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Development of Extender and Techniques for Frozen Turkey Semen
Development of Extender and Techniques for Frozen Turkey Semen. 1. Development1 E. F. GRAHAM, D. S. NELSON, and M.K.L. SCHMEHL Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication July 2, 1980)
1982 Poultry Science 61:550-557 INTRODUCTION D e v e l o p m e n t of an e x t e n d e r system tliat allows frozen-thawed t u r k e y s e m e n t o m a i n t a i n t h e vigorous swirling motility d e m o n s t r a t e d by nonfrozen semen is complicated. C o m m o n c r y o p r o t e c t a n t s for semen such as egg y o l k (Bajpai and Brown, 1963a), milk (Bajpai and Brown, 1 9 6 3 b ) , and glycerol (Brown and Graham, 1 9 7 1 ) , have detrimental effects on t u r k e y semen a n d fertility. Also, S e x t o n et al. (1979) reported that dimetiiylsulfoxide (Me^SO) levels of 10% or higher significantly depressed fertility of fresh t u r k e y semen. T h e p u r p o s e of this study was t o investigate ext e n d e r systems and techniques for m a x i m a l survival of s p e r m a t o z o a after freezing. MATERIALS AND METHODS Seven l a b o r a t o r y trials were c o n d u c t e d and
'Scientific Paper No. 11,282 of the Minnesota Agricultural Experiment Station, St. Paul, MN 55108. 2 [N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonic acid].
repeated four t o six times. Semen samples were collected by a b d o m i n a l massage (Burrows and Q u i n n , 1 9 3 7 ) at 22 C from Nicholas Large White and Wrolstad Medium White t u r k e y s (Meleagris gallopavo). Collected semen was pooled and gently m i x e d . Aliquots (.1 ml) were placed directly into e x t e n d e r s (.4 ml), held at 0 or 22 C, mixed 3 sec at m e d i u m speed on a vortex mixer, and equilibrated at 0 C. Ext e n d e r s were adjusted t o 3 7 0 m O s m / k g a t p H 7.2 e x c e p t Trial 7 ( 3 1 0 t o 5 8 0 m O s m / k g ) . Semen was frozen in 75 jul pellets o n solid C 0 2 (Nagase et al., 1964), held 10 min, and stored in liquid nitrogen. Pellets were t h a w e d individually in plastic vials a t 22 C, and percentages of motile s p e r m a t o z o a were assessed using closed circuit television microscopy. Samples were r a n d o m l y coded before motility estimates were m a d e by t w o trained technicians. T h e d a t a was d e c o d e d after c o m p l e t i o n of t h e e x p e r i m e n t s . Each trial was r e p e a t e d six times (different days) e x c e p t Trial 6 with only four replicates. Also, t h e 2 hr, 0 C p o s t t h a w samples in Trial 4 were repeated only three times. D a t a were subjected t o analysis of variance (randomized c o m p l e t e block design) and significant m e a n differences were de-
550
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ABSTRACT An extender for turkey semen with a frozen-thawed recovery of >50% motile spermatozoa and a vigorous swirl was developed. The effect of glucose, a comparison of 10 different sugars in the extender, the use of sodium acetate and potassium acetate, the ratio of dimethylsulfoxide to ethylene glycol, the percent total cryoproteetant, equilibration time, and osmotic pressure were all tested. Replacement of approximately half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other carbohydrates did not enhance recovery of frozenthawed semen. The proportion of sodium and potassium acetates to TESNaK2 in the extender had no effect on motility. Approximately equal proportions of ethylene glycol to dimethylsulfoxide, with a final concentration of 11.2% after dilution, produced at or near optimal recovery of spermatozoal motility after freezing. Twenty to 90 min equilibration times before freezing yielded significantly higher recovery of motile sperm postthaw than shorter periods. A wide range of extender osmotic pressures were compatible with recovery of spermatozoal motility postthaw. The final extender consisted of 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES, 3 .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H2 O q.s. 1000 ml, 370 mOsm/kg, pH 7.2. Ethylene glycol and dimethylsulfoxide were added (1:1 ratio) for a final concentration of 11.2% cryoproteetant after dilution. Semen was held undiluted for 6 min, extended 1:4 at 22 C, and equilibrated for 30 min at 0 C before pellet freezing. (Key words: turkey semen, semen extender, glucose, cryoproteetant, sperm motility)
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termined by Tukey's W procedure (Steel and Torrie, 1960). Trial 1. Effect of Glucose in the Extender. Eleven extenders consisting of 10.0 to 0:10 ratios of extender A (Table 1) and glucose solution (64.0 g/1, 370 mOsm/kg) were prepared. Dimethylsulfoxide (Me2 SO) and ethylene glycol (3:2 ratio) were added to the extenders yielding a final concentration of 8.3% after dilution. Pooled semen was diluted 1:5, equilibrated 10 min and frozen. Pellets were thawed and transferred to a 0 C water bath. Percentages of motile spermatozoa were assessed immediately for frozen-thawed semen and after 1, 5, and 8 hr of 0 C storage for fresh semen. Trial 2. Effect of Various Carbohydrates. Ten extenders were prepared consisting of one part extender D (Table 1) and one part of the following solutions (370 mOsm/kg): i-erythritol, D (—) arabinose, D (—) ribose, D (+) xylose, D (—) fructose, D (+) galactose, D (+) glucose, i-inositol, D (+) mannose, and D (+) lactose. Me 2 SO and ethylene glycol were added to each extender (1:1 ratio, 8.2% v/v after dilution). Semen was collected, diluted 1:4, equilibrated 35 min, and frozen. Pellets were thawed, assessed immediately, and assessed after 3 hr storage at 0 C. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 3. Ratio of Sodium and Potassium Acetates to TESNaK. Eleven extenders were prepared consisting of 10:0 to 0:10 ratios of TESNaK (Brown et al, 1972) to sodiumpotassium acetate solution (13.44 g sodium acetate and 9.69 g/1 potassium acetate, 370 mOsm/kg). One part of glucose solution (3 70 mOsm/kg) was added to one part of each extender. Cryoprotectant (Me2 SO and ethylene glycol, 1:1 ratio) was added at a concentration of 8.2% after dilution. Semen was collected, diluted 1:4 in extender, and frozen after 20 and 35 min equilibration. Pellets were thawed and motilities assessed. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 4. Ratio ofMe2SO to Ethylene Glycol. Eleven mixtures consisting of 10:0 to 0:10 ratios of Me 2 SO and ethylene glycol were added (8.3% v/v after dilution) to extender B (Table 1). Semen was collected, diluted 1:5, equilibrated 15 min and frozen. Semen was thawed, assessed immediately, and assessed again after 2 hr storage at 0 C. Fresh semen motilities were assessed after 5 hr storage at 0 C.
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GRAHAM ET AL. TABLE 2. Effect of ratio of glucose solution (370 mOsm/kg) to TESNaK acetate extender1 (370 mOsm/kg) on frozen and fresh turkey semen % Motility Fresh storage, 1 hr, O C2
Fresh storage, 5 hr, O C
Fresh storage, 8 hr, O C
Postthaw, 3 22 C
0:10 1:9 2:8 3:7 4:6 5:5 6:4 7:3 8:2 9:1 10:0
51.0ab 4 50.7 a b 55.2 a b 52.2 a b 51.8 a b 57.3 a 58.5 a 53.0 a b 51.3ab 44.8bc 38.0C
9.8d 4 16.0 c d 18.0 c d 18.0 c d 32.oabc 38.8ab 45.0 a 47.0 a 50.0 a 32.0abc 2i.obcd
7. 4 e 4 8.4 e 170cde 170cde 20.0cde 27.4bcd 37.4 a b 43.0ab 46.0 a 31.0abc 26.0bcd
21.8bc" 21.0 b c 27 2 a b c 28.5abc 29.3ab 32.8 a 29.0abc 26.7abc 29.3ab 23.7abc 20.0 C
Mean SD
51.4 8
29.8 16
25.0 15
26.3 8
3. h o (i t*
' ' ' ' Means in the same vertical line not followed by the same letter are significantly different (P<.05).
1
Extender formula: 12.10 g sodium acetate, 8.72 g potassium acetate, 6.15 g TES, .235 g sodium hydroxide, .328 g potassium hydroxide, H 2 0 q.s. 1000 ml. 2 Diluted 1:5 in 370 mOsm/kg and pH 7.2 extender with dimethylsulfoxide (5.0% final volume) and ethylene glycol (3.3% final volume). 3
As 75-jul pellets frozen on solid CO, and stored at —196 C.
"Mean of 6 replicates.
TABLE 3. Effect of adding various carbohydrate solutions (1.1 ratio) to acetate-TESNaK extender1 on frozen and fresh turkey semen2 % Motility Postthaw 3 storage, 3 hr, O C
Fresh storage, 1 hr, O C
Fresh storage, 5 hr, O C
i-Erythritol D(—) Arabinose D ( - ) Ribose D(+) Xylose D(—) Fructose D(+) Galactose D(+) Glucose i-lnositol D(+) Mannose D(+) Lactose
50.8 b " 61.2 a 60.3 a 61.7 a 64.2 a 63.7 a 60.0 a 61.5 a 62.8 a 62.3 a
14.3 b 4 40.7 a 38.7 a 42.0 a 46.5 a 48.2 a 48.5 a 51.5 a 47.3 a 40.0 a
21.8 b " 28.0 a b 30.2 a b 28.5ab 33.5* 34.7 a 29.2ab 28.5ab 28.2ab 27.5ab
5.7b4 12.7 a b 12.3ab 12.7 a b 15.7ab 17.3 a 17.5 a 22.0 a 18.2 a 19.0 a
Mean SD
61.5 5
41.7 13
29.1 7
15.3 6
Carbohydrate
Postthaw, 3 22 C
' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 1
Extender formula: 9.41 g sodium acetate, 6,78 g potassium acetate, 18.45 g TES, .706 g sodium hydroxide, .984 g potassium hydroxide, H2 O q.s. 1000 ml. 2 Diluted 1:4 in 370 mOsm/kg and pH 7.2 extender with 8.2% final cryoprotectant (dimethylsulfoxide and ethylene glycol, 1:1 ratio). 3
Semen was equilibrated 35 min, frozen in 75 ul pellets on solid CO a , and stored at —196 C.
4
Mean of 6 replicates.
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Ratio of glucose solution to extender
FROZEN TURKEY SEMEN EXTENDER
semen was held 6 min (22 C), diluted 1:4, mixed, equilibrated 30 min and frozen. Pellets were thawed and assessed immediately and after 1 hr storage at 0 C. Fresh semen was assessed after 1.5 and 5 hr storage at 0 C. RESULTS Trial 1. A 5:5 ratio of glucose solution to the extender (Table 2) supported significantly higher (P<.05) recovery of motility by frozenthawed spermatozoa than 0:10, 1:9, or 10:0. Also, 5:5 and 6:4 glucose solution to extender ratios yielded the highest percent motile spermatozoa after storage of fresh semen for 1 hr at 0 C, while 8:2, 7:3, and 6:4 glucose solution ratios supported the highest percent motile spermatozoa after storage of fresh semen for 5 hr at 0 C. Therefore, a 5:5 ratio of glucose solution (6.7 g %) to extender was used for die remaining trials. Trial 2. Replacement of glucose in the diluent with different carbohydrates (Table 3) did not significantly improve recovery of motility by frozen-thawed spermatozoa, nor
TABLE 4. Effect of ratio of cryoprotective agents added to glucose-acetate-TESNaK extender1 on frozen and fresh turkey semen Cryoprotective agents (ratio) Dimethylsulfoxide 10 9 8 7 6 5 4 3 2 1 0 Mean SD
% Motility Ethylene glycol 0 1 2 3 4 5 6 8 8 9 10
Fresh storage, 5 hr, O C
Postthaw, 2 22 C
Postthaw 2 storage, 2 hr, O C
13.8 C " 17.2bc 23.7 a bc 23.5 a bc 29.3 a b 34.3 a 33.3 a 28.7 a b 32.8 a 26.5 a bc 34.0 a
23.5abc 4 25.8abc 26.8 a b 27.oabc 25.7 a b 30.7 a 27.2ab 19gabc 18.0bc 14.3 C 13.8 C
1 2 . 3aa b 3 18.3 b 14.7ab 17.3ab 25.0 a 24.7 a 20.0ab 16.7ab 14.0 a b 11.3ab 6.7 b
27.0 14
23.0 9
16.5 7
' ' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 'Diluted 1:5 in 370 mOsm/kg extender, pH 7.2, 8.3% total cryoprotectant after dilution. Extender formula: 6.05 g sodium acetate, 4.36 g potassium acetate, 3.08 g TES, .118 g sodium hydroxide, .164 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml. 2 As 75-jul pellets frozen on solid C0 2 and stored at —196 C. 3 Mean of 3 replicates. 4 Mean of 6 replicates.
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Trial 5. Total Percent of Me2SO and Ethylene Glycol (1.1 ratio). Extender C (Table 1) was prepared with 11 concentrations of cryoprotectant added, ranging from 0 to 16.0% v/v after dilution. Semen was collected, diluted 1:4, equilibrated 35 min, and frozen. Pellets were thawed, assessed immediately, and assessed after 1 hr storage at 0 C. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 6. Equilibration Time. Semen was collected, diluted 1:4 in extender B, and equilibrated 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 min before freezing. Cryoprotectant (Me 2 SO and ethylene glycol, 1:1 ratio) was added for a final concentration of 8.2% v/v after dilution. Pellets were thawed and motilities assessed immediately and after 1 hr storage at 0 C. Trial 7. Osmotic Pressure. Extender C was prepared at the following osmotic pressures: 580, 550, 520, 490, 460, 430, 400, 370, 340, and 310 mOsm/kg and cryoprotectant (Me 2 SO and ethylene glycol, 1:1 ratio) was added for a 9.6% concentration after dilution. Pooled
553
554
GRAHAM ET AL. Trial 7. Results indicated (Table 7) that osmotic pressure over a wide range supported motility in frozen-thawed spermatozoa. Osmotic pressures of 550 and 580 mOsm/kg severely decreased percent motile spermatozoa (P<.01) in fresh semen samples stored for 1.5 and 5 hr and in both the 0 and 1 hr postthaw samples. DISCUSSION A high percentage of glucose in an extender for freezing turkey semen has been used. Watanabe and Kato (1970) studied the use of 5, 7, and 9 g % glucose as a diluent for freezing semen and reported good recoveries of frozenthawed semen with less abnormal spermatozoa with the 7 g % glucose diluent. Watanabe and Miyashima (1973) reported 3.8% fertility with semen frozen in glucose (6.5 g %) egg yolkglycerol diluent and stored for 2 years in liquid nitrogen. Our results indicated that incorporating some glucose in the extender formula was beneficial. However, 6.4 g % glucose produced lower postthaw recovery of motile spermatozoa than glucose used with Extender A (Table 1). The 3.2 g % glucose proportion
TABLE 5. Effect of adding various percents of cryoprotectant (dime thy Isulfoxide and ethylene glycol, 1:1 ratio) to glucose-acetate-TESNaK extender1 on frozen and fresh turkey semen Cryoprotectant
% in Buffer 0 2 4 6 8 10 12 14 16 18 20 Mean SD
% After dilution 0 1.6 3.2 4.8 6.4 8.0 9.6 11.2 12.8 14.4 16.0
% Motility Fresh storage, 1 hr, O C
Fresh storage, 5hr, OC
Postthaw 3 22 C
Postthaw 3 storage, 1 hr, O C
65.7a" 65.2ab 65.2ab 64.3ab 64.3ab 61.7ab 61.3ab 63.7ab 64.3ab 64.3ab 59.2 b
59.8 a " 59.5 a 56.2abc 58.2 a 57.8 a b 57.8 a b 57.3abc 58.7 a 55.3abc 50.3bc 49.8C
1.0C4 4.5C 13.3 C 27.3 b 28.7 b 40.3ab 45.8 a 49.2 a 46.2 a 44.2 a 42.5 a
1.2d4 3.7 d 13.5 c d 24.8abc 27.8ab 30.8 a b 30.0 a b 37.5 a 27.3abc 25.0abe 20.8 b c
63.3 4
56.4 5
31.2 18
22.0 13
' ' ' Mean in the same vertical line not followed by the same letter are significantly different (P<,05). Extender formula: 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 gTES, .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 glucose, H 2 0 q.s. 1000 ml. 2 Diluted 1:4 in extender (22 C) with 370 mOsm/kg and pH 7.2. 3 Semen was equilibrated 35 min, frozen in 75 p\ pellets on solid C0 2 , and stored at —196 C. 4 Mean of 6 replicates. 1
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did they improve motility in fresh stored semen samples. Trial 3. There was no significant difference in diluents using different proportions of sodium and potassium acetate to TESNaK. Average motilities of all treatments ranged from 57 to 65% for fresh samples stored 1 hr, 41 to 52% for fresh samples stored 5 hr, and 22 to 29% for postthaw semen samples. Trial 4. Results (Table 4) indicated approximately equal proportions of Me 2 SO, and ethylene glycol produced recovery of frozenthawed spermatozoa equal to or better than other proportions, so a ratio of 1:1 Me 2 SO to ethylene glycol was adopted as our standard cryoprotective agent. Trial 5. Results indicated (Table 5) that 11.2% v/v of cryoprotectant after dilution yielded at or near maximal motility for postthaw frozen spermatozoa for both times tested. Results were variable, however. Trial 6. The results (Table 6) indicated no differences over a wide range of times. In general, recovery of motility by frozen-thawed spermatozoa was poorest at the extremes of the times tested.
FROZEN TURKEY SEMEN EXTENDER TABLE 6. Effect of equilibration time with cryoprotective agents1 in glucose-acetate-TESNaK extender2 on frozen turkey semen % Motility
i
Equilibration time prior to freeze (rain)
Mean SD
Postthaw 3 storage, 1 hr, O C
7.0b 4 19.3ab 23.5 a 25.3 a 29.3a 28.5 a 19.8 a b 21.oa 16.3 a b 16.3 a b
4.8b 4 13.5 a b 19.0 a 21.5a 23.5 a 17.8 a 20.0 a 16.5ab 13.5 a b 13.Sab
20.1 9
16.4 8
a ' b Mean in the same vertical line not followed by the same letter are significantly different (P<.01). 1 8.2% Cryoprotectant after dilution (dimethylsulfoxide, ethylene glycol, 1:1 v/v). 2 Diluted 1:4 in extender with 370 mOsm/kg and pH 7.2. Extender formula: 6.05 g sodium acetate, 4.36 g potassium acetate, 3.08 g TES, .118 g sodium hydroxide, .164 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml. 3 As 75 /il pellets frozen on solid C0 2 and stored at - 1 9 6 C. 4
Mean of 4 replicates.
was chosen because it showed the highest postthaw recovery of motile spermatozoa. Replacement of glucose in our extender with different carbohydrates did not enhance recovery of motile spermatozoa. Others have reported using different carbohydrates with some degree of success. Oderkirk and Buckland (1977) compared various extenders which contained fructose, raffinose, or lactose. Percent fertility ranged from 3.7 to 20.7, depending on extender, technique, and cryoprotectant used. The 20.7% fertility was obtained using an extender containing lactose. Schefels (1978) reported 63% overall fertility (95 eggs) using a fructose-yolk-glycerol extender with 84% fertility of eggs laid after the first insemination with frozen semen. Further studies regarding the effects of carbohydrates are needed. Acetate was selected for use in the semen
diluent because it is an intermediate in many metabolic reactions and provides a substrate for spermatozoa. Preliminary studies indicated a 1:1 mixture of sodium acetate and potassium acetate solutions (370 mOsm/kg) maintained a high percent of motile spermatozoa. Since acetates are weak buffers, TESNaK was added to stabilize the pH of the diluent. Also, according to Sexton (1979a), omission of TES, sodium acetate, and other salts from extenders increased cytotoxicity of Me 2 SO to turkey spermatozoa. The proportions of sodium and potassium acetates and TESNaK in extender C (Table 1) maintained a stable pH of 7.2 with a high percentage of motile spermatozoa. Various cryoprotectants have been used by others. Macpherson et al. (1969) used a combination of 1.7% glycerol with 8.3% ethylene glycol and 1.7% glycerol with 3.3% N, N-dimethylacetamide by volume before diluting 1:6. The latter, with dialysis to remove the cryoprotectant, yielded 24% fertility with frozen semen. Zavos et al. (1976) reported 26.0% fertility for frozen semen with 6% ethylene glycol and 6% Me 2 SO by volume before diluting 1:4. Sexton (1980) reported fertility with semen frozen to —20 C increased from 27% (1st week) to 50% (4th week) over a 4-week egg production period. Their techniques utilized 4% Me 2 SO in Beltsville Poultry Semen Extender. Brown and Graham (1971) reported 6% glycerol in extended semen (diluted 1:2, 1/30 ml insemination dose) decreased fertility in fresh turkey semen with 10.2% fertility vs. 81.0% for the control. Fertility for ethylene glycol and Me 2 SO under the same conditions were 74.8 and 91.0%, respectively. In our laboratory, motility recovery of frozen-thawed semen with the ethylene glycol, Me 2 SO combination equaled glycerol (unpublished data). Optimum equilibration times before the freezing of semen have been reported. Rajamannan (1968) tested equilibration times of 5, 10, and 15 min before vapor (straw), spin (ampule), and film freezings and reported 10 min yielded the highest recovery of motile spermatozoa. Sexton (1979b) tested equilibration times of 10 and 60 min with 10 min being the optimum for his procedures. Our data indicated no differences within the range 10 to 90 min, but extender composition, prefreeze handling, and freezing techniques were different in our experiments than in those used by the above researchers. The optimum osmotic pressure for turkey
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0 10 20 30 40 50 60 70 80 90
Postthaw, 3 22 C
555
GRAHAM ET AL.
556
TABLE 7. Effect of osmotic pressure of extenders1
on frozen and fresh turkey semen % Motility
Osmotic pressure mOsm/kg
Fresh2 storage, 1.5 hr, O C
Fresh storage, 5 hr, O C
Postthaw, 3 22C
Postthaw 3 storage, 1 hr, 0 C
10.0C" 30.3 b 58.2 a 58.3 a 58.8 a 60.3 a 60.0 a 59.2a 58.7 a 58.3 a
8.7c4 22.3bc 44.8a 49.0a 42.0ab 45.3a 40.7ab 48.2a 40.3 a b 37.0ab
4.0d 4 5.7^ 13.7" 1 2iybcd 30.8abc 39.0ab 40.8 a 43.7a 36.7ab 29.5abc
4.7C4 11.2 b c 28.3ab 28.5 a b 37.8 a 37.8 a 39.8a 33.7a 28.7ab 24.2 a b
Mean SD
51.2 18
37.8 16
26.6 16
27.5 15
' ' ' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 'Extender formula: 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES, .353 sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml, pH 7.2, with 9.6% cryoprotectant after dilution (dimethylsulfoxide and ethylene glycol v/v, 1:1 ratio). 2
Holding time of 6 min (22 C), diluted 1:4 in extenders (22 C) mixed and equilibrated 30 min (O C).
3
As 75 Ail pellets frozen on solid C0 2 and stored at —196 C.
4
Mean of 6 replicates.
s p e r m a t o z o a is n o t well defined. Graham and Brown ( 1 9 7 1 ) r e p o r t e d n o difference in fertility with 300, 3 2 5 , 350, 4 0 0 , or 4 5 0 m O s m / kg for fresh e x t e n d e d semen b u t r e p o r t e d a significantly higher p e r c e n t hatch of fertile eggs with 90.6% (325 m O s m / k g ) vs. 73.7% ( 3 5 0 m O s m / k g ) vs. 7 1 . 5 % ( 3 0 0 m O s m / k g ) . Watanabe and K a t o ( 1 9 7 0 ) reported greater recovery of motile s p e r m a t o z o a with 5% glucose ( 3 1 7 m O s m / k g ) t h a n with 7% ( 4 4 1 m O s m / k g ) or 9% glucose ( 5 7 5 m O s m / k g ) , b u t 7% glucose p r o vided t h e lowest p e r c e n t of a b n o r m a l sperm a t o z o a . Our s t u d y indicated t h a t osmotic pressures of 5 5 0 and 5 8 0 m O s m / k g decreased t h e p e r c e n t m o t i l e cells for b o t h fresh and frozen-thawed semen, while there were n o significant differences for t h e o t h e r o s m o t i c pressures studied ( 3 1 0 t o 5 2 0 m O s m / k g range). T h e definitive assay of semen quality is t h e fertilizing capacity of spermatozoa, b u t fertility trials are expensive and t i m e consuming. We, therefore, utilized l a b o r a t o r y studies t o test a t o t a l of 74 t r e a t m e n t s for t h e seven trials analyzed. Many studies showed n o differences over several of t h e t r e a t m e n t s tested, b u t w e consistently i n c o r p o r a t e d t h e highest ranking
t r e a t m e n t for frozen-thawed semen. This enabled u s t o develop an e x t e n d e r system and techniques for freezing t u r k e y semen t h a t allowed frozen-thawed t u r k e y semen t o maintain a vigorous swirling motility similar t o unfrozen turkey semen. F u t u r e research requires evaluating this frozen-thawed semen with fertility trials.
REFERENCES Bajpai, P. K., and K. I, Brown, 1963a. The effect of egg yolk on semen characteristics of turkeys. Poultry Sci. 42:888-893. Bajpai, P. K., and K. I. Brown, 1963b. The effect of some diluents on semen characteristics of turkeys. Poultry Sci. 4 2 : 8 8 2 - 8 8 8 . Brown, K. I., and E. F. Graham, 1971. Effect of some cryophylactic agents on turkey spermatozoa. Poultry Sci. 50:832-835. Brown, K. I., E. F. Graham, and B. G. Crabo, 1972. Effect of some hydrogen ion buffers on storage and freezing of turkey spermatozoa. Poultry Sci. 51:840-849. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Graham, E. F., and K. I. Brown, 1971. Effect of
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580 550 520 490 460 430 400 370 340 310
FROZEN TURKEY SEMEN EXTENDER
Sexton, T. J., 1979b. Effect of prefreeze procedure on the fertility of frozen turkey semen. Poultry Sci. 58:1106. Sexton, T. J., 1980. Recent advances in semen storage of the fowl and turkey. IX Int. Congr. Anim. Reprod. A. I., Madrid, 2:527-533. Sexton, T. J., M. R. Bakst, and H. Cecil, 1979. Reproductive efficiency of turkeys. In; Annu. Rep. Western Reg. Coop. Turkey Res. Proj. Steel, R.G.D. and J. H. Torrie, 1960. Principles and procedures of statistics. McGraw-Hill, New York, NY. Watanabe, M. and S. Kato, 1970. Studies on deep freezing preservation of turkey semen. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 9:133—138. Watanabe, M., and Y. Miyashima, 1973. Fertility of turkey spermatozoa frozen for two years. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 12:117— 120. Zavos, P. M., R. Hanson, and E. F. Graham. 1976. The effects of the degree of supercooling on motility and fertility of frozen turkey spermatozoa. Poultry Sci. 55:2110.
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osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Macpherson, J. W., S. Chatterjee, and G. W. Friars, 1969. Frozen turkey semen. Can. J. Comp. Med. 33:37-38. Nagase, H., E. F. Graham, and T. Niwa, 1964. Pelleted semen: The effect of glycerol level and composition of thawing solution on fertility of bovine spermatozoa. V. Int. Congr. Anim. Reprod. A. I., Trento, 4:404-409. Oderkirk, A.H.F., and R. B. Buckland, 1977. A comparison of diluents and cryopreservatives for freezing turkey semen. Poultry Sci. 56:1861— 1867. Rajamanan, A.H.J., 1968. A new media and technique for freezing turkey semen. VI Int. Congr. Anim. Reprod. A. I., Paris, 2:1641-1643. Schefels, W., 1978. Freezing and storage of turkey semen. Zuchthygiene 13:81. Sexton. T. J., 1979a. Cytotoxicity of DMSO as related to components of a turkey semen extender. Poultry Sci. 58:1024.
557
1982 Poultry Science 61:550-557 INTRODUCTION D e v e l o p m e n t of an e x t e n d e r system tliat allows frozen-thawed t u r k e y s e m e n t o m a i n t a i n t h e vigorous swirling motility d e m o n s t r a t e d by nonfrozen semen is complicated. C o m m o n c r y o p r o t e c t a n t s for semen such as egg y o l k (Bajpai and Brown, 1963a), milk (Bajpai and Brown, 1 9 6 3 b ) , and glycerol (Brown and Graham, 1 9 7 1 ) , have detrimental effects on t u r k e y semen a n d fertility. Also, S e x t o n et al. (1979) reported that dimetiiylsulfoxide (Me^SO) levels of 10% or higher significantly depressed fertility of fresh t u r k e y semen. T h e p u r p o s e of this study was t o investigate ext e n d e r systems and techniques for m a x i m a l survival of s p e r m a t o z o a after freezing. MATERIALS AND METHODS Seven l a b o r a t o r y trials were c o n d u c t e d and
'Scientific Paper No. 11,282 of the Minnesota Agricultural Experiment Station, St. Paul, MN 55108. 2 [N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonic acid].
repeated four t o six times. Semen samples were collected by a b d o m i n a l massage (Burrows and Q u i n n , 1 9 3 7 ) at 22 C from Nicholas Large White and Wrolstad Medium White t u r k e y s (Meleagris gallopavo). Collected semen was pooled and gently m i x e d . Aliquots (.1 ml) were placed directly into e x t e n d e r s (.4 ml), held at 0 or 22 C, mixed 3 sec at m e d i u m speed on a vortex mixer, and equilibrated at 0 C. Ext e n d e r s were adjusted t o 3 7 0 m O s m / k g a t p H 7.2 e x c e p t Trial 7 ( 3 1 0 t o 5 8 0 m O s m / k g ) . Semen was frozen in 75 jul pellets o n solid C 0 2 (Nagase et al., 1964), held 10 min, and stored in liquid nitrogen. Pellets were t h a w e d individually in plastic vials a t 22 C, and percentages of motile s p e r m a t o z o a were assessed using closed circuit television microscopy. Samples were r a n d o m l y coded before motility estimates were m a d e by t w o trained technicians. T h e d a t a was d e c o d e d after c o m p l e t i o n of t h e e x p e r i m e n t s . Each trial was r e p e a t e d six times (different days) e x c e p t Trial 6 with only four replicates. Also, t h e 2 hr, 0 C p o s t t h a w samples in Trial 4 were repeated only three times. D a t a were subjected t o analysis of variance (randomized c o m p l e t e block design) and significant m e a n differences were de-
550
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ABSTRACT An extender for turkey semen with a frozen-thawed recovery of >50% motile spermatozoa and a vigorous swirl was developed. The effect of glucose, a comparison of 10 different sugars in the extender, the use of sodium acetate and potassium acetate, the ratio of dimethylsulfoxide to ethylene glycol, the percent total cryoproteetant, equilibration time, and osmotic pressure were all tested. Replacement of approximately half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other carbohydrates did not enhance recovery of frozenthawed semen. The proportion of sodium and potassium acetates to TESNaK2 in the extender had no effect on motility. Approximately equal proportions of ethylene glycol to dimethylsulfoxide, with a final concentration of 11.2% after dilution, produced at or near optimal recovery of spermatozoal motility after freezing. Twenty to 90 min equilibration times before freezing yielded significantly higher recovery of motile sperm postthaw than shorter periods. A wide range of extender osmotic pressures were compatible with recovery of spermatozoal motility postthaw. The final extender consisted of 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES, 3 .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H2 O q.s. 1000 ml, 370 mOsm/kg, pH 7.2. Ethylene glycol and dimethylsulfoxide were added (1:1 ratio) for a final concentration of 11.2% cryoproteetant after dilution. Semen was held undiluted for 6 min, extended 1:4 at 22 C, and equilibrated for 30 min at 0 C before pellet freezing. (Key words: turkey semen, semen extender, glucose, cryoproteetant, sperm motility)
FROZEN TURKEY SEMEN EXTENDER
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termined by Tukey's W procedure (Steel and Torrie, 1960). Trial 1. Effect of Glucose in the Extender. Eleven extenders consisting of 10.0 to 0:10 ratios of extender A (Table 1) and glucose solution (64.0 g/1, 370 mOsm/kg) were prepared. Dimethylsulfoxide (Me2 SO) and ethylene glycol (3:2 ratio) were added to the extenders yielding a final concentration of 8.3% after dilution. Pooled semen was diluted 1:5, equilibrated 10 min and frozen. Pellets were thawed and transferred to a 0 C water bath. Percentages of motile spermatozoa were assessed immediately for frozen-thawed semen and after 1, 5, and 8 hr of 0 C storage for fresh semen. Trial 2. Effect of Various Carbohydrates. Ten extenders were prepared consisting of one part extender D (Table 1) and one part of the following solutions (370 mOsm/kg): i-erythritol, D (—) arabinose, D (—) ribose, D (+) xylose, D (—) fructose, D (+) galactose, D (+) glucose, i-inositol, D (+) mannose, and D (+) lactose. Me 2 SO and ethylene glycol were added to each extender (1:1 ratio, 8.2% v/v after dilution). Semen was collected, diluted 1:4, equilibrated 35 min, and frozen. Pellets were thawed, assessed immediately, and assessed after 3 hr storage at 0 C. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 3. Ratio of Sodium and Potassium Acetates to TESNaK. Eleven extenders were prepared consisting of 10:0 to 0:10 ratios of TESNaK (Brown et al, 1972) to sodiumpotassium acetate solution (13.44 g sodium acetate and 9.69 g/1 potassium acetate, 370 mOsm/kg). One part of glucose solution (3 70 mOsm/kg) was added to one part of each extender. Cryoprotectant (Me2 SO and ethylene glycol, 1:1 ratio) was added at a concentration of 8.2% after dilution. Semen was collected, diluted 1:4 in extender, and frozen after 20 and 35 min equilibration. Pellets were thawed and motilities assessed. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 4. Ratio ofMe2SO to Ethylene Glycol. Eleven mixtures consisting of 10:0 to 0:10 ratios of Me 2 SO and ethylene glycol were added (8.3% v/v after dilution) to extender B (Table 1). Semen was collected, diluted 1:5, equilibrated 15 min and frozen. Semen was thawed, assessed immediately, and assessed again after 2 hr storage at 0 C. Fresh semen motilities were assessed after 5 hr storage at 0 C.
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GRAHAM ET AL. TABLE 2. Effect of ratio of glucose solution (370 mOsm/kg) to TESNaK acetate extender1 (370 mOsm/kg) on frozen and fresh turkey semen % Motility Fresh storage, 1 hr, O C2
Fresh storage, 5 hr, O C
Fresh storage, 8 hr, O C
Postthaw, 3 22 C
0:10 1:9 2:8 3:7 4:6 5:5 6:4 7:3 8:2 9:1 10:0
51.0ab 4 50.7 a b 55.2 a b 52.2 a b 51.8 a b 57.3 a 58.5 a 53.0 a b 51.3ab 44.8bc 38.0C
9.8d 4 16.0 c d 18.0 c d 18.0 c d 32.oabc 38.8ab 45.0 a 47.0 a 50.0 a 32.0abc 2i.obcd
7. 4 e 4 8.4 e 170cde 170cde 20.0cde 27.4bcd 37.4 a b 43.0ab 46.0 a 31.0abc 26.0bcd
21.8bc" 21.0 b c 27 2 a b c 28.5abc 29.3ab 32.8 a 29.0abc 26.7abc 29.3ab 23.7abc 20.0 C
Mean SD
51.4 8
29.8 16
25.0 15
26.3 8
3. h o (i t*
' ' ' ' Means in the same vertical line not followed by the same letter are significantly different (P<.05).
1
Extender formula: 12.10 g sodium acetate, 8.72 g potassium acetate, 6.15 g TES, .235 g sodium hydroxide, .328 g potassium hydroxide, H 2 0 q.s. 1000 ml. 2 Diluted 1:5 in 370 mOsm/kg and pH 7.2 extender with dimethylsulfoxide (5.0% final volume) and ethylene glycol (3.3% final volume). 3
As 75-jul pellets frozen on solid CO, and stored at —196 C.
"Mean of 6 replicates.
TABLE 3. Effect of adding various carbohydrate solutions (1.1 ratio) to acetate-TESNaK extender1 on frozen and fresh turkey semen2 % Motility Postthaw 3 storage, 3 hr, O C
Fresh storage, 1 hr, O C
Fresh storage, 5 hr, O C
i-Erythritol D(—) Arabinose D ( - ) Ribose D(+) Xylose D(—) Fructose D(+) Galactose D(+) Glucose i-lnositol D(+) Mannose D(+) Lactose
50.8 b " 61.2 a 60.3 a 61.7 a 64.2 a 63.7 a 60.0 a 61.5 a 62.8 a 62.3 a
14.3 b 4 40.7 a 38.7 a 42.0 a 46.5 a 48.2 a 48.5 a 51.5 a 47.3 a 40.0 a
21.8 b " 28.0 a b 30.2 a b 28.5ab 33.5* 34.7 a 29.2ab 28.5ab 28.2ab 27.5ab
5.7b4 12.7 a b 12.3ab 12.7 a b 15.7ab 17.3 a 17.5 a 22.0 a 18.2 a 19.0 a
Mean SD
61.5 5
41.7 13
29.1 7
15.3 6
Carbohydrate
Postthaw, 3 22 C
' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 1
Extender formula: 9.41 g sodium acetate, 6,78 g potassium acetate, 18.45 g TES, .706 g sodium hydroxide, .984 g potassium hydroxide, H2 O q.s. 1000 ml. 2 Diluted 1:4 in 370 mOsm/kg and pH 7.2 extender with 8.2% final cryoprotectant (dimethylsulfoxide and ethylene glycol, 1:1 ratio). 3
Semen was equilibrated 35 min, frozen in 75 ul pellets on solid CO a , and stored at —196 C.
4
Mean of 6 replicates.
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Ratio of glucose solution to extender
FROZEN TURKEY SEMEN EXTENDER
semen was held 6 min (22 C), diluted 1:4, mixed, equilibrated 30 min and frozen. Pellets were thawed and assessed immediately and after 1 hr storage at 0 C. Fresh semen was assessed after 1.5 and 5 hr storage at 0 C. RESULTS Trial 1. A 5:5 ratio of glucose solution to the extender (Table 2) supported significantly higher (P<.05) recovery of motility by frozenthawed spermatozoa than 0:10, 1:9, or 10:0. Also, 5:5 and 6:4 glucose solution to extender ratios yielded the highest percent motile spermatozoa after storage of fresh semen for 1 hr at 0 C, while 8:2, 7:3, and 6:4 glucose solution ratios supported the highest percent motile spermatozoa after storage of fresh semen for 5 hr at 0 C. Therefore, a 5:5 ratio of glucose solution (6.7 g %) to extender was used for die remaining trials. Trial 2. Replacement of glucose in the diluent with different carbohydrates (Table 3) did not significantly improve recovery of motility by frozen-thawed spermatozoa, nor
TABLE 4. Effect of ratio of cryoprotective agents added to glucose-acetate-TESNaK extender1 on frozen and fresh turkey semen Cryoprotective agents (ratio) Dimethylsulfoxide 10 9 8 7 6 5 4 3 2 1 0 Mean SD
% Motility Ethylene glycol 0 1 2 3 4 5 6 8 8 9 10
Fresh storage, 5 hr, O C
Postthaw, 2 22 C
Postthaw 2 storage, 2 hr, O C
13.8 C " 17.2bc 23.7 a bc 23.5 a bc 29.3 a b 34.3 a 33.3 a 28.7 a b 32.8 a 26.5 a bc 34.0 a
23.5abc 4 25.8abc 26.8 a b 27.oabc 25.7 a b 30.7 a 27.2ab 19gabc 18.0bc 14.3 C 13.8 C
1 2 . 3aa b 3 18.3 b 14.7ab 17.3ab 25.0 a 24.7 a 20.0ab 16.7ab 14.0 a b 11.3ab 6.7 b
27.0 14
23.0 9
16.5 7
' ' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 'Diluted 1:5 in 370 mOsm/kg extender, pH 7.2, 8.3% total cryoprotectant after dilution. Extender formula: 6.05 g sodium acetate, 4.36 g potassium acetate, 3.08 g TES, .118 g sodium hydroxide, .164 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml. 2 As 75-jul pellets frozen on solid C0 2 and stored at —196 C. 3 Mean of 3 replicates. 4 Mean of 6 replicates.
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Trial 5. Total Percent of Me2SO and Ethylene Glycol (1.1 ratio). Extender C (Table 1) was prepared with 11 concentrations of cryoprotectant added, ranging from 0 to 16.0% v/v after dilution. Semen was collected, diluted 1:4, equilibrated 35 min, and frozen. Pellets were thawed, assessed immediately, and assessed after 1 hr storage at 0 C. Fresh semen was assessed after 1 and 5 hr storage at 0 C. Trial 6. Equilibration Time. Semen was collected, diluted 1:4 in extender B, and equilibrated 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 min before freezing. Cryoprotectant (Me 2 SO and ethylene glycol, 1:1 ratio) was added for a final concentration of 8.2% v/v after dilution. Pellets were thawed and motilities assessed immediately and after 1 hr storage at 0 C. Trial 7. Osmotic Pressure. Extender C was prepared at the following osmotic pressures: 580, 550, 520, 490, 460, 430, 400, 370, 340, and 310 mOsm/kg and cryoprotectant (Me 2 SO and ethylene glycol, 1:1 ratio) was added for a 9.6% concentration after dilution. Pooled
553
554
GRAHAM ET AL. Trial 7. Results indicated (Table 7) that osmotic pressure over a wide range supported motility in frozen-thawed spermatozoa. Osmotic pressures of 550 and 580 mOsm/kg severely decreased percent motile spermatozoa (P<.01) in fresh semen samples stored for 1.5 and 5 hr and in both the 0 and 1 hr postthaw samples. DISCUSSION A high percentage of glucose in an extender for freezing turkey semen has been used. Watanabe and Kato (1970) studied the use of 5, 7, and 9 g % glucose as a diluent for freezing semen and reported good recoveries of frozenthawed semen with less abnormal spermatozoa with the 7 g % glucose diluent. Watanabe and Miyashima (1973) reported 3.8% fertility with semen frozen in glucose (6.5 g %) egg yolkglycerol diluent and stored for 2 years in liquid nitrogen. Our results indicated that incorporating some glucose in the extender formula was beneficial. However, 6.4 g % glucose produced lower postthaw recovery of motile spermatozoa than glucose used with Extender A (Table 1). The 3.2 g % glucose proportion
TABLE 5. Effect of adding various percents of cryoprotectant (dime thy Isulfoxide and ethylene glycol, 1:1 ratio) to glucose-acetate-TESNaK extender1 on frozen and fresh turkey semen Cryoprotectant
% in Buffer 0 2 4 6 8 10 12 14 16 18 20 Mean SD
% After dilution 0 1.6 3.2 4.8 6.4 8.0 9.6 11.2 12.8 14.4 16.0
% Motility Fresh storage, 1 hr, O C
Fresh storage, 5hr, OC
Postthaw 3 22 C
Postthaw 3 storage, 1 hr, O C
65.7a" 65.2ab 65.2ab 64.3ab 64.3ab 61.7ab 61.3ab 63.7ab 64.3ab 64.3ab 59.2 b
59.8 a " 59.5 a 56.2abc 58.2 a 57.8 a b 57.8 a b 57.3abc 58.7 a 55.3abc 50.3bc 49.8C
1.0C4 4.5C 13.3 C 27.3 b 28.7 b 40.3ab 45.8 a 49.2 a 46.2 a 44.2 a 42.5 a
1.2d4 3.7 d 13.5 c d 24.8abc 27.8ab 30.8 a b 30.0 a b 37.5 a 27.3abc 25.0abe 20.8 b c
63.3 4
56.4 5
31.2 18
22.0 13
' ' ' Mean in the same vertical line not followed by the same letter are significantly different (P<,05). Extender formula: 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 gTES, .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 glucose, H 2 0 q.s. 1000 ml. 2 Diluted 1:4 in extender (22 C) with 370 mOsm/kg and pH 7.2. 3 Semen was equilibrated 35 min, frozen in 75 p\ pellets on solid C0 2 , and stored at —196 C. 4 Mean of 6 replicates. 1
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did they improve motility in fresh stored semen samples. Trial 3. There was no significant difference in diluents using different proportions of sodium and potassium acetate to TESNaK. Average motilities of all treatments ranged from 57 to 65% for fresh samples stored 1 hr, 41 to 52% for fresh samples stored 5 hr, and 22 to 29% for postthaw semen samples. Trial 4. Results (Table 4) indicated approximately equal proportions of Me 2 SO, and ethylene glycol produced recovery of frozenthawed spermatozoa equal to or better than other proportions, so a ratio of 1:1 Me 2 SO to ethylene glycol was adopted as our standard cryoprotective agent. Trial 5. Results indicated (Table 5) that 11.2% v/v of cryoprotectant after dilution yielded at or near maximal motility for postthaw frozen spermatozoa for both times tested. Results were variable, however. Trial 6. The results (Table 6) indicated no differences over a wide range of times. In general, recovery of motility by frozen-thawed spermatozoa was poorest at the extremes of the times tested.
FROZEN TURKEY SEMEN EXTENDER TABLE 6. Effect of equilibration time with cryoprotective agents1 in glucose-acetate-TESNaK extender2 on frozen turkey semen % Motility
i
Equilibration time prior to freeze (rain)
Mean SD
Postthaw 3 storage, 1 hr, O C
7.0b 4 19.3ab 23.5 a 25.3 a 29.3a 28.5 a 19.8 a b 21.oa 16.3 a b 16.3 a b
4.8b 4 13.5 a b 19.0 a 21.5a 23.5 a 17.8 a 20.0 a 16.5ab 13.5 a b 13.Sab
20.1 9
16.4 8
a ' b Mean in the same vertical line not followed by the same letter are significantly different (P<.01). 1 8.2% Cryoprotectant after dilution (dimethylsulfoxide, ethylene glycol, 1:1 v/v). 2 Diluted 1:4 in extender with 370 mOsm/kg and pH 7.2. Extender formula: 6.05 g sodium acetate, 4.36 g potassium acetate, 3.08 g TES, .118 g sodium hydroxide, .164 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml. 3 As 75 /il pellets frozen on solid C0 2 and stored at - 1 9 6 C. 4
Mean of 4 replicates.
was chosen because it showed the highest postthaw recovery of motile spermatozoa. Replacement of glucose in our extender with different carbohydrates did not enhance recovery of motile spermatozoa. Others have reported using different carbohydrates with some degree of success. Oderkirk and Buckland (1977) compared various extenders which contained fructose, raffinose, or lactose. Percent fertility ranged from 3.7 to 20.7, depending on extender, technique, and cryoprotectant used. The 20.7% fertility was obtained using an extender containing lactose. Schefels (1978) reported 63% overall fertility (95 eggs) using a fructose-yolk-glycerol extender with 84% fertility of eggs laid after the first insemination with frozen semen. Further studies regarding the effects of carbohydrates are needed. Acetate was selected for use in the semen
diluent because it is an intermediate in many metabolic reactions and provides a substrate for spermatozoa. Preliminary studies indicated a 1:1 mixture of sodium acetate and potassium acetate solutions (370 mOsm/kg) maintained a high percent of motile spermatozoa. Since acetates are weak buffers, TESNaK was added to stabilize the pH of the diluent. Also, according to Sexton (1979a), omission of TES, sodium acetate, and other salts from extenders increased cytotoxicity of Me 2 SO to turkey spermatozoa. The proportions of sodium and potassium acetates and TESNaK in extender C (Table 1) maintained a stable pH of 7.2 with a high percentage of motile spermatozoa. Various cryoprotectants have been used by others. Macpherson et al. (1969) used a combination of 1.7% glycerol with 8.3% ethylene glycol and 1.7% glycerol with 3.3% N, N-dimethylacetamide by volume before diluting 1:6. The latter, with dialysis to remove the cryoprotectant, yielded 24% fertility with frozen semen. Zavos et al. (1976) reported 26.0% fertility for frozen semen with 6% ethylene glycol and 6% Me 2 SO by volume before diluting 1:4. Sexton (1980) reported fertility with semen frozen to —20 C increased from 27% (1st week) to 50% (4th week) over a 4-week egg production period. Their techniques utilized 4% Me 2 SO in Beltsville Poultry Semen Extender. Brown and Graham (1971) reported 6% glycerol in extended semen (diluted 1:2, 1/30 ml insemination dose) decreased fertility in fresh turkey semen with 10.2% fertility vs. 81.0% for the control. Fertility for ethylene glycol and Me 2 SO under the same conditions were 74.8 and 91.0%, respectively. In our laboratory, motility recovery of frozen-thawed semen with the ethylene glycol, Me 2 SO combination equaled glycerol (unpublished data). Optimum equilibration times before the freezing of semen have been reported. Rajamannan (1968) tested equilibration times of 5, 10, and 15 min before vapor (straw), spin (ampule), and film freezings and reported 10 min yielded the highest recovery of motile spermatozoa. Sexton (1979b) tested equilibration times of 10 and 60 min with 10 min being the optimum for his procedures. Our data indicated no differences within the range 10 to 90 min, but extender composition, prefreeze handling, and freezing techniques were different in our experiments than in those used by the above researchers. The optimum osmotic pressure for turkey
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0 10 20 30 40 50 60 70 80 90
Postthaw, 3 22 C
555
GRAHAM ET AL.
556
TABLE 7. Effect of osmotic pressure of extenders1
on frozen and fresh turkey semen % Motility
Osmotic pressure mOsm/kg
Fresh2 storage, 1.5 hr, O C
Fresh storage, 5 hr, O C
Postthaw, 3 22C
Postthaw 3 storage, 1 hr, 0 C
10.0C" 30.3 b 58.2 a 58.3 a 58.8 a 60.3 a 60.0 a 59.2a 58.7 a 58.3 a
8.7c4 22.3bc 44.8a 49.0a 42.0ab 45.3a 40.7ab 48.2a 40.3 a b 37.0ab
4.0d 4 5.7^ 13.7" 1 2iybcd 30.8abc 39.0ab 40.8 a 43.7a 36.7ab 29.5abc
4.7C4 11.2 b c 28.3ab 28.5 a b 37.8 a 37.8 a 39.8a 33.7a 28.7ab 24.2 a b
Mean SD
51.2 18
37.8 16
26.6 16
27.5 15
' ' ' Means in the same vertical line not followed by the same letter are significantly different (P<.01). 'Extender formula: 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES, .353 sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H 2 0 q.s. 1000 ml, pH 7.2, with 9.6% cryoprotectant after dilution (dimethylsulfoxide and ethylene glycol v/v, 1:1 ratio). 2
Holding time of 6 min (22 C), diluted 1:4 in extenders (22 C) mixed and equilibrated 30 min (O C).
3
As 75 Ail pellets frozen on solid C0 2 and stored at —196 C.
4
Mean of 6 replicates.
s p e r m a t o z o a is n o t well defined. Graham and Brown ( 1 9 7 1 ) r e p o r t e d n o difference in fertility with 300, 3 2 5 , 350, 4 0 0 , or 4 5 0 m O s m / kg for fresh e x t e n d e d semen b u t r e p o r t e d a significantly higher p e r c e n t hatch of fertile eggs with 90.6% (325 m O s m / k g ) vs. 73.7% ( 3 5 0 m O s m / k g ) vs. 7 1 . 5 % ( 3 0 0 m O s m / k g ) . Watanabe and K a t o ( 1 9 7 0 ) reported greater recovery of motile s p e r m a t o z o a with 5% glucose ( 3 1 7 m O s m / k g ) t h a n with 7% ( 4 4 1 m O s m / k g ) or 9% glucose ( 5 7 5 m O s m / k g ) , b u t 7% glucose p r o vided t h e lowest p e r c e n t of a b n o r m a l sperm a t o z o a . Our s t u d y indicated t h a t osmotic pressures of 5 5 0 and 5 8 0 m O s m / k g decreased t h e p e r c e n t m o t i l e cells for b o t h fresh and frozen-thawed semen, while there were n o significant differences for t h e o t h e r o s m o t i c pressures studied ( 3 1 0 t o 5 2 0 m O s m / k g range). T h e definitive assay of semen quality is t h e fertilizing capacity of spermatozoa, b u t fertility trials are expensive and t i m e consuming. We, therefore, utilized l a b o r a t o r y studies t o test a t o t a l of 74 t r e a t m e n t s for t h e seven trials analyzed. Many studies showed n o differences over several of t h e t r e a t m e n t s tested, b u t w e consistently i n c o r p o r a t e d t h e highest ranking
t r e a t m e n t for frozen-thawed semen. This enabled u s t o develop an e x t e n d e r system and techniques for freezing t u r k e y semen t h a t allowed frozen-thawed t u r k e y semen t o maintain a vigorous swirling motility similar t o unfrozen turkey semen. F u t u r e research requires evaluating this frozen-thawed semen with fertility trials.
REFERENCES Bajpai, P. K., and K. I, Brown, 1963a. The effect of egg yolk on semen characteristics of turkeys. Poultry Sci. 42:888-893. Bajpai, P. K., and K. I. Brown, 1963b. The effect of some diluents on semen characteristics of turkeys. Poultry Sci. 4 2 : 8 8 2 - 8 8 8 . Brown, K. I., and E. F. Graham, 1971. Effect of some cryophylactic agents on turkey spermatozoa. Poultry Sci. 50:832-835. Brown, K. I., E. F. Graham, and B. G. Crabo, 1972. Effect of some hydrogen ion buffers on storage and freezing of turkey spermatozoa. Poultry Sci. 51:840-849. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Graham, E. F., and K. I. Brown, 1971. Effect of
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Sexton, T. J., 1979b. Effect of prefreeze procedure on the fertility of frozen turkey semen. Poultry Sci. 58:1106. Sexton, T. J., 1980. Recent advances in semen storage of the fowl and turkey. IX Int. Congr. Anim. Reprod. A. I., Madrid, 2:527-533. Sexton, T. J., M. R. Bakst, and H. Cecil, 1979. Reproductive efficiency of turkeys. In; Annu. Rep. Western Reg. Coop. Turkey Res. Proj. Steel, R.G.D. and J. H. Torrie, 1960. Principles and procedures of statistics. McGraw-Hill, New York, NY. Watanabe, M. and S. Kato, 1970. Studies on deep freezing preservation of turkey semen. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 9:133—138. Watanabe, M., and Y. Miyashima, 1973. Fertility of turkey spermatozoa frozen for two years. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 12:117— 120. Zavos, P. M., R. Hanson, and E. F. Graham. 1976. The effects of the degree of supercooling on motility and fertility of frozen turkey spermatozoa. Poultry Sci. 55:2110.
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osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Macpherson, J. W., S. Chatterjee, and G. W. Friars, 1969. Frozen turkey semen. Can. J. Comp. Med. 33:37-38. Nagase, H., E. F. Graham, and T. Niwa, 1964. Pelleted semen: The effect of glycerol level and composition of thawing solution on fertility of bovine spermatozoa. V. Int. Congr. Anim. Reprod. A. I., Trento, 4:404-409. Oderkirk, A.H.F., and R. B. Buckland, 1977. A comparison of diluents and cryopreservatives for freezing turkey semen. Poultry Sci. 56:1861— 1867. Rajamanan, A.H.J., 1968. A new media and technique for freezing turkey semen. VI Int. Congr. Anim. Reprod. A. I., Paris, 2:1641-1643. Schefels, W., 1978. Freezing and storage of turkey semen. Zuchthygiene 13:81. Sexton. T. J., 1979a. Cytotoxicity of DMSO as related to components of a turkey semen extender. Poultry Sci. 58:1024.
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