Alteration of Seminal Proteins during Freeze-Drying of Bovine Semen1

Alteration of Seminal Proteins During Freeze-Drying of Bovine Semen 1 R. S. JEYENDRAN, A. G. HUNTER, and E. F. GRAHAM Department of Animal Science University of Minnesota St. Paul 55108

ABSTRACT

Bovine semen in TEST-yolk extender was frozen, freeze-dried to 50, 25, 12, 6, and less than 2% residual moisture, and stored at - 1 9 6 ° C . The freeze-dried semen was rehydrated, sampled for protein analysis, and used to inseminate cattle. Agar gel electrophoresis revealed no seminal protein alteration until residual moisture was reduced to less than 6%. At that point, the percent of cationic proteins and neutral proteins decreased with a concurrent increase in anionic migrating proteins. Immunodiffusion data with antisera against spermatozoa and seminal plasma revealed no difference in formation of precipitin lines if residual moisture was at least 6%. However, semen freezedried to 2% residual moisture was modified antigenically as certain precipitin lines were lost, new lines appeared, and concentration of other seminal antigens decreased. The absence of fertility with semen freeze-dried to 2% residual moisture is hypothesized to be from alteration of the tertiary structure of certain essential seminal proteins. INTRODUCTION

Protein stability in an aqueous medium is dependent in part upon its bound water content (19). Removal of this water can lead to alteration in the protein's structure and function (6, 7). The first report on freeze-dried bovine spermatozoa was by Leidl (10). Since then several attempts to freeze-dry bovine spermatozoa have been made with limited success (1, 2, 4, 9, 11, 12, 14, 17, 18, 21).

Received November 12, 1981. aPaper No. 12,O03 of the Journal Series of the Minnesota Agricultural Experiment Station. 1983 J Dairy Sci. 66:887-891

Recently Jeyendran et al. (5) reported a 29% pregnancy rate with bovine semen freeze-dried to 6% residual moisture. However, no pregnancies were obtained with semen freeze-dried to less than 2% residual moisture. Based on results of that fertility trial and with freeze-dried semen from that fertility trial, a study was undertaken to determine if the stability of seminal proteins had been altered during freeze-drying.

MATERIALS AND METHODS Preparation of Freeze-Dried Semen

Samples were portions of those used by Jeyendran et al. (5) for fertility determinations of dehydrated bull semen. The semen had been collected from three Holstein bulls by artificial vagina, diluted 1 : 1 with glycerol-free TEST-yolk extender (3), and adjusted to a concentration of 200 × 106 spermatozoa per ml. The pellet freezing (13) and freeze-drying of these samples were described in (5). The 18 samples from three bulls consisted of semen that contained 100 (control), 50, 25, 12, 6, and less than 2% residual moisture. Each original ejaculate provided samples for each of the moistures. The frozen pelleted semen (400 mg sample) yielded 87% volatile material dehydrated. The residual moisture content of the sample was based on the fraction of removable volatiles. A 25% residual moisture sample, therefore, contained approximately 87 mg volatiles and 53 mg nonvolatiles. The frozen control semen was stored at - 5 0 ° C for the same time as the freeze-dried semen (approximately 3 wk). The control semen was thawed at 37°C and was rehydrated by placement of the pellets into 1.6 ml of 325 mOsm sodium citrate solution in a heavy aluminum tube at 37°C. The freeze-dried semen was rehydrated by addition of 2.25 ml of 230 mOsm sodium citrate. Osmolarity of rehydrated 887

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samples ranged from 300 to 335 mOsm and contained a total of 160 × 106 cells in approximately 2 ml. A portion of each thawed semen sample was used to inseminate cattle as part of a fertility study (5), and the remainder (18 sampIes) was analyzed for alteration of seminal protein.

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ao Electrophoretic Analysis

Agar-gel electrophoresis was run in duplicate on controls and semen freeze-dried to various residual moistures as described by Wieme (20) and quantitated by densitometry. The electrophoresis procedure was modified by substituting the TEST extender buffer (without egg yolk) for barbital buffer to maintain a pH of 7. Antigenic Analysis

Semen was collected from five Holstein bulls by artificial vagina, pooled, centrifuged at 6,620 x g for 5 rain, and seminal plasma removed. Spermatozoa were washed once with TEST extender (3) and adjusted to 1 × 109 sperm/ml. The seminal plasma was recentrifuged at 10,300 × g for 10 min. Antisera were induced in rabbits (two rabbits/antigen) against the one-time washed bovine spermatozoa, bovine seminal plasma, and chicken egg yolk. Normal serum was obtained from rabbits before immunization. The immunization scheme consisted of intradermal injections of .5 ml of antigen mixed with .5 ml of Freund's complete adjuvant into five sites in the rabbit's scapular region. This was followed by four weekly injections of .5 ml of antigen mixed with .5 ml of Freund's

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Figure 2. Effect of freeze-drying on electrophoretic composition of the proteins in bovine semen extended in TEST-yolk buffer (pH 7.0). All three bulls had similar composition changes which differed by no more than ±1%.

incomplete adjuvant. One week after the fourth injection, each rabbit was bled by venipuncture. Serum was recovered and frozen. The double diffusion m e t h o d of Ouchterlony (15) was used to visualize and semiquantitate antigens. The 18 semen samples extended in TEST-yolk buffer, that had been dehydrated to various residual moistures, were diluted 1:0, 1:1, 1:2, 1:4, 1:8, and 1:16 with 145 mM NaCI and reacted with the above antisera. Each antiserum was reacted twice with the 108 samples (3 bulls, 6 moistures, 6 dilutions). The highest dilution of antigen yielding a visible precipitin line with a constant amount of antisera was determined. RESULTS AND DISCUSSION

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O:o= (7 !: Figure 1. Electrophoretic ~attern of bovine semen extended in TEST-yolk buffer (pH 7.0). All three bulls had the same profile. Journal of Dairy Science Vol. 66, No. 4, 1983

The electrophoretic profile of bovine semen extended in TEST-yolk buffer consisted of seven protein-containing zones (Figure 1). This profile was the same for each of the three bulls. Prior to freeze-drying, composition of this semen at pH 7 was 55% anionic protein (zones 1, 2, and 3), 30% neutral protein (zones 4 and 5), and 15% cathodic migrating protein (zones 6 and 7). As water was removed from the semen during lyophilization, the percentage composition of the various proteins was not affected (P>.05) until the residual moisture was reduced to less than 6% (Figure 2). At 2% or less residual

FREEZE-DRIED SEMEN moisture, there was more (P<.01 by Student's t test) anionic migrating protein and less (P<.01) cathodic migrating protein than in the 100% residual moisture samples. Thus, the protein composition of the extended semen was altered by freeze-drying. Agar gel electrophoresis separates proteins by differences of electrical charge. The net charge on a protein is the sum of all reactive groups (i.e., NH3 +, C O O - , sialic acid residues, etc.) on the protein. Based on the data in Figure 2, we suggest that the secondary structure of certain proteins was altered by water removal. Because the agar gel buffer (TEST) was the identical buffer used to freeze the semen, the protein alteration occurred during freeze-drying and not during analysis. With antisera to bovine washed spermatozoa, the antigenic profile of control or freeze-dried bovine extended semen dehydrated to 6% residual moisture consisted of seven antigens, and six antigens when dehydrated to less than 2% residual moisture (Figure 3). The concentration of antigens 1, 2, 4, 5, and 7 was not affected by freeze-drying. However, antigen 3 was not detected in extended semen dried to <2% residual moisture. In addition, the concentration of antigen 6 decreased in the extended semen dehydrated to <2% residual moisture. With antisera to bovine seminal plasma, the antigenic profile of control or freeze-dried bovine extended semen dehydrated to 6% residual moisture consisted of four antigens versus seven antigens when dehydrated to less than 2% residual moisture (Figure 4). Antigen I was the only seminal plasma protein not affected by freeze-drying. Seminal plasma antigens 5, 6, and 7 were detected only in the semen freeze-dried to <2% moisture. The reaction of once frozen and thawed bovine seminal plasma with antisera to bovine seminal plasma produced seven precipitin lines. These lines were identical to those on semen dehydrated to less than 2% residual moisture. This suggests that seminal plasma antigens on extended semen had been masked and were inaccessible either because of their location in the molecule or by being hidden because of egg yolk proteins coating the spermatozoa. The dehydration process led to their exposure and may help to explain why egg yolk did not have a cryoprotective action at <2% residual mois-

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3 4 5 6 7 ANTIGEN Figure 3. Changes detected with antisera to bovine washed spermatozoa in the antigenic profile of bovine semen extended in TEST-yolk buffer after dehydration to less than 2% residual moisture. • 100% residual moisture, o<2% residual moisture. The antigenic profile of semen dehydrated to 50, 25, 12, and 6% residual moisture was identical to that at 100% residual moisture. Bars represent highest dilution of antigen yielding a visible precipitin line with a constant amount of antisera.

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ANTIGEN Figure 4. Changes detected with antisera to bovine seminal plasma in the antigenic profile of bovine semen extended in TEST-yolk buffer after dehydration to less than 2% residual moisture. • 100% residual moisture, u<2% residual moisture. The antigenic profile of semen dehydrated to 50, 25, 12, and 6% residual moisture was identical to that at 100% residual moisture.

ture. With antisera to chicken egg yolk, the antigenic profile of control and freeze-dried bovine extended semen containing at least 6% residual water consisted of eight antigens (Figure 5). This antisera did not crossreact with any seminal protein. Although further dehydration unmasked another antigen, our freeze-drying conditions did not alter greatly the antigenic properties of egg yolk. By comparison, spermatozoal and seminal plasma proteins were more sensitive to denaturation than egg yolk proteins. Journal of Dairy Science Vol. 66, No. 4, 1983

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ANTIGEN Figure 5. Changes detected with antisera to chicken egg yolk in the antigenic profile of bovine semen extended in TEST-yolk buffer after dehydration to less than 2% residual moisture. • 100% residual moisture, D<2% residual moisture. The antigenic profile of semen dehydrated to 50, 25, 12, and 6% residual moisture was identical to that at 100% residual moisture.

Immunological reactivity of a protein antigen is d e p e n d e n t upon the tertiary structure of the reacting antigen (16). Figure 3 and 4 s h o w that immunological properties of bovine semen were altered by removal of water. Hence, the tertiary structure of s o m e of the seminal proteins was damaged by dehydration. The principal c o n t r i b u t o r to stability of the native c o n f o r m a t i o n of a protein is the t e n d e n c y of its n o n p o l a r groups to avoid c o n t a c t with water (19) - that is, to engage in h y d r o p h o b i c interactions (nonpolar groups fold inward). As the t e m p e r a t u r e around the protein is lowered, stabilization by h y d r o p h o b i c interactions decreases (19). F u r t h e r m o r e , as water is r e m o v e d f r o m around the proteins by dehydration, the t e n d e n c y to f o r m n o n p o l a r interactions decreases. Therefore, freeze-drying could weaken this f o r m of structure stabilization. Transconf o r m a t i o n or denaturation would be an e x p e c t e d response by the protein to such stresses (8). Evidence in the form of changes in the electrophoretic and antigenic properties of seminal protein led to the conclusion that our conditions for d e h y d r a t i o n (freeze-drying) altered seminal protein. The semen f r o m the three bulls on which these protein analyses were run p r o d u c e d a 55% (12/22) pregnancy rate with 100% residual moisture semen (5). This did n o t differ statistically from the 29% (5/17) pregnancy rate obtained with semen d e h y d r a t e d to 6% residual moisture (5). The electrophoretic and antigenic properties of semen dehydrated d o w n to 6% Journal of Dairy Science Vol. 66, No. 4, 1983

residual moisture were also similar to control semen. However, e l e c t r o p h o r e t i c and antigenic differences were m a r k e d in semen d e h y d r a t e d to <2% moisture (Figures 3, 4). No pregnancies (0/45) were obtained f r o m this semen (5). Based on these results, we suggest that major protein denaturation occurred b e t w e e n 6% residual moisture and <2% residual moisture. Hence, further i m p r o v e m e n t s in this freezedrying technique must focus on h o w to stabilize spermatozoal and seminal plasma proteins at low moisture.

ACKNOWLEDGMENTS

We appreciate the c o o p e r a t i o n of the MN, Wl bull studs, w i t h o u t which this investigation would not have been possible. Special thanks are e x t e n d e d to Marilyn Dunshee for technical assistance.

REFERENCES

1 Anderson, M. M., and C. P. Merilan. 1960. Dehydration studies on bovine spermatozoa. Univ. Missouri Agric. Exp. Sm. Res. Bull. 736. 2 Bialy, G., and V. R. Smith. 1957. Freeze-drying of bovine spermatozoa. J. Dairy Sci. 40:739. 3 Graham, E. F., B. G. Crabo, and K. I. Brown. 1972. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. I. Bull. J. Dairy Sci. 55:372. 4 Graham, E. F., E. V. Larson, and B. G. Crabo. 1974. Freezing and freeze-drying bovine spermatozoa. Proc. 5th Tech. Conf., Pages 14-20 in. A.I. Reprod: Natl.*Assoc. Anim. Breeders, Chicago, IL. 5 Jeyendran, R. S., E. F. Graham, and M.K.L. Schmehl. 1981. Fertility of dehydrated bull semen. Cryobiology 18:292. 6 Kendrew, J. C. 1962. Side chain interactions in myoglobin. Brookhaven Syrup. Biol. 15:216. 7 Kuntz, I. D. 1971. Hydration of macromolecules. IV. Polypeptide conformation in frozen solutions. J. Am. Chem. Soc. 93:516. 8 Kuntz, I. D., and W. Kauzmann. 1974. Hydration of proteins and polypeptides. Adv. Protein Chem. 28:239. 9 Larson, E. V., and E. F. Graham. 1977. Freezedrying of spermatozoa. Dev. Biol. Stand. 36:343. 10 Leidl, W. 1956. Experiments in freeze-drying of bull sperm. Proc. 3rd Int. Congr. Anita. Reprod. A.I., Cambridge 3: 39. 11 Merym~n, H. T. 1960. Drying of living mammalian cells. Ann. New York Acad. Sci. 85:729. 12 Meryman, H. T., and E. Kafig. 1963. Freeze-drying of bovine spermatozoa J. Reprod. Fert. 5:87. 13 Nagase, H., E. F. Graham, and T. Niwa. 1964. Pellet semen: The effect of glycerol level and

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composition of thawing solutions on fertility of bovine spermatozoa. 5th Int. Congr. Anim. Reprod. A.I., Trento 4:404. Nei, T., and H. Nagase. 1961. Attempts to freezedry bull spermatozoa. Low Temp. Sci. Ser. B19:107. Ouchterlony, P. 1958. Diffusion-in-gel methods for immunological analysis. Prog. Allergy 5:1. Roitt, I. 1974. Essential immunology. 2nd ed. Blackwell Sci. Publ., London. Saacke, R. G., and J. O. Almquist. 1961. Freezedrying of bovine spermatozoa. Nature (London) 192:995.

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18 Singh, S. G., and D. J. Roy. 1967. Freeze-drying of bovine semen. Indian J. Vet. Sci. 37:1. 19 Taborsky, G. 1979. Protein alterations at low temperatures: An overview. Pages 1--26 in Proteins at low temperatures. Advances in chemistry series 180. O. Fennema, ed. Am. Chem. Soc., Washington, DC. 20 Wieme, R. J. 1965. Agar gel electrophoresis. Elsevier, Amsterdam. 21 Yuschchenko, N. P. 1957. Proof of the possibility of preserving mammalian spermatozoa in a dried state. Proc. Lenin Acad. Agric. Sci. 22:37.

Journal of Dairy Science Vol. 66, No. 4, 1983