- Email: [email protected]
A New Poultry Semen Extender
RESEARCH NOTES A New Poultry Semen Extender. 5. Relationship of Diluent Components to Cytotoxic Effects of Dimethylsulfoxide on Turkey Spermatozoa T.J. SEXTON US Department of Agriculture, SEA-AR, Avian Physiology Laboratory, Animal Science Laboratory, Beltsville, Maryland 20705 (Received for publication April 16, 1979)
INTRODUCTION Dimethysulfoxide (DMSO) has been used in media for freezing and thawing of avian spermat o z o a (Sexton 1 9 7 5 ; S e x t o n and Fewlass, 1 9 7 8 ; Sexton and Gee, 1978). T h e degree of c r y o p r o tection DMSO imparts o n s p e r m a t o z o a during freezing has been s h o w n to vary and this variation probably relates t o species specificity a n d / o r differences associated with t h e freeze procedure (e.g. Mitchell et al., 1 9 7 5 ; Sexton and Gee, 1 9 7 8 ) . However, DMSO, like m a n y o t h e r c r y o p r o t e c t a n t s , reduces the fertilizing capacity of avian s p e r m a t o z o a before freezing (Sexton, 1 9 7 5 ; S c h r a m m , 1 9 7 6 ) . S o m e progress has been m a d e in reducing the c y t o t o x i c effect of 4% DMSO in chicken semen (Sexton, 1975) b y giving special a t t e n t i o n to m e t h o d of DMSO addition, t e m p e r a t u r e of DMSO at time of addition, and t y p e of semen extender. The purpose of this report was t o explore t h e relationship of c o m p o n e n t s in BPSE to t h e c y t o t o x i c effects of DMSO on t u r k e y spermatozoa. MATERIALS AND METHODS Semen from Large White t u r k e y males was pooled, mixed, and divided into equal p o r t i o n s . T h e samples were diluted 1:4 with either Beltsville Poultry Semen E x t e n d e r (BPSE) or modified BPSE (lacking one c o m p o n e n t ) within 10 m i n of collection. T h e c o m p o s i t i o n of t h e BPSE and t h e modified BPSE are outlined in Table 1. Details o n e x t e n d e r p r e p a r a t i o n , adjustments of pH (range 6.8 to 7.5), and osmolarity (range 358 to 379), m O s m / k g H 2 0 )
have been (1978).
outlined
by Sexton and
Fewlass
Immediately after dilution, each sample was further divided into two equal parts. Both aliquots were held at 10 C for 15 min, followed b y t h e addition of 4% DMSO (v/v), in bulk, to one aliquot. T h e remaining aliquot containing 0% DMSO served as a control. Both aliquots were held an additional 10 min at 10 C before being used for inseminations. T w o h u n d r e d Large White hens were inseminated weekly for 5 weeks with .1 cc of diluted semen containing 2 0 0 million sperm per insemination dose. M e t h o d s for egg collection and statistical evaluation for the d a t a have been described previously (Sexton and Fewlass, 1978). Fertility was d e t e r m i n e d on day 7 of incubation and was expressed as t h e percentage of viable e m b r y o s . RESULTS AND DISCUSSION T h e results presented in Table 2 clearly show t h a t omission of any of t h e BPSE c o m p o n e n t s in the control e x t e n d e r (0% DMSO) did n o t alter t h e fertilizing capacity of t u r k e y spermatozoa. It is also apparent from the results presented in Table 2 t h a t holding semen at 10 C and t h a t t h e addition of DMSO (0 vs. 4% in BPSE with no missing c o m p o n e n t ) did n o t adversely affect fertilizing capacity. These results indicate t h a t t u r k e y s p e r m a t o z o a react similarly to chicken s p e r m a t o z o a when e x t e n d e d in BPSE with 0 t o 4% DMSO. The omission of either buffer (TES or potassium phosphates) or the neutral salts
1142
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014
ABSTRACT The relationship of components of the Beltsville Poultry Semen Extender (BPSE) to the cytotoxic effects of 4% dimethylsulfoxide (DMSO) on turkey spermatozoa was studied. Fertilizing capacity was depressed by the omission of any buffer (TES or potassium phosphates) or neutral salt (citrate, acetate, chloride), whereas omitting fructose or sodium glutamate had no significant effect. 1980 Poultry Science 59:1142-1144
1143
RESEARCH NOTE TABLE 1. Modifications of the Beltsville Poultry Semen Extender Trial #1 Constituent2
Control
2 12.70 8.67 5.00 4.30
Trial # 2 3
12.70 8.67
4.30 1.95 .64 .64 .65 .65 .34 .34 850 800 7.7 7.5 361 379
Trial # 3
4 12.70
5.00 4.30 1.95 .64 .65 .34 670 7.5 358
5 b 8.67 5.00 4.30 1.95 .64
.34 540 6.8 358
6 12.70 8.67 5.00
7 12.70 8.67 5.00 4.30 1.95
1.95 .64 .65 .65 .34 .34 720 770 7.5 7.5 375 379
12.70 8.67 5.00 4.30 1.95 .64 .65 770 7.5 374
Grams per volume indicated. Constituent omitted from medium. N-tris[hydroxymethyl) methyl-2-aminoethane sulfonic acid).
TABLE 2. Relationship of diluent component and DMSO on the fertilizing capacity of turkey spermatozoa11
Trial
Extender no.
Fertility (%) at Missing component
0% DMSO
4% DMSO
None TES Fructose None Sodium glutamate Potassium p h o s p h a t e s None Sodium acetate Potassium citrate Magnesium chloride
87 90 81 86 91 82 79 85 83 85
79 ± 54*± 79 ± 90 ± 90 + 70* + 75 ± 71*± 42*± 69*±
± 6 +2 ± 5 ± 3 ± 2 ± 3 ± 2 ± 3 ±4 ± 5
3 8 5 3 4 4 2 4 9 8
Mean and SEM of five inseminations. 'Significantly (P<.05) lower than the control (0% DMSO) on the same row.
(acetate, citrate, or chloride) from the BPSE containing 4% DMSO resulted in a significant decline in the fertilizing capacity of spermatozoa (Table 2). The mode of action of these components is unknown, but these findings support an earlier report by this author (Sexton, 1975) and later confirmed by Schramm (1976) that the degree of cytotoxicity of DMSO is influenced by the type of extender. Sexton (1975) and Schramm (1976) reported that the presence of biologies (milk, egg yolk) or the lack of a strong buffer in the extender increased the cytotoxicity of DMSO towards chicken spermatozoa. This phenomenon could partly explain
the varied degree of cryoprotection provided by DMSO during freezing and thawing of chicken spermatozoa. ACKNOWLEDGMENT
The author wishes to express appreciation to D. M. Harper for her technical assistance.
REFERENCES Mitchell, R. L., R. B. Buckland, L. Forgrave, and R. D. Baker, 1975. A simple controlled rate freezer apparatus as applied to freezing poultry semen. Poultry Sci. 54:1796.
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014
Dipotassium p h o s p h a t e • 3 H 2 O 12.70 Sodium g l u t a m a t e 8.67 Fructose 5.00 Sodium acetate • 3 H 2 0 4.30 TESC 1.95 Potassium citrate .64 Monopotassium phosphate .65 Magnesium chloride • 6 H 2 0 .34 Distilled water (ml) 900 PH 7.5 Osmotic pressure (mOsm/kg- H 2 0 ) 366
1
1144
SEXTON
Schramm, G. P., 1976. Action of cryoprotectives glycerin, ethylene glycol and dimethylsulphoxide on fertilization potential of cock semen. Monatsh Meterinaermed 20:770-773. Sexton, T. J., 1975. Relationship of the method of addition and temperature of cryoprotective agents to the fertilizing capacity of cooled chicken spermatozoa. Poultry Sci. 54:845—848.
Sexton, T. J., and T. A. Fewlass, 1978. A new poultry semen extender. 2. Effect of diluent components on the fertilizaing capacity of chicken semen stored at 5°C. Poultry Sci. 57:277-284. Sexton, T. J., and G. F. Gee, 1978. A comparative study on the cryogenic preservation of semen from the sandhill crane and the domestic fowl. Symp. Zool. Soc. London 4 3 : 8 9 - 9 5 .
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014
INTRODUCTION Dimethysulfoxide (DMSO) has been used in media for freezing and thawing of avian spermat o z o a (Sexton 1 9 7 5 ; S e x t o n and Fewlass, 1 9 7 8 ; Sexton and Gee, 1978). T h e degree of c r y o p r o tection DMSO imparts o n s p e r m a t o z o a during freezing has been s h o w n to vary and this variation probably relates t o species specificity a n d / o r differences associated with t h e freeze procedure (e.g. Mitchell et al., 1 9 7 5 ; Sexton and Gee, 1 9 7 8 ) . However, DMSO, like m a n y o t h e r c r y o p r o t e c t a n t s , reduces the fertilizing capacity of avian s p e r m a t o z o a before freezing (Sexton, 1 9 7 5 ; S c h r a m m , 1 9 7 6 ) . S o m e progress has been m a d e in reducing the c y t o t o x i c effect of 4% DMSO in chicken semen (Sexton, 1975) b y giving special a t t e n t i o n to m e t h o d of DMSO addition, t e m p e r a t u r e of DMSO at time of addition, and t y p e of semen extender. The purpose of this report was t o explore t h e relationship of c o m p o n e n t s in BPSE to t h e c y t o t o x i c effects of DMSO on t u r k e y spermatozoa. MATERIALS AND METHODS Semen from Large White t u r k e y males was pooled, mixed, and divided into equal p o r t i o n s . T h e samples were diluted 1:4 with either Beltsville Poultry Semen E x t e n d e r (BPSE) or modified BPSE (lacking one c o m p o n e n t ) within 10 m i n of collection. T h e c o m p o s i t i o n of t h e BPSE and t h e modified BPSE are outlined in Table 1. Details o n e x t e n d e r p r e p a r a t i o n , adjustments of pH (range 6.8 to 7.5), and osmolarity (range 358 to 379), m O s m / k g H 2 0 )
have been (1978).
outlined
by Sexton and
Fewlass
Immediately after dilution, each sample was further divided into two equal parts. Both aliquots were held at 10 C for 15 min, followed b y t h e addition of 4% DMSO (v/v), in bulk, to one aliquot. T h e remaining aliquot containing 0% DMSO served as a control. Both aliquots were held an additional 10 min at 10 C before being used for inseminations. T w o h u n d r e d Large White hens were inseminated weekly for 5 weeks with .1 cc of diluted semen containing 2 0 0 million sperm per insemination dose. M e t h o d s for egg collection and statistical evaluation for the d a t a have been described previously (Sexton and Fewlass, 1978). Fertility was d e t e r m i n e d on day 7 of incubation and was expressed as t h e percentage of viable e m b r y o s . RESULTS AND DISCUSSION T h e results presented in Table 2 clearly show t h a t omission of any of t h e BPSE c o m p o n e n t s in the control e x t e n d e r (0% DMSO) did n o t alter t h e fertilizing capacity of t u r k e y spermatozoa. It is also apparent from the results presented in Table 2 t h a t holding semen at 10 C and t h a t t h e addition of DMSO (0 vs. 4% in BPSE with no missing c o m p o n e n t ) did n o t adversely affect fertilizing capacity. These results indicate t h a t t u r k e y s p e r m a t o z o a react similarly to chicken s p e r m a t o z o a when e x t e n d e d in BPSE with 0 t o 4% DMSO. The omission of either buffer (TES or potassium phosphates) or the neutral salts
1142
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014
ABSTRACT The relationship of components of the Beltsville Poultry Semen Extender (BPSE) to the cytotoxic effects of 4% dimethylsulfoxide (DMSO) on turkey spermatozoa was studied. Fertilizing capacity was depressed by the omission of any buffer (TES or potassium phosphates) or neutral salt (citrate, acetate, chloride), whereas omitting fructose or sodium glutamate had no significant effect. 1980 Poultry Science 59:1142-1144
1143
RESEARCH NOTE TABLE 1. Modifications of the Beltsville Poultry Semen Extender Trial #1 Constituent2
Control
2 12.70 8.67 5.00 4.30
Trial # 2 3
12.70 8.67
4.30 1.95 .64 .64 .65 .65 .34 .34 850 800 7.7 7.5 361 379
Trial # 3
4 12.70
5.00 4.30 1.95 .64 .65 .34 670 7.5 358
5 b 8.67 5.00 4.30 1.95 .64
.34 540 6.8 358
6 12.70 8.67 5.00
7 12.70 8.67 5.00 4.30 1.95
1.95 .64 .65 .65 .34 .34 720 770 7.5 7.5 375 379
12.70 8.67 5.00 4.30 1.95 .64 .65 770 7.5 374
Grams per volume indicated. Constituent omitted from medium. N-tris[hydroxymethyl) methyl-2-aminoethane sulfonic acid).
TABLE 2. Relationship of diluent component and DMSO on the fertilizing capacity of turkey spermatozoa11
Trial
Extender no.
Fertility (%) at Missing component
0% DMSO
4% DMSO
None TES Fructose None Sodium glutamate Potassium p h o s p h a t e s None Sodium acetate Potassium citrate Magnesium chloride
87 90 81 86 91 82 79 85 83 85
79 ± 54*± 79 ± 90 ± 90 + 70* + 75 ± 71*± 42*± 69*±
± 6 +2 ± 5 ± 3 ± 2 ± 3 ± 2 ± 3 ±4 ± 5
3 8 5 3 4 4 2 4 9 8
Mean and SEM of five inseminations. 'Significantly (P<.05) lower than the control (0% DMSO) on the same row.
(acetate, citrate, or chloride) from the BPSE containing 4% DMSO resulted in a significant decline in the fertilizing capacity of spermatozoa (Table 2). The mode of action of these components is unknown, but these findings support an earlier report by this author (Sexton, 1975) and later confirmed by Schramm (1976) that the degree of cytotoxicity of DMSO is influenced by the type of extender. Sexton (1975) and Schramm (1976) reported that the presence of biologies (milk, egg yolk) or the lack of a strong buffer in the extender increased the cytotoxicity of DMSO towards chicken spermatozoa. This phenomenon could partly explain
the varied degree of cryoprotection provided by DMSO during freezing and thawing of chicken spermatozoa. ACKNOWLEDGMENT
The author wishes to express appreciation to D. M. Harper for her technical assistance.
REFERENCES Mitchell, R. L., R. B. Buckland, L. Forgrave, and R. D. Baker, 1975. A simple controlled rate freezer apparatus as applied to freezing poultry semen. Poultry Sci. 54:1796.
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014
Dipotassium p h o s p h a t e • 3 H 2 O 12.70 Sodium g l u t a m a t e 8.67 Fructose 5.00 Sodium acetate • 3 H 2 0 4.30 TESC 1.95 Potassium citrate .64 Monopotassium phosphate .65 Magnesium chloride • 6 H 2 0 .34 Distilled water (ml) 900 PH 7.5 Osmotic pressure (mOsm/kg- H 2 0 ) 366
1
1144
SEXTON
Schramm, G. P., 1976. Action of cryoprotectives glycerin, ethylene glycol and dimethylsulphoxide on fertilization potential of cock semen. Monatsh Meterinaermed 20:770-773. Sexton, T. J., 1975. Relationship of the method of addition and temperature of cryoprotective agents to the fertilizing capacity of cooled chicken spermatozoa. Poultry Sci. 54:845—848.
Sexton, T. J., and T. A. Fewlass, 1978. A new poultry semen extender. 2. Effect of diluent components on the fertilizaing capacity of chicken semen stored at 5°C. Poultry Sci. 57:277-284. Sexton, T. J., and G. F. Gee, 1978. A comparative study on the cryogenic preservation of semen from the sandhill crane and the domestic fowl. Symp. Zool. Soc. London 4 3 : 8 9 - 9 5 .
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 28, 2014