A study on freeze-drying as a method of preserving mouse sperm

This review describes the study of freeze-dried mouse sperm for practical application in preserving and transporting genetic resources. Freeze-dried sperm can be used to preserve and transport genetic resources; however, there still remain many areas which need to be studied. In particular, it is essential to assure long-term preservation over several decades or centuries. Recently, the theory of accelerated degradation kinetics to freeze-dried mouse sperm has been applied, and found that long-term preservation by conventional methods requires temperatures lower than -80 C. When the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse sperm was examined, a pressure of 0.37 mbar at primary drying significantly improved the developmental rate to the blastocyst stage. In addition, it has been shown that freeze-dried sperm stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. Sperm chromatin structure assay (SCSA) was applied to mouse sperm freeze-dried under several conditions and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. Furthermore, SCSA might be useful for estimation of developmental potential of fertilized eggs derived from ICSI using freeze-dried sperm in mice.     

Long-term preservation of mouse spermatozoa as frozen testicular sections

We previously demonstrated that testicular spermatozoa can be preserved as frozen testicular sections, allowing us to preserve male gametes in less space than conventional methods. However, it remains unclear whether the testicular spermatozoa can be preserved for a long period using this procedure. In this study, we examined the function of testicular spermatozoa preserved as frozen testicular sections for l year at -30 or -80 C. Testicular spermatozoa were successfully retrieved from frozen testicular sections preserved at either -30 or -80 C, and their function was assessed using intracytoplasmic sperm injection (ICSI). Over 90% of the oocytes injected with long-term preserved testicular spermatozoa formed pronuclei, which was a frequency similar to that obtained with spermatozoa preserved for a short term, indicating that the testicular spermatozoa retained oocyte activation factor(s). Approximately 70% of the fertilized oocytes developed to 2-cell stage embryos, and 9.3 to 12.8% of the embryos developed to term after transfer into pseudopregnant females, regardless of the preservation temperatures examined. These results indicate that the birthrates of progeny did not differ between the preservation temperatures examined. They also indicate that male gametes can be preserved in testicular frozen sections for at least 1 year without loss of function.     

Nuclear transfer preserves the nuclear genome of freeze-dried mouse cells

Mouse spermatozoa can be freeze dried without losing genetic integrity and reproductive potential. However, it is not known if freeze-dried mouse cells similarly maintain their genetic integrity and developmental potential following nuclear transfer. Here, we investigated the developmental capacity and embryonic stem (ES) cell derivation of reconstructed oocytes by nuclear transfer using freeze-dried cumulus or ES cells. Cumulus and ES cells were lyophilized overnight and stored at 4 C for up to 1 week. After rehydration, all cells showed membrane damage and were unviable. However, following nuclear transfer, 1-4% of the reconstructed oocytes developed to the blastocyst stage. A total of five nuclear transfer ES (ntES) cell lines were generated from blastocysts and morulae. All ntES cell lines had normal karyotypes and were positive for the ES-cell-specific markers (alkaline phosphatase, Oct3/4 and Nanog). After aggregation of ntES cells with fertilized embryos, chimeric mice with a high level of coat color chimerism were generated. Our findings show that the genomic integrity of cells can be maintained after freeze-drying and that it is possible to produce offspring from the cells using nuclear transfer techniques.     

Challenging endeavour for preservation of freeze-dried mammalian spermatozoa

Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials and the potential risk of total loss of the frozen stock. Since freeze-dried spermatozoa after rehydration lose their motility, which is an essential requirement to complete physiological fertilization, a relatively difficult microinsemination technique must be applied to rehydrated spermatozoa. Theoretically, it has been supposed that freeze-dried spermatozoa could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator temperature. However, sufficient yield of transferable blastocysts and production of live offspring derived from freeze-dried sperm samples are still subjects to be challenged and overcome in large domestic species.     

The freeze-drying of Cowdria ruminantium

Lyophilized tissues of mice and blood of sheep, infected with either the Kümm, the Welgevonden or the Ball 3 stock of Cowdria ruminantium, remain infective to mice and sheep after storage at 4 degree C for 90 days. Freeze-dried tissues stored at -18 degrees C and -28 degrees C are still infective after 6 months and 2 years, respectively.     

Piezo-assisted in vitro fertilization of mouse oocytes with spermatozoa retrieved from epididymides stored at 4 degree C

This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa.     

Nuclear Transfer of Freeze-Dried Somatic Cells into Enucleated Sheep Oocytes

Lyophilization has been used since long time to preserve yeast and bacteria strains. Subsequently, a great deal of efforts has been dedicated to the preservation in a dry state of red blood cells and platelets. However, despite more than 30 years passed by, no significant progress has been achieved. Recently, it has been reported that freeze-dried mice spermatozoa were able to generate normal offspring following injection into the mature mice oocytes. In this work, we prompted to apply the lyophilization protocol developed for mice spermatozoa to sheep somatic cells (lymphocytes and granulosa cells). More than 350 enucleated sheep oocytes were injected with granulosa cells, and freeze dried using the protocol developed for mice sperm cells. Transplanted nuclei organized large pronuclei with fragmented DNA, but none of them entered the first mitosis. In the second part of the experiments, trehalose and EGTA were found to reduce significantly the extent of nuclear damage (65% and 55% intact nuclei in lymphocyte and granulosa cells, respectively) following freeze drying. Granulosa cells lyophilized with EGTA/trehalose and stored at room temperature for 3 years were used for nuclear transfer, and the injected oocytes were cultured in vitro for 7 days. Approximately 16% of the oocyte injected with freeze-dried cells developed into blastocysts. To conclude, we demonstrated for the first time that nucleated cells maintain genomic integrity after prolonged storage in a dry state, and we were able to achieve early embryonic development following injection of these cells into enucleated sheep oocytes.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐SM and 15‐SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5‐SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐SM and 15‐SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5‐SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.     

Boar variability affects sperm metabolism activity in liquid stored semen at 5 degrees C

Metabolic activity of boar spermatozoa, liquid stored for three days at 5 degrees C, was measured using bioluminescence for ATP content, fluorescent assay (JC fluorochrome) of mitochondrial activity and oxygen consumption. Sperm motility and plasma membrane integrity (PMI) were simultaneously analyzed. Apart from the statistically significant effect (P < 0.001) of semen storage time, the importance of the individual source of the ejaculate for the analyzed parameters of metabolic efficiency of spermatozoa was shown. This phenomenon was manifested in the interaction of the individual source of the ejaculate with spermatozoa motility, integrity of their membranes and metabolic activity with the passing time of semen preservation. Recorded results indicate that the individual factor may have a significant influence on the technological usefulness of boar spermatozoa for liquid storage. Quality analyses conducted on boar semen stored at 5 degrees C may be used for pre-selection of boars producing sperm with an enhanced tolerance to cold shock.     

Hypothermic storage of equine isolated hepatocytes

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.     

Suitability of canine herpesvirus as a vector for oral bait vaccination of foxes

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.     

Comparison of Ham's F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H

Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embryos cultured in Dulbecco's phosphate buffered saline + 10% FCS and transferred less than 1 h after collection were used as controls. Pregnancy rates were higher (P less than .05) for embryos stored in Ham's F10 + CO2 (70%, 55%) than for embryos stored in Ham's F10 + Hepes (20%, 15%) at 14 and 35 d, respectively. At 14 d, pregnancy rates for control embryos (90%) were similar (P greater than .05) to pregnancy rates for embryos cultured in Ham's F10 + CO2 (70%); however, by 35 d, pregnancy rates were higher (P less than .05) for controls (80%) than for embryos stored in Ham's F10 + CO2 (55%). It was concluded that Ham's F10 + CO2 was superior to Ham's F10 + Hepes for short-term storage of equine embryos at 5 C, and that satisfactory pregnancy rates could be obtained from transfer of embryos stored in Ham's F10 + CO2 at 5 C for 24 h.     

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.     

Storage of Fresh Bovine Semen in a Diluent Based on the Ionic Composition of Cauda Epididymal Plasma

For artificial insemination (AI) in cattle, much lower insemination doses can be applied when fresh semen is used instead of frozen-thawed semen. However, a particular disadvantage of fresh semen is its limited shelf life. As bovine spermatozoa can be stored for several weeks in the cauda epididymis without negative effects on their fertilizing capacity, it is an interesting organ to serve as a model in order to prolong the shelf life of fresh semen. First, the storage capacity of a diluent [cauda epididymal plasma (CEP-1)] with the same ionic composition, pH and osmolarity as the bovine CEP was compared with a Tris diluent for extended preservation of fresh ejaculated bovine semen. Secondly, the ionic composition of the CEP-1 diluent was modified (CEP-2) and its storage capacity was compared with this of the CEP-1 and Tris diluent. Finally, the effect of addition of different polyols (sorbitol, glycerol, mannitol) and egg yolk concentrations (5, 10 and 20%) to the CEP-2 diluent was assessed. Sperm quality decreased rapidly in the CEP-1 diluent. The quality and especially progressive motility of spermatozoa stored in the CEP-2 diluent were better those in the CEP-1 and Tris diluent. No significant effects of different sugars or egg yolk concentrations on the quality of fresh bovine semen in the CEP-2 diluent were observed. In conclusion, the CEP-2 diluent with 10% egg yolk and 1 g/l sorbitol may be used for extended preservation of fresh bovine semen at 5 degrees C up to 6 days.     

Viability and nematophagous activity of the freeze-dried fungus Arthrobotrys robusta against Ancylostoma spp. infective larvae in dogs

Viability and in vitro and in vivo activities of freeze-dried conidia of the predatory fungus Arthrobotrys robusta (I-31) were evaluated against infective larvae (L(3)) of Ancylostoma spp. in dogs. A. robusta conidia were lyophilized and stored at 4°C for a month. Freeze-dried conidia were diluted to 1×10(3)conidia/ml and tested in vivo. The treated group consisted of a solution containing conidia (1ml) and 1000 Ancylostoma spp. (L(3)) placed on Petri dishes plated with 2% water-agar (2% WA), at 25°C, in the dark for 10 days. The control group consisted of 1000 Ancylostoma spp. L(3), plated on 2% WA. After 10 days, Ancylostoma spp. L(3) from both the treated and the control groups were recovered and counted. The in vivo test was performed on two dogs by administering a single oral dose of freeze-dried conidia (1.5×10(5)) in aqueous solution to one animal and only water to the other. Fecal samples were collected at 12, 24 and 48h after the treatments, plated 2% WA plates and incubated at 25°C for 15 days. A thousand Ancylostoma spp. L(3) larvae were spread on these plates. At day 15, infective L(3) recovered from the treated and control groups were counted. In the in vitro test, A. robusta was able to survive the freeze-drying process, grow in the plates, form traps and capture Ancylostoma spp. L(3). There was a 75.38% decrease in the number of infective larvae recovered from the treated group. The in vivo test showed that freeze-dried A. robusta conidia survived the passage through the gastrointestinal tract of the treated dog, was able to grow in the plates and capture Ancylostoma spp. L(3), reducing the number of recovered L(3) (p<0.01). Freeze-drying can be an alternative method for conservation of conidia of nematophagous fungi.     

干扰素制剂冻干技术研究进展

干扰素制剂具有广谱的抗病毒和免疫调节活性,在医学和兽医临床上具有广阔的应用前景。但在实际生产中,液体剂型干扰素制剂存在热稳定性差、不易长期保存和远距离运输等问题,冻干后则可延长保存期、增加稳定性。作者主要对冻干技术的原理、干扰素冻干保护剂和冻干工艺的筛选等进行了综述。     

Effect of chilling temperature on the long-term survival of rabbit spermatozoa held either in a tris-based or a jellified extender

As the preservation of the fertilizing capacity of rabbit spermatozoa for several days after semen collection remains a major target for the artificial insemination programs of rabbit breeding, a study was conducted to compare the efficacy of 5 or 15°C as holding temperature in lengthening the preservability of rabbit semen quality during 192 h of storage both in a solid (Cunigel) and a liquid (Tris-Citric acid-Glucose; TCG) extender. Six pooled semen samples (two ejaculates/male; two-three males/pool) were taken and made four aliquots: two aliquots were tenfold diluted with the TCG extender, whereas the other two were tenfold diluted with the Cunigel extender. One aliquot per diluent was stored at 5°C and the second one at 15°C. Sperm motility (light microscope), viability (SyBr-PI staining), plasma membrane functional integrity (Hypo-osmotic swelling test) and acrosome integrity (PSA-FITC staining) were recorded at 0, 48, 120 and 192 h of storage. In liquid-stored spermatozoa, mass motility and viability were significantly higher (p ≤ 0.05) in samples stored at 5°C than at 15°C at all the storage times; at 5°C resulted also higher (p ≤ 0.05) the percentages of both forward motility at 48 h and sperm functional integrity at 120 and 192 h of storage, whereas chilling temperature did not affect acrosome integrity. With the Cunigel extender, all the semen qualitative parameters were significantly higher in sample stored at 5 than 15°C over storage time (p ≤ 0.05); only acrosome integrity at 192 h was not different according to the chilling temperatures. In conclusion, 5°C were better than 15°C for the long-term storage of rabbit semen both in the TCG and Cunigel extender.     

A Comparison of the Ability of Three Commercially Available Diluents to Maintain the Motility of Cold Stored Stallion Semen

The objective of this study was to compare the ability of three commercially available extenders to promote poststorage motility of stallion spermatozoa stored at 5°C with and without centrifugation to remove the seminal plasma. Diluents tested included skim milk glucose (SKMG), INRA 96, and VMD-Z. All diluents were tested with (-SP) and without (+SP) centrifugation to remove most of the seminal plasma. In experiment I, after 48 and 72 hours of storage, total (TM) and progressive (PM) motility values were higher (P ≤.05) for those aliquots subjected to the INRA 96-SP as compared with either SKMG treatment. After 72 hours of storage, PM of spermatozoa stored in VMD-Z-SP was superior to that of spermatozoa stored in SKMG regardless of the presence of seminal plasma (P ≤.05). In the second experiment, after 48 hours of storage, PM of spermatozoa subjected to the INRA 96-SP and VMD-Z-SP treatments were superior (P ≤.05) to those for all treatments that had been stored without removal of seminal plasma. Removal of the seminal plasma and resuspension of the sperm pellet with either INRA 96 or VMD-Z resulted in TM after 48 hours of storage that were similar to those obtained after 24 hours of storage.     

Virulence stability in Flavobacterium psychrophilum after storage and preservation according to different procedures

Experimental infections and lethal dose 50% (LD 50) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 degrees C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 degrees C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 degrees C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD0. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 degrees C were reliable methods for long-term preservation of virulence.