Development of Extender and Techniques for Frozen Turkey Semen. 2. Fertility Trials 1

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1 Development of Extender and Techniques for Frozen Turkey Semen. 2. Fertility Trials 1 E. F. GRAHAM, D. S. NELSON, and M. K. L. SCHMEHL Department of Animal Science, University of Minnesota, St. Paul, Minnesota (Received for publication July 2, 1980) ABSTRACT The effects of cryoprotectant concentrations, redilution, centrifugation, and dialysis techniques on fertility of fresh and frozen-thawed turkey semen were tested. Fresh extended semen with final concentrations of 4.8, 5.6, 9.6, and 11.2% cryoprotectant (dimethylsulfoxide and ethylene glycol, 1:1 ratio) showed a significant (P<.05) decrease in fertility when compared to semen without cryoprotectant. Fertility of fresh semen extended with 4.8% cryoprotectant when centrifuged or rediluted and centrifuged, was unaffected when compared to cryoprotectant treated semen. Fertility of frozen-thawed semen after redilution and centrifugation was low, but samples rediluted one part semen for two parts extender yielded significantly (P<.05) higher fertility than semen rediluted 1:1. Fresh semen extended with 11.2% cryoprotectant showed a highly significant (P<.01) decrease in fertility despite dialysis, while semen with 9.6% cryoprotectant showed no difference when compared to dialyzed semen without cryoprotectant. Fertility of frozen-thawed semen dialyzed against blood plasma CVpH 7.9) and blood plasma adjusted with TES 2 to ph 7.4 or ph 7.2 was not significantly different. Fertility of frozen-thawed semen dialyzed for 30 min was significantly higher (P<.01) than semen dialyzed for 15 min or 0 min. Frozen-thawed semen dialyzed with a semen to dialysate ratio of 1:15 and 1:24 supported significantly higher (P<.01) fertility than at 1:50 ratio. The AGPT 3 dialysate yielded significantly higher (P<.01) fertility than blood plasma adjusted to ph 7.2 with TES. (Key words: turkey semen, cryoprotectant, ph, dialysis, fertility) INTRODUCTION Many different approaches to obtaining acceptable fertility with frozen-thawed turkey semen were reported in recent years. Zavos et al. (1976) reported 50.0% fertility with semen cooled to 20 C with nucleation at 4 versus 26.0% for semen frozen to 196 C. Both treatments utilized 6% dimethylsulfoxide Me 2 SO and 6% ethylene glycol as cryoprotectants. Schefels (1978) reported 63% fertility (95 eggs) using fructose-yolk-glycerol diluent for frozen semen. Sexton et al. (1979) reported 50% fertility with the 4th week of insemination for semen frozen to 20 C with 4% Me 2 SO. The objective of this study was to test the fertility of turkey semen frozen with the extender and techniques developed by Graham 'Scientific paper no. 11,282 of the Minnesota Agricultural Experiment Station, St. Paul, MN [N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonic acid]. 'Amino acids-glucose-phosphate-tesnak Poultry Science 61: et al. (1981) with recovery of >50% motility of frozen thawed semen. METHODS AND MATERIALS Source and Handling of Turkeys. Mature Wrolstad Medium White Turkeys were caged individually, fed ad libitum, and were exposed to 14 hr artificial light per day. Hens were intravaginally artificially inseminated with approximately 300 million spermatozoa (.15 ml). All fresh semen treatments were inseminated within 2 hr after semen collection. All frozen semen treatments were inseminated within 1 hr after thawing. Inseminations were done on days 1, 4, 8, and 15. Eggs were collected and set daily for 3 weeks. Percent fertility was determined by candling after 10 days incubation. Semen was collected from individual toms and held undiluted for 6 min (22 C), diluted 1:4 in extender with cryoprotectant (1:1 ratio of Me 2 SO to ethylene glycol), equilibrated 30 min (0 C), frozen in pellets on solid C0 2 (Nagase et al, 1964), and stored at -196 C. One pellet was thawed at 22 C for 5 min in plastic vials 558

2 FERTILITY AND FROZEN SEMEN EXTENDER 559 and placed in a 0 C bath. Motilities were assessed immediately and after 1 hr. Motility estimates of >50% with visual swirls were required before using the frozen semen for the fertility trials. Fresh pooled semen was also held, diluted, mixed, and equilibrated 30 min (0 C) before insemination. Extender. The extenders used consisted of 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES: [N-tris (hydroxymethyl) methyl-2-aminoethane-sulfonic acid],.353 g sodium hydroxide,.492 g potassium hydroxide, g glucose, and water q.s ml, ph7.2, 370mOsm/kg. Redilution. Semen was rediluted by adding 100 jul aliquots of extender without cryoprotectant to semen gently swirling on magnetic mixers at 5 C. Semen was inseminated or centrifuged and inseminated immediately after redilution. Centrifugation. Semen was put in IMV 4 Turkey insemination straws, which were sealed on one end with crito caps (size K) 5. Samples were centrifuged at 2,750 g for 10 min in a hematocrit centrifuge. The supernatant was removed using a syringe with needle and the packed cells were gently mixed. All semen handling was performed at 5 C. Semen was inseminated immediately. Dialysis. Three dialysates were prepared using heparinized turkey blood that had been centrifuged for 20 min at 13,200 g. Blood plasma (BP) only (335 mosm/kg, ph ) along with titrations of TES:N-tris (Hydroxymethyl) methyl-2 aminoethane sulfonic acid, 320 mosm/kg, ph 3.5, to a final ph of 7.2 and 7.4 were tested. Pellets were thawed individually and pooled immediately before dialysis. Also, amino acids-glucosephosphate-tesnak dialysate (AGPT) was prepared consisting of: 7.02 g glutamic acid;.35 g each of aspartic acid, serine, glycine;.26 g each of arginine, threonine, alanine;.18 g each of leucine, lysine;.07 g each of histidine, methionine, tyrosine, phenylalanine, isoleucine;.80 g potassium phosphate (monobasic); 4.56 g sodium phosphate (dibasic, heptahydrate); g glucose; 6.15 g TES:N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid; 4 IMV International Corp., 6120 Earl Brown Drive, Minneapolis, MN Brunswick Company, St. Louis, MO g sodium hydroxide, and.3 3 g potassium hydroxide. Various methods of dialysis were tested in preliminary studies. The method used for all dialysis treatments was as follows. Pellets were thawed individually and pooled immediately before dialysis. Cellulose dialysis tubing (25 mm flat width) was washed in buffer before filling with semen and knotting the ends. Bags of semen sealed in vials containing the dialysate were placed on a rotary mixer (5 C). Dialysate was changed at 5 min intervals. Semen to dialysate ratios were 1:15, 1:24 or 1:50. Experimental Treatments Percent Cryoprotectant in Fresh Semen. Fresh semen was collected and diluted 1:4 in extender with either 0, 4.8, 5.6, 9.6, or 11.2% cryoprotectant after dilution. Hens were inseminated with.15 ml of extended semen. Redilution and Centrifugation. Semen was extended 1:4 for a final cryoprotectant level of 4.8% (v/v) for both fresh and frozen semen. Treatments were: 1) fresh semen control; 2) fresh semen, centrifuged; 3) fresh semen, rediluted 1:1, centrifuged; 4) frozen-thawed semen, rediluted 1:1, centrifuged; and 5) frozen-thawed semen, rediluted 1:2 parts extender, centrifuged. Dialysis of Fresh Semen. Fresh semen with 0, 9.6, and 11.2% cryoprotectant was dialyzed against blood plasma dialysate before insemination. Dialysate ph. Frozen semen was prepared as TABLE 1. Effect of cryoprotectant 1 extender on fertility of fresh turkey semen Final % cryopro- % Fertectant eggs tility a b b C d ' ' ' Values in the same vertical line not followed by the same letter are significantly different (P<.05). ratio). 1 Dimethylsulfoxide and ethylene glycol (1:1 in

3 560 GRAHAM ET AL. TABLE 2. The effect of re dilution and centrifugation on fertility of fresh and frozen-thawed extender turkey semen with 4.8% cry oprotectant 1 Treatment Fresh semen Control, no centrifugation Centrifuged Rediluted 1:1, centrifuged % Fertility N S Frozen semen Rediluted 1:1, centrifuged Rediluted 1:2, centrifuged ab ' Values in the same vertical line not followed by the same letter are significantly different (P<.05). 1 Dimethylsulfoxide and ethylene glycol (1:1 ratio). above and dialyzed against blood plasma dialysates, ph 'W.9, and blood plasma was adjusted to ph 7.2 and 7.4 with TES. Semen was dialyzed, 1 part semen against 15 parts dialysate, for 15 and 30 min. Time of Dialysis of Frozen Semen. Frozen semen pellets were individually thawed in plastic beakers. The semen was pooled, and aliquots were dialyzed against blood plasma-tes for 0, 15, and 30 min before insemination. Semen to Dialysate Ratio. Frozen-thawed semen was dialyzed against blood plasma-tes dialysates for 30 min at semen to dialysate ratios of 1:15, 1:24, and 1:50. Inseminations were done immediately after dialysis. Dialysate Composition. Frozen-thawed semen was dialyzed against blood plasma-tes and APGT dialysates at a 1:50 ratio of semen TABLE 3. Effect of cry oprotectant 1 in extender on fertility of fresh turkey semen utilizing turkey plasma dialysate Final % cryoprotectant Fertility 2 81.l a 82.6 a 3.9 b a b ' Values in the same vertical line not followed by the same letter are significantly different (P<.01). 'Dimethylsulfoxide and ethylene glycol (1:1 ratio) oa 10.5 b to dialysate. Hens were inseminated immediately after 30 min of dialysis at 5 C. Experimental Evaluation. Fertility results were obtained by 10 day candling of eggs. Data were subjected to chi square analysis (Steele andtorrie, 1960). RESULTS Percent cryoprotectant in the extender (Table 1) significantly (P<.05) affected fertility of fresh extended turkey semen. A significant decrease (P<.05) was observed with 4.8 and 5.6% added cryoprotectant after dilution and a highly significant (P<.01) decrease in the percentage fertility was seen with 9.6 and 11.2% added cryoprotectant. Centrifugation and redilution followed by centrifugation (Table 2) did not change the percent fertility obtained with fresh extended semen with 4.8% cryoprotectant after dilution. Redilution of frozen-thawed semen (1:2 ratio) followed by centrifugation did yield significantly higher (P<.05) fertility than redilution, 1:1 ratio. However, the quality of semen frozen at this concentration of cryoprotectant was poor, and a low percentage of fertility was obtained. Fresh semen with 9.6% cryoprotectant (Table 3) showed no decrease in fertility when dialyzed against turkey blood plasma. A highly significant decrease (P<.01) in fertility was observed when semen with 11.2% cryoprotectant was dialyzed. There was no significant difference in fertility (Table 4) when frozen-thawed semen was

4 FERTILITY AND FROZEN SEMEN EXTENDER 561 TABLE 4. Effect of pll of blood plasma dialysate 1 on fertility of frozen-thawed and dialyzed turkey semen % FerpH eggs tility NS ^ 'TES:N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid was used to adjust the ph of blood plasma to 7.2 or 7.4. dialyzed against blood plasma ('vph 7.9) or blood plasma titrated to ph 7.2 or 7.4 with TES. However, the data suggests that adjusting the blood plasma ph may have some beneficial effects on fertility. Fertility of frozen-thawed semen improved with increased dialysis time (Table 5). Fertility after 30 min of dialysis was significantly better (P<.01) than no dialysis or 15 min of dialysis. The ratio of semen to dialysate affected fertility of frozen-thawed and dialyzed turkey semen (Table 6). A highly significant (P<.01) decrease in fertility was seen with semen dialyzed against 50 parts of dialysate. Composition of dialysate also affected fertility of frozen-thawed and dialyzed turkey semen (Table 7). The AGPT dialysate samples yielded significantly higher fertility (P<.01) than blood TABLE 5. Effect of dialysis time 1 on fertility of frozen-thawed turkey semen Dialysis % Fertime eggs tility 2 0 min a 15 min a 30 min l b ' Values in the same vertical line not followed by the same letters are significantly different (P<.01). 'Blood plasma dialysate. TES:N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid was used to adjust the ph of blood plasma to 7.2 or 7.4. plasma-tes dialysate samples. Unfortunately, this study was done before the detrimental effect of high semen to dialysate ratio was determined, and the 1:50 ratio was used in this study. DISCUSSION Fertility data for fresh extended semen with cryoprotectant generally agreed with results reported by others. Brown and Graham (1971) reported that neither 6% ethylene glycol nor 6% Me 2 SO after dilution, when used individually in extender with a 1/30 ml insemination dose, significantly decreased fertility when compared to inseminations with fresh semen. Sexton et al. (1979) reported that levels of 10% Me 2 SO v/v final concentration and 8% concentration after 20 min equilibration time depressed fertility. Our study indicated that both 9.6% and 11.2% (v/v) cryoprotectant after dilution (Me 2 SO, ethylene glycol, 1:1 ratio) with a.15 ml insemination dose significantly decreased fertility, while 4.8 and 5.6% did not significantly depress fertility. Redilution and centrifugation of frozen turkey semen was used to remove glycerol (Marquez and Ogasawara, 1977a; Oderkirk and Buckland, 1977). They reported obtaining some fertility after treatment. Ogasawara et al. (1979) reported a decrease in motility and normal cells after centrifugation. Marquez and Ogasawara (1977b) reported a loosening of the cytoplasmic membrane and some mitochondrial swelling due to the addition of glycerolated TABLE 6. The effect of semen to dialysate 1 on the fertility of frozen-thawed and dialyzed turkey semen Ratio semen: dialyzate 1:15 1:24 1:50 eggs ratio % Fertility a 41.l a 10.4 b ' Values in the same vertical line not followed by the same letters are significantly different (P<.01). 'Turkey blood plasma adjusted to ph 7.2 with TES. eggs. Eggs are collected for 3 weeks.

5 562 GRAHAM ET AL. TABLE 7. Effect of dialysate composition on fertility of frozen-thawed and dialyzed turkey semen Dialysate % Fertility 1 Blood plasma + TES, ph 7.2 AGPT dialysate * Fertility was determined by 10-day candling of 2 Amino acids-glucose-phosphate-tes NaK. *Significandy different (P<.01). extender to fresh semen before freezing. Bakst and Sexton (1979) reported damage to the ultrastructure of turkey spermatozoa by addition of extender, by cooling to 5 C, and by the addition of Me 2 SO; 80% of the spermatozoa exhibited plasmalemma damage as well as organelle displacement. Damage was much more severe after freezing. Evidently, prefreeze treatment of turkey semen creates greatly weakened cells before freezing and postthaw treatments begin. However, in our study, centrifugation and redilution of semen samples did not cause a decrease in fertility when compared to fresh extended control semen (Table 2). Fertility of frozen semen after redilution and centrifugation did not reflect damage due to freezing when compared to fresh semen treatments. Redilution at a 1:2 ratio, which decreased cryoprotectant levels to 1:6%, yielded significantly higher fertility than redilution at 1:1 ratio (2.4% cryoprotectant). Semen frozen with low cryoprotectant levels (redilution and centrifugation study) did not exhibit vigorously swirling motility postthaw as did semen frozen with 9.6 or 11.2% cryoprotectant after dilution. Therefore, dialysis was chosen as a method for lowering the cryoprotectant level postthaw. Dialysis to remove glycerol from frozen turkey semen has been used with some success. Macpherson et al. (1969) improved fertility of frozen turkey semen from 9 to 24% after reducing cryoprotectant levels by dialysis. Oderkirk and Buckland (1977) compared seven treatments for frozen turkey semen, and five included dialysis procedures. The highest fertility produced was 20.7%. Two extenders were later compared with and without dialysis, but no differences in treatments were seen. However, fertility was low, ranging from 1.4 to 4.6% for all four treatments. Data from trials with frozen and dialyzed semen indicated that dialysis techniques did affect fertility results (Tables 3 to 7). Dialysis time, ph, semen to dialysate ratio, and dialysate composition all influenced fertility data. The fertility decrease observed with the 1:50 semen to dialysate ratio (Table 6) suggests adverse effects of either removing cryoprotectant too rapidly or removing too much cryoprotectant. Possibly the optimum ratio lies between 1:24 and 1:50 and may be a critical factor. Unfortunately, the comparison of blood-piasma-tes and APGT dialysates were done simultaneously with the ratio study and the 1:50 ratio was used. Nevertheless, there was a highly significant difference in fertility for the two dialysates. Future work calls for improved techniques with consistent results. Dialysis could be a viable technique for the future, but optimum dialysate composition ratio and techniques need to be determined. REFERENCES Bakst, M. R., and T. J. Sexton, Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J. Reprod. Fertil. 55:1-7. Brown, K. I., and E. F. Graham, Effect of some cryophylactic agents on turkey spermatozoa. Poultry Sci. 50: Graham, E. F., D. S. Nelson, and M.K.L. Schmehl, Development of extender and techniques for frozen turkey semen. 1. Development. Poultry Sci. 61: Macpherson, J. W., S. Chatterjee, and G. W. Friars, Frozen turkey semen. Can. J. Comp. Med. 33: Marquez, B. J., and F. X. Ogasawara, 1977a. Effects of glycerol on turkey sperm viability and fertilizing capacity. Poultry Sci. 56: Marquez, B. J., and F. X. Ogasawara, 1977b. Ultrastructural changes in turkey spermatozoa after

6 FERTILITY AND FROZEN SEMEN EXTENDER 563 immersion in glycerolyzed media and during various steps used for cryopreservaaon. Poultry Sci. 56: Nagase, H., E. F. Graham, and T. Niwa, 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: Oderkirk, A.H.F., and R. B. Buckland, A comparison of diluents and cryopreservatives for freezing turkey semen. Poultry Sci. 56: Ogasawara, F. X., B. Ferrier, and L. Fuqua, Reproductive efficiency of turkeys. Annu. Rep. Western Reg. Coop. Turkey Res. Proj. Schefels, W., Freezing and storage of turkey semen. Zuchthygiene 13:81. Sexton, T. J., M. R. Bakst, and H. Cecil, Reproductive efficiency of turkeys. Annu. Rep. Western Reg. Coop. Turkey Res. Proj. Steele, R.G.D., and J. H. Torrie, Principles and procedures of statistics. McGraw-Hill, New York, NY. Zavos, P. M., R. Hanson, and E. F. Graham, The effects of the degree of supercooling on motility and fertility of frozen turkey spermatozoa. Poultry Sci. 55:2110. (Abstr.)