REPRODUCTION - Facultad de Ciencias Veterinarias

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840 REPRODUCTION Cryopreservation of Buffalo (Bubalus Bubalis) Spermatogonial Stem Cells Abstract Jayanti Tokas 1 and Gautam Kaul 2 1 Seth Jai Parkash Mukund Lal Institute of Engineering and Technology (JMIT), Radaur-135133 (Yamunanagar). 2 National Dairy Research Institute (NDRI), Karnal-132001 - Email – jiyandri@gmail.com A protocol was developed for cryopreservation of buffalo (Bubalus bubalis) spermatogonial stem cells (SSCs). Spermatogonia isolated by serial enzymatic digestion and then enriched by differential plating and percoll gradient were suspended in freezing media (DMEM/F12 + BSA) to examine the effect of cryoprotectants under two freezing regimes (slow and fast freezing). DMSO was found to be a better cryoprotectant than glycerol. A controlled slow freezing regime resulted in a significantly higher (p
MATERIAL AND METHODS REPRODUCTION Spermatogonia were isolated from the testes of 5-9 months old buffalo (Bubalis bubalis) calves (Idgah slaughterhouse, New Delhi) by enzymic digestion procedure [collagenase (2mg/ml), Trypsin (1mg/ml) and Dnase I] 8 . After further removal of somatic cells, the isolated spermatogiona were purified by discontinuous percoll density gradient purification with suitable modifications 2 . The enriched population of spermatogonia cell suspension in DMEM/F12+BSA supplemented with sodium bicarbonate, penicillin, streptomycin were cryopreserved in eight different combination of cryoprotectants : A) 10% FCS B) 20% FCS C) DMSO +10% FCS D) DMSO + 20% FCS E) DMSO + 10%FCS + 0.07 M sucrose F) DMSO + 10%FCS + 0.14 M sucrose G) DMSO + 10% FCS + 0.21 M sucrose H) Glycerol + 10% FCS. In the two freezing media, DMSO+10%FCS & Glycerol+10%FCS, effect of two freezing regimes was evaluated on viability of spermatoginia after cryopreservation viz., i) fast freezing (direct immersion of the cryovials into the liquid nitrogen (-196 o C) for storage), (ii) Slow freezing (placing cryovials into an ambient temperature freezing chamber containing isopropyl alcohol and then into a -70 o C freezer for 18 h before plunging into liquid nitrogen. Frozen spermatogonia were recovered through two thawing methods (a) rubbing of frozen vial between palms into a back and forth motion until the cell suspension just thawed and placing the vials into crushed ice before further processing, (b) immersing a vial of frozen cell suspension in a 37 o C water bath until the cell suspension just thawed. Cell viability of recovered spermatogonia was assessed with trypan blue staining. The donor cell suspension was washed twice in serum-free medium, resuspended in loading buffer (Sigma) and stained with lipophilic dye, PKH-26 (0.5µl PKH-26 (1mM in ethanol) in 200µl of loading buffer) and the reaction was stopped with 500µl FCS to bind most of the serum proteins to the dye. Cell pellet was obtained by centrifugation and washed twice with 40ml of DMEM/F12 medium containing 10% FCS to remove all of the PKH-26. The cells were then resuspended @1 x 10 6 cells/ml and cultured for a month with four subculture passages in sterile 96 well gelatin coated culture plates. Cell proliferation was assessed by CD9 labelling 8 . Buffalo spermatogonial stem cells were transplanted into seminiferous tubules obtained from of mice treated with Busulfan @36 mg/kg body weight for four weeks as per the standard procedures 2 . Successful transplantation of the donor cells into the mouse seminiferous tubules was monitored by PKH-26 and then CD9 staining respectively. The data was subjected to analysis of variance using SYSTAT 7.0 statistical software. Significance was considered at P< 0.05 or mentioned otherwise. RESULTS AND DISCUSSION We examined eight different freezing media to define the best freezing media for the cryopreservation of buffalo spermatogonial stem cell. The viabilities of spermatogonia in different freezing media are presented in Table 1. Viability of freshly isolated buffalo spermatogonia was 80%. Freezing media composition influenced the viability of cells after freezing and thawing. Increasing FCS to 20% did not improve cell survival. Only 30% of the cells frozen in DMEM/F-12 containing 10% FCS survived after cryopreservation. However, a significantly higher percentage of living cells was observed when cells were frozen in medium containing 10% FCS and 1.4 M glycerol or DMSO as compared to medium with only FCS. DMSO provided significantly better cell survival (P < 0.001) than glycerol and the viability of cells was significantly high in slow freezing than fast freezing (Table 2). Addition of sucrose (0.07M, 0.14M, or 0.21 M) in freezing media significantly improved the viability of cells (Table 1). Amongst the three concentrations of sucrose studied, 0.07M was found to be the most effective. Survival and proliferation in culture, was significantly higher in spermatogonia that had been frozen in the media containing sucrose than those without sucrose. This was similar to that of freshly cultured cells, indicating that the proliferative activity of spermatogonia in culture was not influenced by the freeze/thaw Buenos Aires, Abril 2010 841
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Page 3 and 4: Semi quantitative RT PCR REPRODUCTI
Page 5 and 6: DISCUSSION REPRODUCTION The results
Page 7 and 8: REPRODUCTION Although MTT assay has
Page 9 and 10: DISCUSSION REPRODUCTION The data ge
Page 11 and 12: REPRODUCTION REFERENCES Adeel, M.,
Page 13 and 14: REPRODUCTION Comparative Efficacy o
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Page 17 and 18: Abstract REPRODUCTION Correlations
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Page 31 and 32: REPRODUCTION Composition of TRIS (h
Page 33 and 34: Composition of CEBRAN II diluter (R
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Page 43 and 44: REPRODUCTION REFERENCES 1.Boonkusol
Page 45 and 46: Table-1: Composition of dilution pr
Page 47 and 48: Abstract REPRODUCTION Effect of dif
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RESULTS AND DISCUSSION REPRODUCTION
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REPRODUCTION The Cumulus-oocyte com
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INTRODUCTION REPRODUCTION Normal sp
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REPRODUCTION Table 3 - Means ± SEM
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REPRODUCTION The visualization of a
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REPRODUCTION nipulation system. The
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Acknowledgements. REPRODUCTION This
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INTRODUCTION REPRODUCTION Histopath
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REPRODUCTION Sixty days after the i
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REPRODUCTION The Ovsynch protocol,
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REPRODUCTION - Facultad de Ciencias Veterinarias

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