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The Relation of Spermatozoa to the Glandular Tissue in the Storage Sites of the Hen Oviduct*
1462
E. T. MORAN, JR. AND J. MCGINNIS
SUMMARY Turkey poults were fed rations containing either corn or barley as the cereal grain from hatching to 4 weeks of age. The pancreas of the barley fed birds appeared to be smaller than that observed in the corn-fed birds; there were no differences in the intestine weights. Supplementation with oleandomycin, regardless of grain caused increased pancreas weights; with enzyme supplementation this increase was apparent only with the birds fed the corn rations. A reduced intestine
weight was noted for the poults given the corn rations supplemented with oleandomycin. REFERENCES Arscott, G. H., D. C. Hutto and P. Rachapaetayakom, 1965. Use of barley in high-efficiency broiler rations. 7. Pancreatic enlargement in chicks fed barley containing diets. Poultry Sci. 44: 432-434. Burnett, G. S., 1966. Studies of viscosity, as the probable factor involved in the improvement of certain barleys for chickens by enzyme supplementation. Brit. Poultry Sci. 7: 55-75. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Federer, W. T., 1955. Experimental Design. Macmillan Co., New York. Fisher, R. A., and F. Yates, 1963. Statistical Tables for Biological, Agricultural and Medical Research. Hapner Publishing Co., New York. Jukes, H. G., D. C. Hill and H. D. Branion, 1956. Effect of feeding antibiotics on the intestinal tract of the chick. Poultry Sci. 35: 716-723. Moran, E. T., and J. McGinnis, 1965. The effect of a cereal grain and energy level of the diet on the response of turkey poults to antibiotic and enzyme supplements. Poultry Sci. 44: 12531261. Moran, E. T., and J. (McGinnis, 1966. A comparison of corn and barley for the developing turkey and the effect of antibiotic and enzyme supplementation. Poultry Sci. 45: 636-639.
The Relation of Spermatozoa to the Glandular Tissue in the Storage Sites of the Hen Oviduct* H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND ORA KEMPENICH The Volcani Institute of Agricultural Research, Rehovot, Israel (Received for publication March 20, 1967)
T
HE storage sites of spermatozoa in the laying hen's oviduct appear to be the glands of either the uterovaginal junction or the caudal part of the infundibulum, depending on the method of insemination. Van Drimmelen (1946) located sper* Contribution from The National and University Institute of Agriculture, Rehovot, Israel. 1967 Series; No. 1142-E.
matozoa in the mucosal folds of the infundibulum after intraperitoneal inseminations. Bobr et al. (1964) found spermatozoa in the glands of the uterovaginal junction after intravaginal insemination and in both the uterovaginal glands and infundibular glands after intrauterine insemination. Also in our laboratory (unpublished data) spermatozoa were found in the utero-
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ment, growth rate and probably chronological age, not to mention the various possible interactions. Unpublished data from this laboratory with respect to a lysine deficiency in the growing chick showed that the gut continued to grow regardless of the deficiency as, to a lesser extent, did the pancreas while the muscle mass growth was retarded. Because of the many known and probably unknown factors which can influence pancreas size it is obvious that considerable care must be taken before the statement "hypertrophy," "atrophy," etc. of the pancreas can be made.
SPERMATOZOA IN STORAGE SITES
MATERIALS AND METHODS White Leghorn roosters and laying hens were used. They were kept in individual cages kept in an open shed. Intravaginal inseminations were performed by depositing 0.2 ml. of untreated semen into the vagina, which was exposed by abdominal pressure. Intramagnal inseminations were performed after exposing one of the loops of the magnum by laparotomy. After cleaning the feathers from the left abdominal side, local anaesthesia was applied with Lidocain (Teva Ltd., Jerusalem). An incision was made in the skin beneath the pubic bone and parallel to it, beginning at a distance of about 1 cm. from its posterior end and continuing about 1.5 cm. in the cranial direction. After penetration of the underlying muscle layer and the peritoneum, the magnum was easily reached. Subcutaneous and abdominal fat was removed when necessary. 0.2 ml. of a washed sperm suspension (Schindler and Hurwitz, 1966) was injected into one of the loops of the magnum. The peritoneum and the muscles were then sutured and the skin was closed with clamps,
which were removed after 5 days. Care was taken to minimize the duration of the operation and to avoid undue stress on the hen. After three days of incubation, eggs were broken open and checked for the presence of an embryo. After intramagnal inseminations, segments about 1 cm. in length were sampled from all regions of the oviduct and fixed in 4% formaldehyde. After intravaginal insemination, only the uterovaginal junction served for the examinations. Sections were made from different parts of the segments and stained with haematoxylin-eosin. For electron microscope examinations segments about 1 cm. in length were removed from the posterior part of the infundibulum or from the uterovaginal junction and immediately transferred to 1% glutaraldehyde in isotonic phosphate buffer (pH 7.2) for primary fixation. The segments were slit open (while immersed in the glutaraldehyde solution) and pieces of about 1-2 mm.3 were sliced from the inside of the duct, excluding the muscular layer. The cubes were left in the glutaraldehyde solution for 1 hr. in the cold followed by five washings with phosphate buffer at 10minute intervals. They were then transferred to 1% osmic acid in phosphate buffer and kept in the cold for 1 hr. After washing out the osmic acid with phosphate buffer (five washings at 10-minute intervals) the preparations were dehydrated gradually in acetone and embedded in Vestopal W (Martin Jaeger, Vesenaz, Switzerland), according to the manufacturer's instructions. In order to identify the spermatozoacontaining regions in the block before sectioning for electron microscopy, preliminary sections of 2-4 \x. thickness were prepared, and stained with toluidine blue, essentially according to the technique described by Lynn (1965), except for the following modifications: (a) the sections were
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vaginal junction only after intravaginal insemination. On the other hand, Fujii and Tamura (1963) and Takeda (1964) found spermatozoa after intravaginal inseminations in the infundibular as well as in the uterovaginal glands. After deposition of spermatozoa in the infundibulum (Takeda, 1966) or in the magnum (Van Krey et al., 1966), large accumulations of spermatozoa were found in the infundibular glands and a low incidence of spermatozoa in the uterovaginal junction. The present study was undertaken in order to obtain information on the exact position of the fowl spermatozoa in the storage glands and their association with the glandular tissue at various times after insemination.
1463
1464
H. SCHINDLER, E. BEN-DAVID, S. HUEWITZ AND O. KEMPENICH
RESULTS
Egg production and fertility after intramagnal insemination. This experiment was conducted in order to evaluate the fertilizing capacity of the spermatozoa after the intramagnal inseminations. Washed spermatozoa were injected into the magnum of 10 hens several hours after oviposition; three hens served as controls and were inseminated intravaginally with the same sperm suspension. Seven of the operated hens returned to egg production within seven days after operation and the remain-
ing hens did so by the twelfth day. It can be seen from Table 1 that although egg production was low during the first 11 days after the operation, the fertilization rate was about the same as in the control group. The fertilization rate in the third week was much higher in the treated group than in the control, but most of the embryos in the former group died at an early stage. Location of spermatozoa after deposition of semen in the magnum. In another experiment the sites where spermatozoa were present following intramagnal insemination, were identified. Semen was deposited in the magnum of 11 hens which had laid several hours prior to insemination or had a shellegg in the uterus. The hens were killed after intervals as indicated in Table 2. The results of the histological examinations are presented in Table 2. Spermatozoa were always present in the caudal part (chalaziferous region) of the infundibulum, except in the hen which was killed after 13 days in which no spermatozoa were detected at all. Only in one hen killed one day after the operation, were large concentrations of spermatozoa found in the uterovaginal glands, whereas in two other hens only a few spermatozoa were found in this place. Position of spermatozoa in the storage sites. Shortly after intramagnal inseminaTABLE 1.—Egg production and fertility of hens inseminated in the magnum Daysj after insemination 2-5
6-11
12-13
14-20
Intramagnal insemination Hen days N o . of eggs N o . of fertilized eggs
40 9 5
60 31 28
20 19 14
70 61 35»
Intravaginal insemination Hen days N o . of eggs N o . of fertilized eggs
12 11 9
18 12 11
6 6 1
* 23 dead embryos. f All embryos dead.
21 17
3t
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affixed to slides with albumen-and-glycerin by carefully holding them at a distance over a small open flame for about 1 minute; (b) the necessary exposure to high temperature during the staining process was found to be a little longer than that recommended by Lynn; (c) decolorization was achieved with acetone (95%); and (d) Vestopal served as mounting medium. These preliminary sections were examined with a phase contrast for the presence of spermatozoa, and whenever such were located the corresponding portion of the same block was sectioned for electron microscopy, using a Dannon Ultramicrotome, at about 500 A thickness. Some of these sections were again screened with a phase contrast after staining with toluidine blue in order to verify that they contained the desired portion of the infundibulum and of the uterovaginal junction, i.e., the glandular grooves and the tubular glands with spermatozoa. The other sections were mounted on copper electron microscope grids (type 200, 3-mm. diameter; Morton Ltd., Harrow, England) which were coated with Formvar 15/95E (Shawinigan Resins Corp., Springfield, Mass., U.S.A.), and then stained with uranyl acetate (Pease, 1964). The sections were screened with an RCA Electron Microscope.
SPERMATOZOA IN STORAGE SITES
1465
TABLE 2.—Presence ( 4 ) or absence ( —) of spermatozoa in the various regions of the hen oviduct after intramagnal insemination
Hen no.
6 7 8 9 10 11
3hr. 3 hr. 20 hr. 20 hr. 22 hr. 22 24 72 72 12 13
hr. hr. hr. hr. days days
Stage of egg formation egg m uterus no egg egg in magnum egg in magnum no egg, after oviposition egg in funnel no egg no egg egg in uterus egg in uterus egg in uterus
Infundibulum Cranial
+
+
Caudal I
+ + I
4
Magnum Upper
Middle
Lower
Isth mus
Uterus
Utero^ tion
+
+
f I-
+ i
4
tion (3-4 hr.) they were present in the lumen as well as in the secondary mucosal folds (glandular grooves) of the infundibulum, where they were dispersed or arranged in bundles, sometimes attached to the epithelial lining of the grooves (Fig. 1). Figure 2 shows bundles of spermatozoa on their way to deeper regions of the mucosal folds. A few tubular glands were penetrated by spermatozoa. After intramural insemination spermato-
m
4
zoa were also found in the folds of the cranial part (funnel) of the infundibulum as well as in the glands of the upper part of the magnum. The presence of spermatozoa in these parts of the oviduct may be regarded as temporary because in all other hens killed one day or more after insemination, no spermatozoa were found there, with the exception of one hen which had spermatozoa in the magnal glands one day after intramagnal insemination. (Fig. 3).
\fcii
1. Caudal part (chalaziferous region) of the infundibulum, three hours after intramagnal insemination Note dispersed spermatozoa in the lumen of the secondary mucosal folds, as well as bundles of spermatozoa near the epithelial lining of one of the grooves. (No egg in the oviduct at time of killing.) FIG.
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I 2 3 4 5
Time after insemination
H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND 0.
1466
KEMPENICH
- fA
mm
f fir
t
^»
s
- *
i J o b *_-»-. . « t > ' H B FIG. 2. Caudal part of the infundibulum, three hours after intramagnal insemination. Note bundles of spermatozoa during passage to the deeper parts of the secondary mucosal folds. (Egg in uterus at time of killing.)
At 24 hours after intravaginal or intramagnal inseminations, large concentrations of spermatozoa were found in the grooves of the mucosal folds and tubular glands of the uterovaginal junction and the infundibulum, respectively, and only a few were present in the lumen (Fig. 4). At two or three days after insemination, spermatozoa
were present in the glands, whereas the folds were almost empty of spermatozoa. At 5 or 12 days after insemination, spermatozoa were found in the glands only. The accumulation of spermatozoa in the tubular glands was highest on the first day after insemination and declined with time, as shown in Table 3. In order to determine whether the migra-
: • • :
&
i f &"'::^-y • • •• 'PA. &%-FIG. 3. Sperm bundles in the glands of the magnum, adjacent to the infundibulum, 24 hours after intramagnal insemination. Longitudinal section. (Egg in uterus at time of killing.)
wop. :•
FIG. 4. Sperm bundles in the chalaziferous glands of the infundibulum, 24 hours after intramagnal insemination. (Egg in the cranial part (funnel) of the infundibulum at time of killing.)
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:
SPERMATOZOA IN STORAGE SITES
The idtrastructiire of the spermatozoa and, of their storage sites. The histological preparations showed, in most cases, a close association between the spermatozoa and the epithelial lining of the glandular grooves or the tubular glands, but it was impossible to determine whether the spermatozoa actually penetrate the cells of the epithelium. In order to elucidate the position of the spermatozoa at these places ultramicroscopic examinations of the sperm storage sites were carried out. The ultrastructural details of the infundibulum were identified according to the description by Aitken and Johnston TAHLE 3.—Sperm contents of tubular glands at various limes after semen deposition Infundibulum* Time after insemination 3-4 hr 1 day 2 days 3 days 5 days 12 days
xT
N o . Of
ne
d?nd examinedt i 46 (2) 96(4)
Uterovaginal junction XT,.
r
N o . Of
Slands %°n£ ^nds °nta™nS e x t S d l ^ ^ ^ c vtt ^ '+ spermatozoa Spermatozoa c
7 61
61_(2)
.ill
23(1)
6
* Intramagnal insemination. f Intravaginal insemination. + In parentheses number of hens
100 (2) 81 (2)
63 37
89(3)
I'll
5%^ #•;
a
M%
(1963). Fig. S shows glandular grooves with many sections of spermatozoa. In Fig. Sa also cross-sections of cilia can be seen, which are easily distinguishable from the cross-sections of sperm tails, in spite of the similar arrangement of their fibrils, because sperm tails are considerably larger. Fig. 6 shows spermatozoa inside tubular glands. In all preparations in which concentrations of spermatozoa were found, groups could be distinguished according to the level at which the spermatozoa were cut; this can be seen in the tubular gland of
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tion of the spermatozoa is dependent upon their own activity, washed spermatozoa which had been killed by exposure to 60°C. were injected into the magnum of six hens which were killed after 3, 24 or 72 hours. Histological examination revealed masses of spermatozoa entangled in the secretion of the magnum of the two hens which were killed three hours after the insemination. No spermatozoa were found in any other part of the oviduct of these hens, nor were spermatozoa detected in any part of the oviduct of the remaining four hens which were killed after 24 and 72 hours. It is clear from these results that only active spermatozoa are capable of migrating and entering the storage sites, whereas immotile spermatozoa are expelled.
1467
1468
H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND O. KEMPENICH
Fig. 6. In one part of the glandular lumen a group of transverse sections of the sperm heads is seen, whereas in another part a
FIG. 7. Section through a tubular gland of the uterovaginal junction. Microvilli are abundant at the luminal surface of the cells. Note cross-sections of spermatozoa and secretory material in the lumen of the gland as well as close to the glandular cells. (Electron micrograph.)
Fie. 8. Section through a secondary mucosal fold of the uterovaginal junction. Note secreting cells and a ciliated cell; secretory material is present in the lumen. (Electron micrograph.)
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FIG. 6. Tubular glands of the infundibulum. Note vacuoles containing flock-like material. Secretory granules are located near the luminal surface. The cells are lower than those of the grooves. Spermatozoa are present in the lumen; cilia are absent. (Electron micrograph.)
group of sections of sperm tails is seen. In the glandular groove shown in Fig. Sb also a group of sperm head sections is seen, but in addition another group of sagittal sections of heads and mid-pieces is seen. This finding shows that the spermatozoa in the storage sites are arranged in bundles, with the corresponding parts of the cells lying parallel to each other. The electron micrographs also reveal that although the spermatozoa are arranged in bundles, some space is left between neighboring sperm cells. In none of the many ultrasections made one or six days after insemination from the infundibulum or the uterovaginal junction have spermatozoa been found to be inserted in the epithelial lining. Most of the sperm sections were found to be free in the lumen; occasionally, sperm sections were found to be very close or attached to the epithelial cells (Figs. Sb, 6, 7). Also in those preparations in which sagittal sections of the anterior part of the sperm head, including the acrosome, were obtained, the sperm head was found to be free
SPERMATOZOA IN STORAGE SITES
DISCUSSION The preliminary experiment carried out in the present study confirms the results obtained by Takeda (1966) and Van Krey et al. (1966) that the infundibular glands are capable of maintaining the fertilizing capacity of the spermatozoa during a period which is at least equal to that of sperm preservation in the uterovaginal glands, and that the conditions prevailing in the infundibulum are conducive to sperm preservation. Since in the present study the hens were
Fie. 9. Non-secreting ciliated cells and mucussecreting cells from a secondary mucosal fold of the chalaziferous region of the infundibulum. (Electron micrograph.)
killed at different intervals after insemination, the migration of the spermatozoa could be followed. Whereas the transit from the magnum to the infundibulum took place within a very short time, the migration of the bulk of the spermatozoa from the lumen of this region into the tubular glands took at least 24 hours, and only after a few days were the mucosal folds empty. With the semen dose in this study, about 60% of the tubular glands of the uterovaginal junction and the infundibulum were filled with spermatozoa one day after intravaginal or intramagnal insemination, respectively. After this time, the number of glands containing sperm declined gradually. It appears that during the process of sperm depletion some glands are emptied entirely and others remain temporarily unaffected, rather than that all glands release part of their sperm reserve synchronously while retaining another part for future release. The migration of the spermatozoa is dependent upon their own activity, as indicated clearly by the fact that immotile spermatozoa did not reach the infundibulum and were soon expelled. This observation agrees with the results of Fujii and Tamura (1963) who demonstrated that spermatozoa deposited in the vagina can pass into the uterovaginal junction only when they are motile, and with those of Allen and Grigg (1957) who had shown previously that only active spermatozoa pass the barrier of the uterovaginal sphincter. The examinations which were carried out with the electron microscope showed that most of the spermatozoa lie free in the lumen of the glands. This position is not changed even after prolonged storage. This finding does not confirm the assumption by Fujii and Tamura (1963) that the spermatozoa penetrate the intercellular spaces of the glandular epithelium. It may be con-
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in the lumen of the gland. Glandular secretion of relatively low electron density could be seen in the lumen of the glands or of the glandular grooves. The group of sperm heads shown in Fig. 6 is embedded in such a secretion. Tissue structure as well as cellular ultrastructure were similar in the uterovaginal junction and the caudal part of the infundibulum (Figs. 8 and 9).
1469
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H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND O. KEMPENICH
parts of the cells lying parallel to each other. Most of the sperm sections were found to be free in the lumen of the glands; occasionally sperm sections were found to be very close or attached to the epithelial cells. Tissue structure as well as cellular ultrastructure were similar in the uterovaginal junction and the caudal part of the infundibulum. ACKNOWLEDGMENTS
This investigation was supported by a grant from the United States Department of Agriculture under P.L. 480. The assistance of Y. Cohen (Division of Virology) in the electron microscopy is gratefully acknowledged. Thanks are also due to Dr. T. A. Nobel (The Veterinary Institute, Bet Dagan) for helpful discussion of the histological findings.
SUMMARY
REFERENCES
The position of the spermatozoa in the storage sites of the hen oviduct was examined at various intervals after intravaginal and intramagnal inseminations. Shortly after intramagnal insemination spermatozoa were present in the lumen as well as in the secondary mucosal folds of the chalaziferous region, but tubular glands were penetrated by spermatozoa only to a small extent. At 24 hours after intravaginal or intramagnal insemination, large concentrations of spermatozoa were still present in the grooves of the mucosal folds of the uterovaginal junction and the chalaziferous region, respectively, but disappeared from there within a few days. Sperm accumulation in the tubular glands of either the uterovaginal junction or the infundibulum was highest one day after insemination and then declined with time. Electron micrographs showed that the spermatozoa in the storage sites are arranged in bundles, with the corresponding
Aitken, R. N. C , and H. S. Johnston, 1963. Observations on the fine structure of the infundibulum of the avian oviduct J. Anat. 97: 87-99. Allen, T. E., and G. W. Grigg, 19S7. Sperm transport in the fowl. Aust. J. Agric. Res. 8: 788799. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1964. Distribution of spermatozoa in the oviduct and fertility in domestic birds. I. Residence sites of spermatozoa in fowl oviducts. J. Reprod. Fertil. 8: 39-47. Fujii, S., and T. Tamura, 1963. Location of sperms in the oviduct of the domestic fowl -with special reference to storage of sperms in the vaginal gland. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 5: 145-163. Lynn, J. A., 1965. Rapid Toluidine Blue staining of epon-embedded and mounted "adjacent" sections. Am. J. Clin. Pathol. 44: 57-58. Pease, D. C , 1964. Histological Techniques for Electron Microscopy. Academic Press, New York. Schindler, H., and S. Hurwitz, 1966. The preservation of sperm motility in different regions of the hen oviduct in vivo. Poultry Sci. 45: 369374. Takeda, A., 1964. Behavior of spermatozoa in the
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eluded that the spermatozoa in the glands derive energy-yielding substances from the glandular secretion and not from intimate association with the cytoplasm of the glandular cells. It also appears from the electron micrographs that the spermatozoa are not as densely packed as may be assumed from the histological preparations. Therefore, sufficient oxygen may diffuse into the spaces between the spermatozoa to support aerobic metabolism. The histological structure and ultrastructure of both storage sites show great similarity. The behavior of the spermatozoa in these places, i.e., their gradual infiltration into the tubular glands from the glandular grooves and the gradual depletion of the glands, is also similar. It may be, therefore, postulated that the factors supporting the survival of the spermatozoa in both sites are similar.
SPERMATOZOA IN STORAGE SITES genital tract of the hen. I. Presence of spermatozoa in the oviduct. Jap. J. Poultry Sci. 1: 19-31. Takeda, A., 1966. Behavior of spermatozoa in the genital tract of the hen. III. Fertility and spermatozoal behavior following infundibulal insemination. Jap. Poultry Sci. 3 : 15-21. Van Drimmelen, G. C , 1946. "Spermnests" in the
1471
oviduct of the domestic hen. J. S. Afr. Vet. Med. Ass. 19: 42-52. Van Krey, H. P., F. X. Ogasawara and F. W. Lorenz, 1966. Distribution of spermatozoa in the oviduct and fertility in domestic birds. IV. Fertility of spermatozoa from infundibular and uterovaginal glands. J. Reprod. Fertil. 11: 257— 262.
F. E. CUNNINGHAM 2 AND HANS LINEWEAVER Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 (Received for publication March 20, 1967)
I
N A STUDY of the heat stability of egg white and egg white proteins, Cunningham and Lineweaver (1965) reported that in egg white heated to 62.5°C. for 10 minutes at pH 9 lysozyme was inactivated extensively, but that the purified protein dissolved in buffer was unaffected by this time-temperature condition. These results confirmed the heat sensitivity of lysozyme in egg white (Sandow, 1926; Cotterill and Winter, 1954; and Seideman, Cotterill and Funk, 1963) and the stability of lysozyme in buffer (Gorini and Felix, 1953), but they do not explain the reason for the marked difference in stability of lysozyme in the two media. Highly basic lysozyme is known to form electrostatic complexes with other substances. It forms complexes with sodium thymus nucleate and yeast nucleate (Klotz and Walker, 1948), bovine plasma albumin (Steiner, 1953) ovomucin (Hawthorne, 1950; Cotterill and Winter, 1955), conalbumin (Ehrenpreis and Warner, 1956), 1 Presented in part at the Poultry Science Association Meeting, Athens, Ga. 1965 and in part at the 2nd International Congress of Food Science and Technology, Warsaw, Poland, Aug. 1966. 2 Present address, Henningsen Foods, Inc., 2501 College St., Springfield, Missouri 65802.
ovalbumin (Forsythe and Foster, 1950; Nichol and Winzor, 1964), and thyroxine or thyroglobulin (Litwack and Sears, 1965). Such complexes occur in egg white (Hawthorne, 1950), but do not cause inactivation of lysozyme at room temperature. The present study concerns a reaction that occurs at temperatures near 60°C. and that appears to occur subsequently to the electrostatic complex formation. METHODS AND MATERIALS 3
The egg white used in these experiments was obtained from commercial shell eggs, broken out in the laboratory, blended and frozen. Samples were thawed at room temperature as needed. The proteins ovalbumin, conalbumin, ovomucoid, avidin and lysozyme were twice crystallized preparations purchased from the Worthington Biochemical Corp., Freehold, New Jersey. Ovomucin was prepared from lysozyme-free egg white as described by Cunningham and Lineweaver (1965). L-cysteine hydrochloride was purchased from the Nutritional Biochemical Corp., Cleveland, Ohio. Solutions 3 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
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Inactivation of Lysozyme by Native Ovalbumin1
E. T. MORAN, JR. AND J. MCGINNIS
SUMMARY Turkey poults were fed rations containing either corn or barley as the cereal grain from hatching to 4 weeks of age. The pancreas of the barley fed birds appeared to be smaller than that observed in the corn-fed birds; there were no differences in the intestine weights. Supplementation with oleandomycin, regardless of grain caused increased pancreas weights; with enzyme supplementation this increase was apparent only with the birds fed the corn rations. A reduced intestine
weight was noted for the poults given the corn rations supplemented with oleandomycin. REFERENCES Arscott, G. H., D. C. Hutto and P. Rachapaetayakom, 1965. Use of barley in high-efficiency broiler rations. 7. Pancreatic enlargement in chicks fed barley containing diets. Poultry Sci. 44: 432-434. Burnett, G. S., 1966. Studies of viscosity, as the probable factor involved in the improvement of certain barleys for chickens by enzyme supplementation. Brit. Poultry Sci. 7: 55-75. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Federer, W. T., 1955. Experimental Design. Macmillan Co., New York. Fisher, R. A., and F. Yates, 1963. Statistical Tables for Biological, Agricultural and Medical Research. Hapner Publishing Co., New York. Jukes, H. G., D. C. Hill and H. D. Branion, 1956. Effect of feeding antibiotics on the intestinal tract of the chick. Poultry Sci. 35: 716-723. Moran, E. T., and J. McGinnis, 1965. The effect of a cereal grain and energy level of the diet on the response of turkey poults to antibiotic and enzyme supplements. Poultry Sci. 44: 12531261. Moran, E. T., and J. (McGinnis, 1966. A comparison of corn and barley for the developing turkey and the effect of antibiotic and enzyme supplementation. Poultry Sci. 45: 636-639.
The Relation of Spermatozoa to the Glandular Tissue in the Storage Sites of the Hen Oviduct* H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND ORA KEMPENICH The Volcani Institute of Agricultural Research, Rehovot, Israel (Received for publication March 20, 1967)
T
HE storage sites of spermatozoa in the laying hen's oviduct appear to be the glands of either the uterovaginal junction or the caudal part of the infundibulum, depending on the method of insemination. Van Drimmelen (1946) located sper* Contribution from The National and University Institute of Agriculture, Rehovot, Israel. 1967 Series; No. 1142-E.
matozoa in the mucosal folds of the infundibulum after intraperitoneal inseminations. Bobr et al. (1964) found spermatozoa in the glands of the uterovaginal junction after intravaginal insemination and in both the uterovaginal glands and infundibular glands after intrauterine insemination. Also in our laboratory (unpublished data) spermatozoa were found in the utero-
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ment, growth rate and probably chronological age, not to mention the various possible interactions. Unpublished data from this laboratory with respect to a lysine deficiency in the growing chick showed that the gut continued to grow regardless of the deficiency as, to a lesser extent, did the pancreas while the muscle mass growth was retarded. Because of the many known and probably unknown factors which can influence pancreas size it is obvious that considerable care must be taken before the statement "hypertrophy," "atrophy," etc. of the pancreas can be made.
SPERMATOZOA IN STORAGE SITES
MATERIALS AND METHODS White Leghorn roosters and laying hens were used. They were kept in individual cages kept in an open shed. Intravaginal inseminations were performed by depositing 0.2 ml. of untreated semen into the vagina, which was exposed by abdominal pressure. Intramagnal inseminations were performed after exposing one of the loops of the magnum by laparotomy. After cleaning the feathers from the left abdominal side, local anaesthesia was applied with Lidocain (Teva Ltd., Jerusalem). An incision was made in the skin beneath the pubic bone and parallel to it, beginning at a distance of about 1 cm. from its posterior end and continuing about 1.5 cm. in the cranial direction. After penetration of the underlying muscle layer and the peritoneum, the magnum was easily reached. Subcutaneous and abdominal fat was removed when necessary. 0.2 ml. of a washed sperm suspension (Schindler and Hurwitz, 1966) was injected into one of the loops of the magnum. The peritoneum and the muscles were then sutured and the skin was closed with clamps,
which were removed after 5 days. Care was taken to minimize the duration of the operation and to avoid undue stress on the hen. After three days of incubation, eggs were broken open and checked for the presence of an embryo. After intramagnal inseminations, segments about 1 cm. in length were sampled from all regions of the oviduct and fixed in 4% formaldehyde. After intravaginal insemination, only the uterovaginal junction served for the examinations. Sections were made from different parts of the segments and stained with haematoxylin-eosin. For electron microscope examinations segments about 1 cm. in length were removed from the posterior part of the infundibulum or from the uterovaginal junction and immediately transferred to 1% glutaraldehyde in isotonic phosphate buffer (pH 7.2) for primary fixation. The segments were slit open (while immersed in the glutaraldehyde solution) and pieces of about 1-2 mm.3 were sliced from the inside of the duct, excluding the muscular layer. The cubes were left in the glutaraldehyde solution for 1 hr. in the cold followed by five washings with phosphate buffer at 10minute intervals. They were then transferred to 1% osmic acid in phosphate buffer and kept in the cold for 1 hr. After washing out the osmic acid with phosphate buffer (five washings at 10-minute intervals) the preparations were dehydrated gradually in acetone and embedded in Vestopal W (Martin Jaeger, Vesenaz, Switzerland), according to the manufacturer's instructions. In order to identify the spermatozoacontaining regions in the block before sectioning for electron microscopy, preliminary sections of 2-4 \x. thickness were prepared, and stained with toluidine blue, essentially according to the technique described by Lynn (1965), except for the following modifications: (a) the sections were
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vaginal junction only after intravaginal insemination. On the other hand, Fujii and Tamura (1963) and Takeda (1964) found spermatozoa after intravaginal inseminations in the infundibular as well as in the uterovaginal glands. After deposition of spermatozoa in the infundibulum (Takeda, 1966) or in the magnum (Van Krey et al., 1966), large accumulations of spermatozoa were found in the infundibular glands and a low incidence of spermatozoa in the uterovaginal junction. The present study was undertaken in order to obtain information on the exact position of the fowl spermatozoa in the storage glands and their association with the glandular tissue at various times after insemination.
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H. SCHINDLER, E. BEN-DAVID, S. HUEWITZ AND O. KEMPENICH
RESULTS
Egg production and fertility after intramagnal insemination. This experiment was conducted in order to evaluate the fertilizing capacity of the spermatozoa after the intramagnal inseminations. Washed spermatozoa were injected into the magnum of 10 hens several hours after oviposition; three hens served as controls and were inseminated intravaginally with the same sperm suspension. Seven of the operated hens returned to egg production within seven days after operation and the remain-
ing hens did so by the twelfth day. It can be seen from Table 1 that although egg production was low during the first 11 days after the operation, the fertilization rate was about the same as in the control group. The fertilization rate in the third week was much higher in the treated group than in the control, but most of the embryos in the former group died at an early stage. Location of spermatozoa after deposition of semen in the magnum. In another experiment the sites where spermatozoa were present following intramagnal insemination, were identified. Semen was deposited in the magnum of 11 hens which had laid several hours prior to insemination or had a shellegg in the uterus. The hens were killed after intervals as indicated in Table 2. The results of the histological examinations are presented in Table 2. Spermatozoa were always present in the caudal part (chalaziferous region) of the infundibulum, except in the hen which was killed after 13 days in which no spermatozoa were detected at all. Only in one hen killed one day after the operation, were large concentrations of spermatozoa found in the uterovaginal glands, whereas in two other hens only a few spermatozoa were found in this place. Position of spermatozoa in the storage sites. Shortly after intramagnal inseminaTABLE 1.—Egg production and fertility of hens inseminated in the magnum Daysj after insemination 2-5
6-11
12-13
14-20
Intramagnal insemination Hen days N o . of eggs N o . of fertilized eggs
40 9 5
60 31 28
20 19 14
70 61 35»
Intravaginal insemination Hen days N o . of eggs N o . of fertilized eggs
12 11 9
18 12 11
6 6 1
* 23 dead embryos. f All embryos dead.
21 17
3t
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affixed to slides with albumen-and-glycerin by carefully holding them at a distance over a small open flame for about 1 minute; (b) the necessary exposure to high temperature during the staining process was found to be a little longer than that recommended by Lynn; (c) decolorization was achieved with acetone (95%); and (d) Vestopal served as mounting medium. These preliminary sections were examined with a phase contrast for the presence of spermatozoa, and whenever such were located the corresponding portion of the same block was sectioned for electron microscopy, using a Dannon Ultramicrotome, at about 500 A thickness. Some of these sections were again screened with a phase contrast after staining with toluidine blue in order to verify that they contained the desired portion of the infundibulum and of the uterovaginal junction, i.e., the glandular grooves and the tubular glands with spermatozoa. The other sections were mounted on copper electron microscope grids (type 200, 3-mm. diameter; Morton Ltd., Harrow, England) which were coated with Formvar 15/95E (Shawinigan Resins Corp., Springfield, Mass., U.S.A.), and then stained with uranyl acetate (Pease, 1964). The sections were screened with an RCA Electron Microscope.
SPERMATOZOA IN STORAGE SITES
1465
TABLE 2.—Presence ( 4 ) or absence ( —) of spermatozoa in the various regions of the hen oviduct after intramagnal insemination
Hen no.
6 7 8 9 10 11
3hr. 3 hr. 20 hr. 20 hr. 22 hr. 22 24 72 72 12 13
hr. hr. hr. hr. days days
Stage of egg formation egg m uterus no egg egg in magnum egg in magnum no egg, after oviposition egg in funnel no egg no egg egg in uterus egg in uterus egg in uterus
Infundibulum Cranial
+
+
Caudal I
+ + I
4
Magnum Upper
Middle
Lower
Isth mus
Uterus
Utero^ tion
+
+
f I-
+ i
4
tion (3-4 hr.) they were present in the lumen as well as in the secondary mucosal folds (glandular grooves) of the infundibulum, where they were dispersed or arranged in bundles, sometimes attached to the epithelial lining of the grooves (Fig. 1). Figure 2 shows bundles of spermatozoa on their way to deeper regions of the mucosal folds. A few tubular glands were penetrated by spermatozoa. After intramural insemination spermato-
m
4
zoa were also found in the folds of the cranial part (funnel) of the infundibulum as well as in the glands of the upper part of the magnum. The presence of spermatozoa in these parts of the oviduct may be regarded as temporary because in all other hens killed one day or more after insemination, no spermatozoa were found there, with the exception of one hen which had spermatozoa in the magnal glands one day after intramagnal insemination. (Fig. 3).
\fcii
1. Caudal part (chalaziferous region) of the infundibulum, three hours after intramagnal insemination Note dispersed spermatozoa in the lumen of the secondary mucosal folds, as well as bundles of spermatozoa near the epithelial lining of one of the grooves. (No egg in the oviduct at time of killing.) FIG.
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I 2 3 4 5
Time after insemination
H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND 0.
1466
KEMPENICH
- fA
mm
f fir
t
^»
s
- *
i J o b *_-»-. . « t > ' H B FIG. 2. Caudal part of the infundibulum, three hours after intramagnal insemination. Note bundles of spermatozoa during passage to the deeper parts of the secondary mucosal folds. (Egg in uterus at time of killing.)
At 24 hours after intravaginal or intramagnal inseminations, large concentrations of spermatozoa were found in the grooves of the mucosal folds and tubular glands of the uterovaginal junction and the infundibulum, respectively, and only a few were present in the lumen (Fig. 4). At two or three days after insemination, spermatozoa
were present in the glands, whereas the folds were almost empty of spermatozoa. At 5 or 12 days after insemination, spermatozoa were found in the glands only. The accumulation of spermatozoa in the tubular glands was highest on the first day after insemination and declined with time, as shown in Table 3. In order to determine whether the migra-
: • • :
&
i f &"'::^-y • • •• 'PA. &%-FIG. 3. Sperm bundles in the glands of the magnum, adjacent to the infundibulum, 24 hours after intramagnal insemination. Longitudinal section. (Egg in uterus at time of killing.)
wop. :•
FIG. 4. Sperm bundles in the chalaziferous glands of the infundibulum, 24 hours after intramagnal insemination. (Egg in the cranial part (funnel) of the infundibulum at time of killing.)
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:
SPERMATOZOA IN STORAGE SITES
The idtrastructiire of the spermatozoa and, of their storage sites. The histological preparations showed, in most cases, a close association between the spermatozoa and the epithelial lining of the glandular grooves or the tubular glands, but it was impossible to determine whether the spermatozoa actually penetrate the cells of the epithelium. In order to elucidate the position of the spermatozoa at these places ultramicroscopic examinations of the sperm storage sites were carried out. The ultrastructural details of the infundibulum were identified according to the description by Aitken and Johnston TAHLE 3.—Sperm contents of tubular glands at various limes after semen deposition Infundibulum* Time after insemination 3-4 hr 1 day 2 days 3 days 5 days 12 days
xT
N o . Of
ne
d?nd examinedt i 46 (2) 96(4)
Uterovaginal junction XT,.
r
N o . Of
Slands %°n£ ^nds °nta™nS e x t S d l ^ ^ ^ c vtt ^ '+ spermatozoa Spermatozoa c
7 61
61_(2)
.ill
23(1)
6
* Intramagnal insemination. f Intravaginal insemination. + In parentheses number of hens
100 (2) 81 (2)
63 37
89(3)
I'll
5%^ #•;
a
M%
(1963). Fig. S shows glandular grooves with many sections of spermatozoa. In Fig. Sa also cross-sections of cilia can be seen, which are easily distinguishable from the cross-sections of sperm tails, in spite of the similar arrangement of their fibrils, because sperm tails are considerably larger. Fig. 6 shows spermatozoa inside tubular glands. In all preparations in which concentrations of spermatozoa were found, groups could be distinguished according to the level at which the spermatozoa were cut; this can be seen in the tubular gland of
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tion of the spermatozoa is dependent upon their own activity, washed spermatozoa which had been killed by exposure to 60°C. were injected into the magnum of six hens which were killed after 3, 24 or 72 hours. Histological examination revealed masses of spermatozoa entangled in the secretion of the magnum of the two hens which were killed three hours after the insemination. No spermatozoa were found in any other part of the oviduct of these hens, nor were spermatozoa detected in any part of the oviduct of the remaining four hens which were killed after 24 and 72 hours. It is clear from these results that only active spermatozoa are capable of migrating and entering the storage sites, whereas immotile spermatozoa are expelled.
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H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND O. KEMPENICH
Fig. 6. In one part of the glandular lumen a group of transverse sections of the sperm heads is seen, whereas in another part a
FIG. 7. Section through a tubular gland of the uterovaginal junction. Microvilli are abundant at the luminal surface of the cells. Note cross-sections of spermatozoa and secretory material in the lumen of the gland as well as close to the glandular cells. (Electron micrograph.)
Fie. 8. Section through a secondary mucosal fold of the uterovaginal junction. Note secreting cells and a ciliated cell; secretory material is present in the lumen. (Electron micrograph.)
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FIG. 6. Tubular glands of the infundibulum. Note vacuoles containing flock-like material. Secretory granules are located near the luminal surface. The cells are lower than those of the grooves. Spermatozoa are present in the lumen; cilia are absent. (Electron micrograph.)
group of sections of sperm tails is seen. In the glandular groove shown in Fig. Sb also a group of sperm head sections is seen, but in addition another group of sagittal sections of heads and mid-pieces is seen. This finding shows that the spermatozoa in the storage sites are arranged in bundles, with the corresponding parts of the cells lying parallel to each other. The electron micrographs also reveal that although the spermatozoa are arranged in bundles, some space is left between neighboring sperm cells. In none of the many ultrasections made one or six days after insemination from the infundibulum or the uterovaginal junction have spermatozoa been found to be inserted in the epithelial lining. Most of the sperm sections were found to be free in the lumen; occasionally, sperm sections were found to be very close or attached to the epithelial cells (Figs. Sb, 6, 7). Also in those preparations in which sagittal sections of the anterior part of the sperm head, including the acrosome, were obtained, the sperm head was found to be free
SPERMATOZOA IN STORAGE SITES
DISCUSSION The preliminary experiment carried out in the present study confirms the results obtained by Takeda (1966) and Van Krey et al. (1966) that the infundibular glands are capable of maintaining the fertilizing capacity of the spermatozoa during a period which is at least equal to that of sperm preservation in the uterovaginal glands, and that the conditions prevailing in the infundibulum are conducive to sperm preservation. Since in the present study the hens were
Fie. 9. Non-secreting ciliated cells and mucussecreting cells from a secondary mucosal fold of the chalaziferous region of the infundibulum. (Electron micrograph.)
killed at different intervals after insemination, the migration of the spermatozoa could be followed. Whereas the transit from the magnum to the infundibulum took place within a very short time, the migration of the bulk of the spermatozoa from the lumen of this region into the tubular glands took at least 24 hours, and only after a few days were the mucosal folds empty. With the semen dose in this study, about 60% of the tubular glands of the uterovaginal junction and the infundibulum were filled with spermatozoa one day after intravaginal or intramagnal insemination, respectively. After this time, the number of glands containing sperm declined gradually. It appears that during the process of sperm depletion some glands are emptied entirely and others remain temporarily unaffected, rather than that all glands release part of their sperm reserve synchronously while retaining another part for future release. The migration of the spermatozoa is dependent upon their own activity, as indicated clearly by the fact that immotile spermatozoa did not reach the infundibulum and were soon expelled. This observation agrees with the results of Fujii and Tamura (1963) who demonstrated that spermatozoa deposited in the vagina can pass into the uterovaginal junction only when they are motile, and with those of Allen and Grigg (1957) who had shown previously that only active spermatozoa pass the barrier of the uterovaginal sphincter. The examinations which were carried out with the electron microscope showed that most of the spermatozoa lie free in the lumen of the glands. This position is not changed even after prolonged storage. This finding does not confirm the assumption by Fujii and Tamura (1963) that the spermatozoa penetrate the intercellular spaces of the glandular epithelium. It may be con-
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in the lumen of the gland. Glandular secretion of relatively low electron density could be seen in the lumen of the glands or of the glandular grooves. The group of sperm heads shown in Fig. 6 is embedded in such a secretion. Tissue structure as well as cellular ultrastructure were similar in the uterovaginal junction and the caudal part of the infundibulum (Figs. 8 and 9).
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H. SCHINDLER, E. BEN-DAVID, S. HURWITZ AND O. KEMPENICH
parts of the cells lying parallel to each other. Most of the sperm sections were found to be free in the lumen of the glands; occasionally sperm sections were found to be very close or attached to the epithelial cells. Tissue structure as well as cellular ultrastructure were similar in the uterovaginal junction and the caudal part of the infundibulum. ACKNOWLEDGMENTS
This investigation was supported by a grant from the United States Department of Agriculture under P.L. 480. The assistance of Y. Cohen (Division of Virology) in the electron microscopy is gratefully acknowledged. Thanks are also due to Dr. T. A. Nobel (The Veterinary Institute, Bet Dagan) for helpful discussion of the histological findings.
SUMMARY
REFERENCES
The position of the spermatozoa in the storage sites of the hen oviduct was examined at various intervals after intravaginal and intramagnal inseminations. Shortly after intramagnal insemination spermatozoa were present in the lumen as well as in the secondary mucosal folds of the chalaziferous region, but tubular glands were penetrated by spermatozoa only to a small extent. At 24 hours after intravaginal or intramagnal insemination, large concentrations of spermatozoa were still present in the grooves of the mucosal folds of the uterovaginal junction and the chalaziferous region, respectively, but disappeared from there within a few days. Sperm accumulation in the tubular glands of either the uterovaginal junction or the infundibulum was highest one day after insemination and then declined with time. Electron micrographs showed that the spermatozoa in the storage sites are arranged in bundles, with the corresponding
Aitken, R. N. C , and H. S. Johnston, 1963. Observations on the fine structure of the infundibulum of the avian oviduct J. Anat. 97: 87-99. Allen, T. E., and G. W. Grigg, 19S7. Sperm transport in the fowl. Aust. J. Agric. Res. 8: 788799. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1964. Distribution of spermatozoa in the oviduct and fertility in domestic birds. I. Residence sites of spermatozoa in fowl oviducts. J. Reprod. Fertil. 8: 39-47. Fujii, S., and T. Tamura, 1963. Location of sperms in the oviduct of the domestic fowl -with special reference to storage of sperms in the vaginal gland. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 5: 145-163. Lynn, J. A., 1965. Rapid Toluidine Blue staining of epon-embedded and mounted "adjacent" sections. Am. J. Clin. Pathol. 44: 57-58. Pease, D. C , 1964. Histological Techniques for Electron Microscopy. Academic Press, New York. Schindler, H., and S. Hurwitz, 1966. The preservation of sperm motility in different regions of the hen oviduct in vivo. Poultry Sci. 45: 369374. Takeda, A., 1964. Behavior of spermatozoa in the
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eluded that the spermatozoa in the glands derive energy-yielding substances from the glandular secretion and not from intimate association with the cytoplasm of the glandular cells. It also appears from the electron micrographs that the spermatozoa are not as densely packed as may be assumed from the histological preparations. Therefore, sufficient oxygen may diffuse into the spaces between the spermatozoa to support aerobic metabolism. The histological structure and ultrastructure of both storage sites show great similarity. The behavior of the spermatozoa in these places, i.e., their gradual infiltration into the tubular glands from the glandular grooves and the gradual depletion of the glands, is also similar. It may be, therefore, postulated that the factors supporting the survival of the spermatozoa in both sites are similar.
SPERMATOZOA IN STORAGE SITES genital tract of the hen. I. Presence of spermatozoa in the oviduct. Jap. J. Poultry Sci. 1: 19-31. Takeda, A., 1966. Behavior of spermatozoa in the genital tract of the hen. III. Fertility and spermatozoal behavior following infundibulal insemination. Jap. Poultry Sci. 3 : 15-21. Van Drimmelen, G. C , 1946. "Spermnests" in the
1471
oviduct of the domestic hen. J. S. Afr. Vet. Med. Ass. 19: 42-52. Van Krey, H. P., F. X. Ogasawara and F. W. Lorenz, 1966. Distribution of spermatozoa in the oviduct and fertility in domestic birds. IV. Fertility of spermatozoa from infundibular and uterovaginal glands. J. Reprod. Fertil. 11: 257— 262.
F. E. CUNNINGHAM 2 AND HANS LINEWEAVER Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 (Received for publication March 20, 1967)
I
N A STUDY of the heat stability of egg white and egg white proteins, Cunningham and Lineweaver (1965) reported that in egg white heated to 62.5°C. for 10 minutes at pH 9 lysozyme was inactivated extensively, but that the purified protein dissolved in buffer was unaffected by this time-temperature condition. These results confirmed the heat sensitivity of lysozyme in egg white (Sandow, 1926; Cotterill and Winter, 1954; and Seideman, Cotterill and Funk, 1963) and the stability of lysozyme in buffer (Gorini and Felix, 1953), but they do not explain the reason for the marked difference in stability of lysozyme in the two media. Highly basic lysozyme is known to form electrostatic complexes with other substances. It forms complexes with sodium thymus nucleate and yeast nucleate (Klotz and Walker, 1948), bovine plasma albumin (Steiner, 1953) ovomucin (Hawthorne, 1950; Cotterill and Winter, 1955), conalbumin (Ehrenpreis and Warner, 1956), 1 Presented in part at the Poultry Science Association Meeting, Athens, Ga. 1965 and in part at the 2nd International Congress of Food Science and Technology, Warsaw, Poland, Aug. 1966. 2 Present address, Henningsen Foods, Inc., 2501 College St., Springfield, Missouri 65802.
ovalbumin (Forsythe and Foster, 1950; Nichol and Winzor, 1964), and thyroxine or thyroglobulin (Litwack and Sears, 1965). Such complexes occur in egg white (Hawthorne, 1950), but do not cause inactivation of lysozyme at room temperature. The present study concerns a reaction that occurs at temperatures near 60°C. and that appears to occur subsequently to the electrostatic complex formation. METHODS AND MATERIALS 3
The egg white used in these experiments was obtained from commercial shell eggs, broken out in the laboratory, blended and frozen. Samples were thawed at room temperature as needed. The proteins ovalbumin, conalbumin, ovomucoid, avidin and lysozyme were twice crystallized preparations purchased from the Worthington Biochemical Corp., Freehold, New Jersey. Ovomucin was prepared from lysozyme-free egg white as described by Cunningham and Lineweaver (1965). L-cysteine hydrochloride was purchased from the Nutritional Biochemical Corp., Cleveland, Ohio. Solutions 3 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
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Inactivation of Lysozyme by Native Ovalbumin1