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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
National Animal Germplasm Program, National Center for Genetic Resources Preservation, Fort Collins, CO 80521
2 Corresponding author: Harvey.Blackburn{at}ars.usda.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05). The highest level (183.6 ± 28.4) of viable PGC per individual embryo was observed for 10% EG with 10E and was significantly higher (P 0.05) than cryopreservation in 2.5% DMSO with 10E and 20 embryos, 2.5% EG with 10E, 5% EG with 10E, and all 0% cryoprotectant treatments. No statistical interaction (P > 0.05) was observed for the percentage of viable PGC. However, the highest percentage (80.6%) was observed at 10% EG with 10E. It was demonstrated that PGC were successfully frozen, and the most effective treatment was 10% EG with 10 embryos/straw.
Key Words: primordial germ cell • cryopreservation • flow cytometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Currently, there are many specialized research lines of poultry threatened with extinction (Fulton and Delany, 2003). In addition, the biosecurity of commercial industry poultry stocks is at risk from economic pressures and potential disease epidemics (Blackburn, 2006). Although poultry genetic resources are contracting, consumer demand for specialized poultry products is increasing, and the industry may need novel genetic resources in the future. For example, there is evidence that consumer preferences for nontraditional turkey varieties is growing with the sale of approximately 50,000 heritage breed turkeys in 2004 and 2005 (D. Bixby, American Livestock Breeds Conservancy, Pittsboro, NC, personal communication). Also, the recent concern about highly virulent strains of avian influenza poses a threat to the global poultry industry. If an outbreak was to occur in a genetically sensitive geographic area for the poultry industry, entire flocks of parent stock may be at risk. Such situations make it imperative to protect the current genetic variation in all poultry stocks.
Currently, cryopreservation of poultry semen has been the only avenue for ex situ conservation (Bakst, 1990; Buss, 1993; Hammerstedt, 1995). However, the fertility of frozen or thawed poultry semen lags behind other species (Chalah et al., 1999) and requires significant improvement for industry use. In addition, a major disadvantage to semen cryoperservation is that mitochondrial DNA and genes on the W chromosome cannot be captured, because the male is the homogametic sex with ZZ chromosomes. Therefore, it is impossible to recreate a chicken line from cryopreserved semen without an extensive and multigenerational breeding program. On the other hand, the cryopreservation of PGC would facilitate the capture of the entire genetics of the stock and would potentially enable the reconstitution of a desired line within 2 generations through the creation of germline chimeras. Germline chimeras can be created from blastodermal cells (Petitte et al., 1990), fresh PGC (Tajima et al., 1993; Naito et al., 1994b), or frozen or thawed PGC (Naito et al., 1994a; Tajima et al., 1998, 2003, 2004). Although the previous work provides evidence that PGC can be harvested, stored, and reintroduced to recipient embryos, the procedures are inefficient. Hence, the exploration of various freezing protocols could lead to a more efficient cryopreservation process. The current study examined the cryoprotectants dimethyl sulfoxide (DMSO) and ethylene glycol (EG), the concentration of these cryoprotectants, and PGC pooled from multiple embryos to develop a reliable method for storing chicken PGC in liquid N.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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White Leghorn chicken embryos from the Hy-Line International (West Des Moines, IA) line W-36 were used in this experiment. Stage 27 embryos (Hamburger and Hamilton, 1951) were collected by making a hole in the blunt end of the egg, removing the embryo from the yolk sac, and placing it in a petri dish containing pH 7.4 PBS (8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 to make 1 L of PBS). Sex of the embryos was not determined. Once the embryo was rinsed, it was placed under a dissection microscope in a petri dish containing black dissecting wax and PBS. The embryos were pinned to the dissection wax, and the gonads were removed with fine-tipped forceps. The gonads were placed into a siliconized 1.5-mL centrifuge tube containing 500 µL of room-temperature Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) containing 10% premium fetal bovine serum (PFBS; Atlanta Biologics, GA). Gonad pairs from 5 (5E), 10 (10E), or 20 (20E) embryos were pooled in the tube. Embryos were pooled due to the low number of PGC usually recovered per embryo, as found by previous investigators [706 ± 41 PGC/embryo (Mozdziak et al., 2005) and ~800 PGC/embryo (Allioli et al., 1994)], and the need to have sufficient amounts of PGC from gonadal pairs for freezing that could be efficiently collected. Tubes were centrifuged at 600 x g for 5 min, and the supernatant was discarded. Room-temperature 0.25% trypsin EDTA (200 µL, Sigma-Aldrich, St. Louis, MO) was added to the tube and incubated at 37°C for 20 min. Gonads were teased until the homogenate remained, which was then subsequently removed. The trypsin was deactivated with 200 µL of DMEM containing 20% PFBS. The suspension was then passed through a 30-µL filter into a clean 1.5-mL siliconized centrifuge tube. The original tube was washed and collected twice with 200 µM of DMEM containing 20% PFBS and was then washed and collected twice with 200 µL of 0% PFBS DMEM. The suspension was centrifuged, the supernatant discarded, and 500 µL of Media 199 (Gibco) containing 20% embryonic stem cell-qualified FBS (ESFBS; Gibco) was added to the tube in preparation for freezing.
PGC Cryopreservation
The entire gonadal cell suspension was placed in a cold room at 4°C for 1 h and then centrifuged (600 x g for 5 min). The supernatant was removed, and the sample was diluted with 150 µL of freeze Media 199 containing 20% ESFBS and one of the following cryoprotectant treatments (final concentrations; vol:vol): 2.5% DMSO, 5% DMSO, 10% DMSO, 2.5% EG, 5% EG, 10% EG, or the control (0% cryoprotectant). The cryopreservation media were added drop-wise to the pellets containing all gonadal cells, including PGC, which resulted in each straw containing 1 of the 7 cryoprotectant treatments and 1 of the 3 embryo (5E-, 10E-, or 20E-donating cells/straw) treatments. The cell suspensions were loaded into labeled 0.5-mL Cryo BioSystem straws (Cryo BioSystem, Paris, France). The straws were sealed and frozen using a programmable freezer (Mini Digitcool UJ40, Cryo BioSystem) that decreased temperature at 1°C/min from 5 to –85°C. The straws were plunged into liquid N for storage. Straws were frozen for 48 h to 5 mo before analysis.
Flow Cytometry
Three straws from each combination of cryoprotectant and embryo number were thawed for sampling for a total of 63 straws analyzed. Straws were thawed in a 37°C water bath for 30 s. The straw was emptied into a clean 1.5-mL siliconized centrifuge tube. Two rinses of 200 µL of room-temperature Media 199 containing 20% ESFBS were passed through each straw, followed by a passage of air to empty the contents of the straw into the tube. The tube was centrifuged (600 x g for 5 min), and 500 µL of cold (5°C) Media 199 containing 20% ESFBS was added to the tube. The mouse monoclonal antibody stage-specific embryonic antigen-1 (SSEA-1; Developmental Studies Hybridoma Bank, Iowa City, IA) IgM was used to identify gonadal PGC by flow cytometry. Stage-specific embryonic antigen-1 is a carbohydrate epitope that is involved with PGC adhesion (Solter and Knowles, 1978; Gooi et al., 1981). Stage-specific embryonic antigen-1 binds specifically to germ cells in chicken embryos beyond Hamburger and Hamilton stage 10 (Pain et al., 1996) and has been successfully used to sort chicken gonadal PGC using fluorescence-activated cell sorting (Mozdziak et al., 2005). Anti-SSEA-1 was added to the cell suspension at a dilution of 1:60 and incubated on ice for 1 h. The tube was then washed once with cold PBS. Goat anti-mouse IgM fluorescein isothyiocynate; (FITC; Sigma-Ald-rich) was added to the tube at a dilution of 1:60 and incubated for 30 min on ice. The suspension was washed twice with cold PBS and then finally resuspended in 500 µL of cold PBS. Propidium iodide (PI; 50 µg/mL in PBS; Sigma-Aldrich) was added to the suspension (1:20) to label all cells with compromised plasma membranes and was incubated for 5 min on ice. Verification of PGC staining was observed under a Nikon Eclipse E600W microscope (Nikon USA, Medville, NY) equipped with a FITC filter set. Flow cytometry was performed on a CyAN ADP flow cytometer (DakoCytomation, Fort Collins, CO) to evaluate all cell suspensions. The flow cytometer was equipped with an Ar laser (488 nm) at 20 mW of power. The entire volume of the sample was analyzed using the FL-1 detector (530-nm band pass filter) to detect FITC and the FL-3 detector (613-nm band pass filter) to detect PI so that the viable population of PGC could be determined. The auto compensation function of the flow cytometer was used.
Statistical Analysis
The GLM procedure of SAS (SAS Institute, 1985) was used to perform a 3 x 7 factorial analysis with 2 main effects: number of embryos contributing PGC (5E, 10E, and 20E) and cryoprotectant level used (2.5% DMSO, 5% DMSO, 10% DMSO, 2.5% EG, 5% EG, 10% EG, and 0% cryoprotectant) and the interaction of the main effects. The interaction and main effect means were separated using least square means, with significance accepted at P 0.05, unless otherwise stated.
For each combination of embryos per straw and cryoprotectant, 3 straws were measured using flow cytometry (Figure 1
). For each straw, the following parameters were determined: the number of viable, nonviable, and total PGC; the number of viable, nonviable, and total PGC per individual embryo; cell concentration (cells/mL) of each straw; and the percentage of viable PGC to overall PGC.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differences among the number of embryos (5E, 10E, 20E) donating PGC/straw were determined for each parameter studied. As expected, 5E produced the lowest (604.4 ± 68.4) total amount of PGC, whereas 20E produced the highest (1,881.9 ± 219.3) number of overall PGC (P < 0.05). The 20E treatment had the highest number of nonviable PGC (522.5 ± 43.3) due to a larger total number of PGC.
It was also important to determine the percentage of all PGC (Figure 2) that was viable to select the best treatment of storage. The 20E treatment was shown to have the highest percentage of viable PGC (67.8% ± 2.9) when compared with the 60.3% ± 2.8 viability provided by the 5E treatment (P < 0.05). The 10E treatment resulted in 64.2% ± 3.3 viable PGC, which was not different (P > 0.05) from the other treatments.
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Cryoprotectant
The highest total number of PGC was 1,887.9 ± 398.8 from 10% EG, which was significantly different (P < 0.05) from the control and 2.5% DMSO. The lowest percentage of viable PGC (Figure 3) was the control with 44.4% ± 3.42, whereas 10% EG (74.3% ± 3.3) and 10% DMSO (76.5% ± 1.5) had the highest percentage of viable PGC and were significantly (P < 0.05) different from the control, 2.5% EG, and 2.5% DMSO.
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Interaction effects between cryoprotectant and the number of embryos donating PGC to the straw were found for the number of viable PGC (P < 0.06), viable PGC per individual embryo (P < 0.05), and total PGC per individual embryo (P < 0.06). The total PGC per individual embryo were highest in the 10% EG 10E (225.3 ± 22.3) and 5% DMSO 10E treatments (225.3 ± 102.2) and were significantly greater (P < 0.05) than the 2.5% DMSO 10E treatment and all of the control treatments (Figure 4, panels A and B). The 10% EG 20E and 10% DMSO 20E treatments produced the highest number of viable PGC, although these treatments did not differ significantly (P < 0.05) from 10% DMSO 10E, 10% EG 10E, 2.5% DMSO 20E, 2.5% EG 20E, 5% DMSO 10E and 20E, and 5% EG 20E (Figure 5, panels A and B). However, the number of viable PGC per individual embryo and the percentage of PGC that are viable may be more informative criteria, because these variables normalize the data and are indicators of cryopreservation efficiency. The highest number of viable PGC per individual embryo was the 10% EG 10E (183.6 ± 28.4) treatment, which was significantly higher (P < 0.05) than 2.5% DMSO 10E (20.6 ± 6.8) and 20E (40.3 ± 12.0), 2.5% EG 10E (40.5 ± 7.9), 5% EG 10E (28 ± 5.1), and all control treatments (Figure 6, panels A and B). No interaction effect (P > 0.05) was observed for the percentage of viable PGC that would demonstrate optimum post-thaw survivability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We also examined 2 different cryoprotectants and a range of concentrations of each. Dimethyl sulfoxide was used due to its wide use as a cryoprotectant and because it was previously used to freeze PGC (Naito et al., 1994a; Tajima et al., 1998, 2003, 2004). Ethylene glycol was used because it was recently determined to be a superior cryoprotectant to DMSO (Kobayashi et al., 2003). The 10% EG treatment consistently produced favorable results and had the second highest percentage of viable PGC (74.3% ± 3.3 vs. 76.8% ± 1.5 for 10% DMSO). Interestingly, similar results for freezing trout PGC with EG have been observed (73% viability; Kobayashi et al., 2003).
The determination of an optimum treatment of freezing chicken PGC is critically important. The number of viable PGC per individual embryo is a parameter that allows the efficiency of each treatment to be uniformly evaluated. Using this parameter, the best option for freezing PGC is the 10% EG 10E treatment. This treatment also resulted in the overall highest percentage of PGC that was viable postthaw; however, there was not a significant interaction effect probably due to the sample size.
Other studies that have frozen chicken PGC used 10% DMSO as the cryoprotectant (Naito et al., 1994a; Tajima et al., 1998, 2003, 2004), and only 1 study (Naito et al., 1994a) determined a postthaw PGC viability. The trypan blue exclusion test is commonly used to determine the viability of frozen or thawed cells. However, as indicated previously, PGC have a tendency to aggregate, and a small percentage of the population examined under a slide, as in the trypan blue exclusion test, may not be indicative of the entire PGC population, because most PGC may not reside in the area of the cell suspension where the sample was taken. Also, using the trypan blue exclusion test, PGC must be identified morphologically. The use of the flow cytometer allows immunohistochemistry to label the PGC and the use of PI exclusion to determine if the labeled cells are viable. Flow cytometry also enables the entire cell population to be examined, which reduces error of evaluating PGC because of their location in suspension.
The overall number of PGC counted per individual embryo in this experiment was lower than the number of gonadal PGC counted in other experiments. For example, in this experiment, 10% EG 10E produced 225.3 ± 22.3 total PGC per individual embryo compared with 706 ± 41 PGC per individual embryo (Mozdziak et al., 2005) and ~800 PGC per individual embryo (Allioli et al., 1994). In the current experiment, PI exclusion was used to separate the viable population of PGC from the moribund and dead cells. The nonviable PGC population was determined to be the population that was dual-stained with both PI and FITC. It is possible that some nonviable PGC were counted in the nondual-stained PI population due to the subjectivity involved in gating cells. Therefore, the total PGC count per individual embryo would appear lower than in previous research. In addition, the studies reporting higher numbers of total PGC per individual embryo were quantified using fresh PGC rather than frozen PGC. The extra steps involved with freezing the PGC and storage within the straw may result in the loss of PGC during handling. Also, during the freezing process, the carbohydrate epitope SSEA-1 found on the surface of PGC may be reduced, resulting in decreased cell labeling. Using the current practice of creating germline chimeras with 100 cells from a cell suspension containing PGC or using 100 hand-picked PGC, 1 straw from the most efficient treatment of producing viable PGC per individual embryo (10% EG 10E) that averages 1,836 viable PGC could be used to inject over 18 embryos.
Another important aspect of this study was the methodology used to prepare the cell suspension for analysis by flow cytometry. This same methodology could be used to sort viable PGC from a frozen or thawed gonadal cell suspension, allowing small numbers of PGC to be frozen. The ability to freeze small numbers of PGC found in a gonadal cell suspension from 10 embryos compared with freezing a pure population of PGC makes it more feasible to store PGC from full siblings when compared with using more embryos per straw. Also, the difference between injecting these PGC vs. hand-picked PGC is that the use of PI exclusion ensures that the PGC being used are viable and may result in an increase in germline production.
It has been demonstrated that using 10% EG containing a gonadal suspension from 10 embryos is the preferred method for cryopreserving chicken PGC. The use of the treatment 10E per straw is the optimum concentration when labor and efficiency of recovering PGC per embryo is the goal. This study also showed that levels of 2.5% or less of cryoprotectant were not effective, and although 5% EG and 5% DMSO were not significantly different than 10% EG for the number of viable PGC, they were numerically lower. The freezing vessel used was a cryopreservation straw, rather than a cryovial, resulting in a cryopreservation storage method that will allow the systematic storage and labeling of cryopreserved PGC in liquid N at a germplasm repository and ease of entry into a database. The importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 25, 2006. Accepted for publication June 5, 2006.
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