice. After killing each mouse, both epididymides and vasa deferentia were removed and placed in a 35-mm sterile plastic dish (Falcon 1008; Becton Dickinson) containing 1 ml of CPA equilibrated beforehand at 37°C. Both the epididymis and vas deferens were used because the epididymides itself has good sperm concentration and motility and the vas deference has low concentration but high motility (unpublished results), and the combination of both seems to improve the overall motility rate. An identical procedure was used for all strains, in which each cauda epididymis along with the vas deferens was cut five to seven times with the edge of a 30-G injection needle. Sperm were allowed to disperse ("swim out") for 10 min in the CPA solution. Each sample was obtained from a single male mouse, and samples were never pooled for any experiment.
Sperm samples for normal control parameters and control IVF dishes were collected under the same conditions but in human tubal fluid (HTF) medium [42] prepared from reagent-grade chemicals (Sigma) in our laboratory.
Sperm Analysis
Concentration, motility, and progressive motility of the "fresh" nonfrozen control (collected and counted in HTF) and of the frozen-thawed sperm sample (collected, frozen, and thawed in CPA; counted in HTF) were determined using a Hamilton Thorn IVOS computerized semen analyzer (Hamilton Thorn, Beverly, MA). The machine was calibrated using a Makler chamber (Sefi-Medical Instruments, Haifa, Israel). The count accuracy was validated using Accu-beads (a quality-control product; 18 M/ml and 35 M/ml latex beads, Hamilton Thorn). Every sperm collection or sample was analyzed twice, counting a total of six fields. The average number of cells counted per sample was approximately 2000. All counts were performed at 37°C. Motility was defined as any movement of the sperm head, and progressive motility was defined as the count of those spermatozoa that moved in a forward, linear direction at a speed of 50 µm/sec.
Freezing and Thawing Procedure
Sperm samples from individual males were distributed in aliquots of 100 µl into nine 1.8-ml cryotubes (Nunc Cryotubes, Roskilde, Denmark). After capping, eight vials were immediately placed in the vapor phase of a liquid nitrogen (LN2) storage container (
-120°C) for 10 min (descending cooling rate of -20 to -40°C per minute). After that time, the tubes were plunged into the liquid nitrogen (-196°C) for storage. The remaining unfrozen vial was used for a prefreeze quality control.
Frozen samples were rapidly thawed by transferring them from liquid nitrogen into a 37°C water bath for 2 min. Subsequently, the sample was centrifuged at 735 x g for 4 min. The supernatant (cryoprotectant) was discarded and replaced with 50 µl of HTF. The resuspended sperm sample was incubated for 10 min at 37°C to allow a minimal "swim up". The sample was gently mixed (by tapping the tube with the fingertips) before a 40-µl aliquot was taken for the IVF assay. The 10-µl remainder was utilized for motility analysis. The difference in the sample volume of frozen sperm (40 µl) compared with fresh sperm (10 µl) used in the IVF was an attempt to have at least a similar, if not a greater, number of frozen-thawed spermatozoa with progressive motility to compensate for the detrimental effect of freezing.
In Vitro Fertilization
Three to five 20- to 23-day-old inbred or hybrid female mice were superovulated by i.p. injection of 2.5 IU of eCG (Sigma) followed by 2.5 IU of hCG (Sigma) 48 h later for each replicate experiment. Thirteen hours after the second injection, the females were killed and their oviducts removed. The oocyte-cumulus complexes were isolated in a sterile culture dish containing 2 ml of HTF medium.
Forty microliters of the thawed sperm sample, with a minimum count of 1 000 000 progressively motile sperm per milliliter, were added to a 250-µl drop of HTF covered with light mineral oil (Sigma; embryo tested). The oocyte-cumulus complexes collected from three to five female mice were placed in each fertilization drop in which the sperm had already been incubated for at least 10 min. Dishes were placed in a sealed, modular incubator chamber (Billups-Rothenberg, Del Mar, CA) gassed with 5% CO2/5% O2 balanced in 90% N2 and maintained at 37°C for 5 h. After that time, eggs were washed to eliminate excess sperm and then cultured overnight in a 250-µl drop of HTF under same conditions. The next morning, the number of two-cell embryos was scored, and the embryos were either transferred to a 200-µl drop of KSOM/AA medium [43] (prepared from reagent-grade chemicals in our laboratory) for culture until the blastocyst stage or surgically transferred into a surrogate mother for in vivo evaluation. For each strain, a control IVF experiment was done using 10 µl of B6D2F1 fresh sperm collected in 1 ml of HTF. Fertility was considered to be the percentage of two-cell embryos produced by IVF scored 24 h after insemination. Each IVF was replicated three to five times using sperm from different males in each experiment.
Embryo Transfer
Forty-five two-cell embryos from each group were surgically transferred into the infundibulum of pseudopregnant CB6F1 female mice. In most cases, a total of 15 embryos per recipient were transferred using both uterine horns, having three female recipients per group. Embryos were allowed to develop to term, and the number of pups born was recorded.
Statistics
The total concentration number and the arcsine transformation of the motility and the progressive motility percentages of sperm from inbred and hybrid strain groups were evaluated by one-way ANOVA (two-tailed test). The analysis was corroborated by the Bartlett test for equal variances. The Dunnett multiple-comparison test was used to compare all inbred strains against the hybrid used as a control. The whiskers on the figures represent the distribution of each group with its corresponding median (50th percentile of the variable) and the extreme values of the population on each side. Fertility, which was considered to be the percentage of two cell-stage embryos scored 24 h after insemination, was transformed into arcsine values and then compared by paired Student's t-test and expressed as the mean ± SEM. Differences were considered to be significant when P < 0.05. All analyses were performed using the GraphPad Prism version 2.0 computer program (GraphPad Software, San Diego, CA).
RESULTS
Total Sperm Concentration Does Not Differ Between Inbred Strains and the Control Hybrid
The spermatozoa concentration from freshly collected sperm samples of eight different male mice of each strain was compared and is presented in Figure 1. The analysis of the means of sperm concentration from all inbred strains against the B6D2 F1 hybrid showed no significant differences (P > 0.05) within strains (Dunnett test). Despite the similar concentrations, variation was noticeable between male mice within same strain. C57BL/6J male mice, for example, showed sperm concentrations ranging from 13 x 106 ml-1 to 45 x 106 ml-1. A comparison between fresh and frozen sperm concentrations was not possible because of the different manipulations that each group received during the experiment.
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Sperm Motility Before and After Freezing Is Strain Dependent
The percentages of sperm motility and progressive motility both before and after freezing in the various strains are shown in Figures 2 and 3. Statistical analysis with arcsine-transformed data showed that the means of prefreeze motility of sperm from the inbred strains tested here are similar to those of the B6D2F1 control (Dunnett test). However, after freezing and thawing, only the sperm from the 129S3/SvImJ strain maintained their motility at a level similar to that of the control (P > 0.05). The motility of FVB/N sperm was marginally (P < 0.05) reduced, but for all other strains, motility was significantly (P 
0.01) reduced compared with that of the hybrid F1 control. The C57BL/6J, DBA/2J, and BALB/cJ strains also differed between their own prefreeze and post-thawed samples.
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Regarding progressive motility, only the means of BALB/cJ and 129S3/SvImJ sperm were different (lower; P 0.01) than the hybrid F1 control, whereas the rest were similar in this capacity before freezing. The analysis of the percentage of progressive motile sperm after thawing, however, showed that only the DBA/2J sperm did not differ from the control, whereas all others strains were significantly reduced in their rate (P 0.01).
In Vitro Fertilization Rates Depend on Genetic Background of the Sperm and Egg
To assess the relative contribution of the genetic background of sperm and oocyte to the strain-specific variations of IVF rates, we performed IVF using freshly collected, inbred sperm to fertilize hybrid oocytes; hybrid sperm to fertilize inbred oocytes; and inbred sperm to fertilize oocytes from the same inbred strain of mice. Figure 4 shows the results of this experiment, displaying the average of the fertilization rate (percentage of zygotes progressing to the two-cell stage) obtained under all possible gamete combinations from a minimum of five IVF tests. Each IVF was done using fresh sperm from a single male mouse and replicated three to five times for each strain. The results show that the IVF rate varied according to the genetic background of both the sperm and the egg. In general, hybrid sperm was better at fertilizing inbred eggs than inbred sperm were at fertilizing hybrid eggs, with the exception of FVB/N sperm. When inbred sperm was combined with oocytes from the same strain, the fertilization rates were the same or lower than the rate obtained with the same sperm inseminating hybrid CB6F1 oocytes, with the exemption of BALB/cJ sperm. Furthermore, not all strains involved in this study had good fertility in vitro; BALB/cJ sperm had the lowest fertility of those inbred strains that were tested.
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In Vitro Fertilization Rates Are Lower with Frozen Sperm from Inbred Strains
Having established a strain-dependent variability in IVF rates with fresh sperm, we next examined how strain differences might affect the ability of frozen sperm to fertilize eggs from the same strain. Because motility after thawing is strain dependent, we normalized our experiments by using the same number of progressively motile sperm from each strain for fertilization. Increasing the number of motile frozen-thawed sperm, however, did not increase the fertilization rate over that obtained with fresh sperm. For example, frozen C57BL/6J sperm had 18.5 x 104 ml-1 progressive motile spermatozoa in an IVF drop, whereas a drop with fresh sperm had 10.2 x 104 ml-1. Nevertheless, the fertility of the frozen sperm was still significantly lower (P < 0.01). These results, represented in Figure 5, show that the fertilization rate of frozen sperm from inbred strains was markedly reduced from that of fresh sperm (P < 0.01). This contrasts markedly with results from the hybrid strain, in which no difference was seen between fresh and frozen sperm.
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In Vitro Culture of Embryos Derived from Fresh vs. Frozen Sperm
Because many embryos are lost during the preimplantation stages after the two-cell cleavage, we examined the percentage of two-cell embryos that progressed to the blastocyst stage in culture for embryos that were produced by IVF with both fresh and frozen sperm (Table 1). Although fertilization rates of fresh and frozen sperm from hybrid F1 mice showed essentially no differences at either the two-cell or blastocyst stage, an effect was seen with the inbred strains. Not only was there a greater failure to develop to the blastocyst stages after fertilization with inbred frozen sperm, but even under fresh, normal control conditions, fewer embryos from inbred strains completed the transition from the two-cell to the blastocyst stage compared with hybrid embryos.
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Newborn Mice Derived from Fresh or Frozen Sperm
To assess whether embryos produced by IVF with either fresh or frozen sperm support development to term, 45 embryos from each strain (when available) were transferred to pseudopregnant CB6F1 foster mothers, and the number of pups born were counted. The following numbers of offspring were produced with fresh and frozen sperm, respectively: CB6F1, 69% (31/45) and 64% (29/45); C57BL/6J, 15% (7/45) and 30% (9/30); DBA/2J, 15% (7/45) and 11% (5/45); BALB/cJ, 44% (20/45) and 28% (4/14); 129S3/SvImJ, 24% (11/45) and 20% (8/40); and FVB/NJ, 40% (18/45) and 13% (6/45).
DISCUSSION
The results of this study show that regardless of the strain, mouse sperm can be successfully cryopreserved, and that motility can be retained after thawing. However, as described for other mammals [44], the number of motile cells was reduced almost 50% after cryopreservation.
Cryopreservation of sperm from inbred mouse strains is not as successful as that with hybrid F1 sperm, perhaps because of the strain-dependent susceptibility to damage by freezing. Compared with those of the hybrid control, the motility and progressive motility rates of inbred sperm diminish after cryopreservation. Furthermore, inbred sperm produce lower IVF rates with frozen than with nonfrozen sperm, and the fertility rate varies depending on the background of the oocytes used. Nevertheless, we could obtain inbred newborns from oocytes fertilized in vitro with frozen sperm from all strains involved in this study.
To our knowledge, few cryobiology studies have been published on inbred mouse sperm [35–39], and in most of these, the inbred sperm were used to inseminate hybrid eggs. Tada et al. [39] described IVF using inbred frozen sperm inseminating inbred oocytes; however, this work was later described as being impossible to repeat [2–4]. The C57BL/6J mouse strain was the most commonly used in those latter studies. The fertility rate obtained when using frozen sperm from this strain is low, between 0% [36] and 40% [37] using hybrid eggs and 12% [45] using C57BL/6J oocytes. The variation of the fertility rate in those studies could relate to the use of different freezing protocols, different cryoprotectant solutions (one contained glycerol and raffinose [36] and one containing raffinose alone [37]), and different IVF protocols. One simple technique to overcome the low in vitro fertility of frozen C57BL/6J sperm was to make an incision in the oocyte zona pellucida [45].
One goal of this study was to determine whether the variability of success in mouse sperm cryopreservation depends more on the mouse strain background than on the freezing methodology. The freezing method, at least under the technical protocol used in this report, seems to affect fertility more than the motility of the mouse spermatozoa. We corroborated that the genetic background of the sperm influences the IVF rates among the different mouse inbred strains [21, 22, 28]. We concluded also that the genetic background of the gametes influences the fertility rate. The insemination of inbred oocytes with hybrid sperm gives higher rates than the reciprocal condition. Some types of experiments must use this combination, for example, in the analysis of the maternal effect on fertilization [46], in which oocytes from 55 mouse strains were tested with the same hybrid sperm. Nevertheless, when hybrid eggs were inseminated with inbred sperm, with the exception of BALB/cJ, the fertility rate obtained was similar to that using both inbred gametes, which is perhaps an indication that the fertility of the strain is determined by the fertility of the spermatozoa.
The use of a skim milk/raffinose cryoprotectant results in a reliable protocol for mouse sperm cryopreservation for all strains, because the motile sperm count obtained after thawing was within the expected range for each strain. A common misinterpretation in sperm cryopreservation is to link the number of surviving spermatozoa after freezing and thawing with fertility, which is a characteristic of the mouse strain [12]. Nonetheless, fertility does not always correlate with the sperm concentration or with their total motile and progressive fraction counts. For example, male mice from the FVB/N mouse strain have a low sperm concentration but high fertility, which is maintained through the freezing process. The opposite is observed in the C57BL/6J strain, in which the fertility rate is not paired even when matching the number of progressive motile sperm between prefreeze and post-thawed samples.
Some inbred strains have poor reproductive performance both in vivo and in vitro; an example is the BALB/cJ stain, in which the problem relates to the elevated number of abnormal spermatozoa [19, 47, 48]. The frozen sperm from the C57BL/6 and 129S3/SvImJ strains, both of which are used extensively for the generation of targeted mutations, produced the lowest IVF rates from all tested strains. Hence, cryopreserved sperm will, at best, maintain—if not diminish—the normal fertility level of a given mouse strain, and by no means will this level be improved without the aid of special techniques. Two such techniques are zona nicking [45], which was reported to increase the fertility of cryopreserved transgenic C57BL/6J mouse sperm from 12% to 70%, and intracytoplasmic sperm injection [2], which is a difficult, assisted reproduction technique that enables use of dead sperm for fertilization. Therefore, when using standard IVF techniques, it is important to note the reproductive parameters of the strains to be preserved before cryopreservation.
In conclusion, the protocol using skim-milk/raffinose is a reliable method for banking sperm from a variety of mouse strains, although sperm from some inbred strains have better survival regardless of the protocol used for cryopreservation [36, 37]. The spermatozoa from all the inbred strains involved in this study retained some degree of fertility after freezing and thawing; however, the fertility rate largely depended on the strain from which the sperm was collected and on the genetic background of the oocyte selected for IVF.
These results add to our knowledge regarding the existence of considerable reproductive differences between strains [12], establishing that the use of frozen sperm for IVF produces a greater variation in fertilization rates than that seen with fresh sperm. These observations warrant continued optimization of IVF conditions for the different inbred strains and, at the same time, characterization of the damage that spermatozoa undergo during cryopreservation.
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ACKNOWLEDGMENTS
The authors thank Dr. Carlisle Landel for review, comments, and discussions on this manuscript and Dr. Kathleen Kerr for guidance and advice on statistical analysis.
FOOTNOTES
1 Supported by National Institutes of Health grants RR09781 and RR01262 and as part of the NICHD/NCRR National Cooperative Program on Mouse Sperm Cryopreservation to L.E.M. through Cooperative Agreement RR15012. 
2 Correspondence: Jorge M. Sztein, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. FAX: 207 288 6149; jms{at}jax.org
Accepted: July 28, 2000.
Received: May 10, 2000.
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 H. Kusakabe, M. A. Szczygiel, D. G. Whittingham, and R. Yanagimachi Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa PNAS, November 9, 2001; (2001) 241517598. [Abstract] [Full Text] [PDF]
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H. Kusakabe, M. A. Szczygiel, D. G. Whittingham, and R. Yanagimachi Inaugural Article: Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa PNAS, November 20, 2001; 98(24): 13501 - 13506. [Abstract] [Full Text] [PDF]
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