最近走査電子顕微鏡による精子の形態学的研究が行なわれるようになり, 哺乳類の精子は凍結保存により先体に著しい悪影響を被ることが知られている。一方家禽精子においても哺乳類の精子と同様に融解精子先体に著しい変化の認められることが報告されているが, 凍結融解操作によってもたらされる一連の精子の損傷について観察した報告は少ない。

本研究は光学顕微鏡と走査電子顕微鏡を用い, 錠剤化凍結法とストロー凍結法により, それぞれ二種類の希釈液を用いて凍結処理し, 融解後の鶏精子の活力および崎形精子の微細構造について観察した。その結果, 走査電子顕微鏡を用いれば, 従来の光学顕微鏡下で通常認められる首曲り畸形精子の他に精子先体の離脱や, 精子頭部末部の膨化が認められた。しかし凍結法, 希釈液によるそれら畸形精子の出現割合の差異は明らかでなかった。Recently the scanning electron microscope (SEM) is currently being used to study the morphology of normal and abnormal spermatozoa. In general, it is known that the acrosome of mammalian spermatozoa is affected by deep freezing preservation. On the other hand, HARRIS et al. also reported that the acrosome of fowl spermatozoa is influenced by deep freezing as well as in that of mammalian spermatozoa. MARQUEZ et al. reported that some swelling or distortion of the mitochondria were caused when the turkey semen has been exposed to a glycerol containing medium. But there are few reports pertaining to the series of the morphological changes in fowl spermatozoa after freezing and thawing.

In the present study, the scanning electron microscope was used to observe the morphological changes observed in fowl spermatozoa before, during and after freezing and thawing of sperm cell

Maeda, Teruo

Terada, Takato

Watanabe, Moriyuki

Name not available

J. Fac. Appl. Biol. Sci., Hiroshima Univ. (1980), 19: 11-20 Scanning Electron Microscopic Studies of Frozen Fowl Semen Teruo MAEDA, Takato TERADA and Moriyuki WATANABE Faculty of Applied Biological Science, Hiroshima University, Fukuyama Received March 25, 1980 (Figs 1-20; Table I) Recently the scanning electron microscope (SEM) is currently being used to study the morphology of normal and abnormal spermatozoa. In general, it is known that the acro-some of mammalian spermatozoa is affected by deep freezing preservation.1)-3) On the other hand, HARRIS et al. 4 ) also reported that the acrosome of fowl spermatozoa is influenced by deep freezing as well as in that of mammalian spermatozoa. MARQUEZ et al. s) reported that some swelling or distortion of the mitochondria were caused when the turkey semen has been exposed to a glycerol containing medium. But there are few reports pertaining to the series of the morphological changes in fowl spermatozoa after freezing and thawing. In the present study, the scanning electron microscope was used to observe the morphological changes observed in fowl spermatozoa before, during and after freezing and thawing of sperm cell. MATERIALS AND METHODS The semen used for the present experiment was collected from 3 White Leghorn cockerels of 10 months old and mixed. The mixed semen was diluted to four fold with a 5. 7% glucose solution, and a 5. 7% glucose solution 85 plus fresh egg yolk 15 which contained 7% glycerol in final concentration. After dilution, the semen samples were frozen for one hour by two freezing methods; one the pelleted freezing method6) and the other the straw freezing method7 ) described in the previous papers. The pre-freezing time of semen samples in straw freezing was 3 minutes. Afterwards, those samples were thawed in 5°C and 3rc respectively. The smear preparation of fresh undiluted semen for studies of spermatozoa by the light microscope was fixed in formalin vapour and stained with carbol-fuchsin-eosin by the routine staining procedure of our laboratory. The fixation of fresh and thawed semen samples for studies of spermatozoa by SEM were carried out for an hour at 4°C with glutaraldehyde solution (2-4%). After the samples were washed two times with a phosphate buffer, the samples were smeared on aluminum-foil and then air-dried. Afterwards, the smear samples were dehydrated in an ethanol for 30 minutes and were coated with gold palladium. Then the morphological changes of specimens were observed. Motility of samples was scored by a scale of five point(+++, ++, +, ±, - ). For estimating the percentage of abnormal spermatozoa, approximately 500 spermatozoa were examined. 12 Teruo MAEDA, Takato TERADA,Moriyuki WATANABE RESULTS AND DISCUSSION MOTIUTY AND ABNORMALITY OF FOWL SPERM CELL The motility and abnormality of undiluted semen and that of the semen samples treated by two freezing methods (pelleted freezing method and straw freezing method) with two diluents (5.7% glucose solution and 5.7% glucose solution 85 plus fresh egg yolk 15) are shown in Table I. The percentages of motility and abnormal spermatozoa of undiluted semen calculated as the control were 95% (over++) and 2.9% respectively. Table 1. Motility and abnormality of fowl spermatozoa after thawing. Freezing method Pelleted freezing method 5. 7% glucose solution 5. 7% glucose solution Diluent plus fresh egg yolk Motility after dilution 95 95 Motility after thawing 70 80 Abnormality after thawing 7.8 8.4 Notes: Motility of fresh undiluted semen 95% (++over) Abnormality of fresh undiluted semen 2.9% Straw freezing method 5. 7% glucose solution 5. 7% glucose solution plus fresh egg yolk 95 95 80 80 10.3 11.1 As shown in Table I, the motility of semen samples after thawing by two freezing methods (pelleted freezing method and straw freezing method) and two diluents (5.7% glucose solution and 5.7% glucose solution 85 plus fresh egg yolk IS) are pretty active ranging from 70 to 80% (over ++) in each division. The abnormalities of the thawed semen which were treated by the above two freezing methods and two diluents were 8.1% on the average ranging from 7.8 to 8.4% in the former and I 0.8% on the average ranging from I 0.3 to ll.l% in the latter respectively. These results are higher than 5% in the former and 7% in the latter compared to those of the undiluted semen (2.9%). But these results are not always as high as in the result of freezing semen. MORPHOLOGY OF FOWL SPERM CELL 1. Observation by the light microscope The morphology of fresh semen by the light microscope is shown in Figs. I and 2. The distortion of the midpiece could rarely be seen in Fig. 1 but it was seen in the central field of Fig. 2. SEM Studies of Frozen Fowl Semen 13 ' Fig. I . Micrograph of fresh semen immediately after collection by the light microscope (LM). Abnormal spermatozoa are rarely seen in this micrograph. LM X400. - · Fig. 2. Micrograph of fresh semen immediately after collection by LM. Neck bending of sperm cell is seen in the central field of this figure. LM X400. This distortion of the midpiece is called "neck-bending of sperm" 8) , "bent sper-matozoa"9) or "crooked-necked spermatizoa". 10) This phenomenon in sperm cells of fresh semen was observed in present fresh semen. (Fig. 2) According to ThuKUNAGA et al. s) , neck-bending of sperm is a response of the live sperm cell to its hypotonic medium; any hypotonic solution whose freezing-point depression is lower than - 1.03°C, owns this particularity to its osmotic pressures. Since the t:::, of the cock's seminal plasma consisting of transparent accessory sex secretions is much lower than this, showing only - 0.58°C, it is no wonder that even fresh undiluted semen exhibits neck-bent sperms gradually increasing in number with the elapse of time. The micrographs of the spermatozoa after the thawing by means o f pelleted freezing and the two diluents (5.7% glucose solution and 5.7% glucose solution 85 plus fresh egg yolk 15), are shown in Figs. 3 and 4. The micrographs of those thawed by straw freezing and two diluents (5.7% glucose solution and 5.7% glucose solution 85 plus fresh egg yolk 15) are shown in Figs. 5 and 6. Fig. 3. Micrograph of the spermatozoa diluted with 5. 7% glucose solution by pelle ted freezing method. In this micrograph , neck-bending spermatozoon is seen. LM X400. . . • 4 Fig. 4 . Micrograph of the spermatozoa diluted with 5. 7% glucose solu tion plus fresh egg yolk after thawing by pelleted freezing mthod. In this micrograph, neck-bending spermatozoon is seen. LM X400. 14 Teruo MAEDA, Takato TERADA, Moriyuki WATANABE < .1 __r \._ / ~ .- ~ ~ ~ . - I I ·< \~ ' 5 Fig. 5. Micrograph of the spermatozoa diluted with 5. 7% glucose solution after thawing by straw freezing method. Neck-bending spermatozoa are seen in this figure. LM X400. . ) l ·~0 .· ·s Fig. 6. Micrograph of the spermatozoa diluted with 5.7% glucose solution plus fresh egg yolk after thawing by straw freezing method. Neck-bending spermatozoon is seen. LM X400. In each micrograph, the neck-bending of the sperm can be seen notwithstanding the freezing method or the diluent but the differences of the occurrence of the neck-bending of the sperm could not be seen among them. Moreover, as there is a credit limit in the light microscope, the detailed observation of sperm cells, especially that in the sperm head could not be obtained. Therefore, we have tried out further observations on sperm cells using SEM. 2. Observations under the scanning electron microscope The ultrastructures of the undiluted (non-frozen) fowl spermatozoa are shown in Figs. 7 (X 1 ,000), 8 (X 2,000), 9 (X 5 ,000) and I 0 (X 1 0,000). Fig. 7. Micrograph of fresh semen immediately after col-lection by scanning ele-ctron microscope (SEM). Normal sperm ceUs are seen in this micrograph. SEM Xl,OOO. SEM Studies of Frozen Fowl Semen 15 Fig. 8 The same as above. SEM X2,000. Fig. 9. A normal spermatozoon, the same as above. SEM xs,ooo. Fig. 10. The same as above. Acro-some is not isolated. SEM XlO,OOO. 16 Teruo MAEDA, Takato TERADA, Moriyuki W ATANABE In these micrographs, the normal fowl spermatozoa with acrosome can be seen clearly. SEM micrographs of the spermatozoa after thawing by the two freezing methods (pelleted freezing method and straw freezing method) and by the two diluents (5. 7% glucose solution and 5.7% glucose solution 85 plus fresh egg yolk 15) are shown in Figs. 11 to 20. The spermatozoa seen in these micrographs show the ultrastructural changes in acrosome (Figs. 11, 13, 14, 15, 16, 17, 18,19 and 20), in theendofthespermneck (Figs.l2, 16 and 18) and in the midpiece (Fig. 17). Thus, it becomes clear that even those sper-matozoa that would be considered as normal in the sweep field of the light microscope, have suffered many serious ultrastructural changes when observed by a scanning electron microscope (SEM). HARRIS et al. 4 > have shown that fowl spermatozoa are damaged ultrastructurally not only by freezing and thawing but also by the cryoprotectants used , e. g. glycerol and dimethylsulphoxide. MARQUEz and 0GASAWARA s) also reported that the turkey spermatozoa are similary affected by the presence of glycerol. Fig. 11. Micrograph of spermatozoa diluted with 5. 7% glucose solution after thawing by pelleted freezing method. Isolation of acrosome are seen in this micrograph. SEM X2,000. Fig. I 2. The same as above. The swelling of the end of sperm head is seen. SEM X5,000 SEM Studies of Frozen Fowl Semen 17 Fig. 13. Micrograph of spermatozoa diluted with 5. 7% glucose solution plus fresh egg yolk after thawing by pelleted freezing method. Isolation of acrosome is seen in sperm cell. SEM X2,000. Fig. 14. The same as above. En-largemen t of the isolation of acrosome in sperm cell. SEM X5,000. Fig. 15. Micrograph of spermatozoa dilu ted with 5.7% glucose solution after thawing by straw freezing method. Isolation of acrosome is seen. SEM X2,000. 18 Teruo MAEDA, Takato TERADA, Moriyuki WATANABE Fig. 16. The same as above. !so-lation of acrosome and the swelling of the end of sperm head are seen in this micrograph. SEM X3,000. Fig. 17. The same as above. Iso-lation of acrosome and the abnormality of midpiece are seen in this micrograph. SEM X5,000. Fig. I 8. The same as above. I so-la tion of acrosome and the swelling of the end of sperm head are seen in this micrograph. SEM XS,OOO. SEM Studies of Frozen Fowl Semen 19 Fig. 19. Micrograph of spermatozoa diluted with 5.7% glucose solution plus fresh egg yolk after thawing by straw freezing method. Isolatt-ons of acrosome are seen in this micrograph. SEM X2,000. Fig. 20. Enlargement of Fig. 19. Isolation of acrosome in sperm cell is seen. SEM xs,ooo. As mentioned above, in the present experiment, fairly morphological changes of neck-bending of the sperm cell in the midpiece were observed, as it was hitherto revealed by the light microscope. 11 ) Moreover the isolation of the acrosomal cap and the swelling at the end of the sperm head were observed in a new way. These ultrastructural changes in the sperm cell could not be observed by the usual light microscope possibly due to the freezing process. Perhaps, these morphological changes in the sperm cell may be responsible for the decrease in fertility. SUMMARY The purpose of the present study was to investigate the motility and the ultrastructure in fowl spermatozoa frozen by the pelleted and straw methods with scanning electron and the light microscopes. In either case of the pelleted and straw methods, the isolated 20 Teruo MAEDA, Takato TERADA, Moriyuki W ATANABE acrosome was observed in freeze-thaw fowl spermatozoa but the differences between the two methods were not clear. Also in the freeze-thaw fowl spermatozoa, the swelling at the end of the sperm head was observed. At the same time the neck-bending of the mid piece could be observed as clearly as in the case of the light microscope. ACKNOWLEDGEMENT The authors wish to express their gratitude towards Dr. Tatsudo TAMURA of Hiroshima University for his generous collaboration in the course of these experiments. REFERENCES 1) FUJITA, T., MIYOSHI , M. and ToKUNA GA, J. : Z. Zellforsch. , I 05, 483- 497 (1970). 2) ZANEVELD, L. J . 0 ., COULD, K. G., HUMPHREYS, N. J . and WILLIAMS, W. L. : 1. Reprod. Med. , 6, 13- 19 (1971). 3) HA FEZ, E. S. E. and KAN AGAWA, H.: Fert. Steril. , 24,776-789 (1973). 4) HA RR IS, G. C. , THURSTON, R. J. and CUNDALL, 1. : 1. Reprod. Fert., 34,389-394 (1973). 5) MARQUEZ, B. T. and OGASAWARA, F. X. : Poult. Sci., 56, 1806- 1813 (1977). 6) TAKEUCHI, D., TERADA , T. , SHIRAKAWA , Y. and WATANABE , M. : lap. J. Zoo tech. Sci., 50, Supplement, p. 41 (1979) . 7) WATANABE, M. and TERADA, T. : Proceed, 8th Inter. Cong. Anim. Reprod. A. I. (Krakow), 4, 1096- 1099(1976). 8) TSUKUNAGA, S. and TAKAHASHI , T. :Bu/l.HiroshimaAgr. Col/., I , 25 - 34 (1961). 9) BAKST, M. R. and SEXTON , T. J .: 1. R eprod. Fert., 55, 1-7 (1979). 10) SAEKI, Y. and BROWN, K. I. : Poult. Sci., 41 , 1096-1100 (1962). 11) WATANABE, M. , MI URA, M. and MooA, Y. :lap. Pou/t. Sci., 7, 23- 29 (1970) . &:.i!f;Ej~t';;rr-TlJlJiiil'dlliLd::..Qfrj-=fO)J~il!'l"J':011iJf'1.'.:1JsrrtJbh.Q J:. '3 1C tJ.lJ, ilffl~L'MjO)frj-=fiiiJl!tEl*:FfKJ:.It) 5\:i*H::~ LHII'd1~~~t& .Q c:. C: :IJsffi Gh L't.' .Q o -:1J~~fi'Hic:::::Jo t.' L' <b~ffi~LJIT:lO)'fi'HC: F6J.f;'fHc:::f,\!Rmf~i-T Jt:i*lc:::~ L H§~'{ t. O)tr,~ Gh.Q c:. C: :IJst!H!f~ h L't.' .Q :IJs, i!J;!tai\i.'l!mtNd'FK J:...., L' bt:. 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鶏凍結精子の走査電子顕微鏡による研究

Hiroshima University Institutional Repository

Scanning Electron Microscopic Studies of Frozen Fowl Semen

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鶏凍結精子の走査電子顕微鏡による研究

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