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Hydrogen Ion Concentration of the Oviduct of the Laying Domestic Fowl1
Hydrogen Ion Concentration of the Oviduct of the Laying Domestic Fowl1 F. X. OGASAWARA, H. P. VAN KREY AND F. W. LORENZ Department of Poultry Husbandry, University of California, Davis (Received for publication May 6, 1963)
A
MATERIALS AND METHODS
Two groups of 12 Single Comb White Leghorn hens from the department flock were used. All hens were in active egg production and had ovulated during the previous 30 hours. Sodium pentobarbital (diluted with three parts 0.9% NaCl) was injected intravenously until the wink reflex was suppressed. The hens were then laparotomized, taking precautions to minimize hemorrhage. A long, thin electrode assembly fTurkman Prr.hP Aggpmhlyr No. 39176) was inserted through incisions approximately 5 mm. long in. the oviduct and positioned at various places on the inner wall by gentle palpation; pH was read on a Beckman pH meter, Model G. Hard shelled eggs in the uterus were expelled manually prior to inserting the 1 This investigation was supported in part by funds from National Science Foundation grant G9837, and in part by a Public Health Service training grant 2G 646 from the Institute of General Medical Sciences, Public Health Service.
electrode; eggs higher in the oviduct were left undisturbed. Four incisions were necessary to explore the entire oviduct. A laboratory thermometer was inserted through each incision immediately after removal of the probe. Temperatures never fell below 35°C. and usually were closer to 39°C. The data for the first group of birds were obtained over a period of several weeks. Measurements of pH were made in 9 locations throughout the oviducts, all within 15 minutes after laparotomy. Because of this time limitation, and in a few instances because of obstructing eggs, not all locations were measured in all birds. In several birds an additional estimate of vaginal pH was made, prior to anesthesia, by inserting the probe into the vagina which had been everted according to the method ordinarily used for artificial insemination (Burrows and Quinn, 1936). All birds survived the above manipulations. Immediately thereafter they were killed with additional sodium pentobarbital. The oviducts were removed, placed between saline-wetted paper towels and left undisturbed for at least 30 minutes. The pH readings were then repeated, with the pH meter readjusted to the lower temperatures (approximately 25°C), to which the tissues had fallen. The variability in the results, described below, led to selection of a second group of birds all of which were laying at high rates. Determinations of pH on this group were made within a period of 2 days, and pH was measured at 14 points. Extra care was
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
S PART of an investigation of the role - the environment in survival of inseminated spermatozoa, information was obtained on pH of the oviduct lumen. A previous study (Buckner and Martin, 1929) with homogenized tissue segments of oviducts of recently killed hens yielded average pH values of 5.8 to 6.4. In the present investigation pH was determined in oviduct lumina of anesthetized hens. These values were compared with values obtained in the same oviducts shortly after excision.
4
F. X. OGASAWARA, H. P. VAN KREY AND F. W. LORENZ
lintundibulum [ junction
mnnnum
i REGION
isthmus
i
litems
| vgqinn junction
i
OF OVIDUCT
taken to place the electrode as precisely as possible at each point within the oviduct, and the positions of any ova present were recorded. Otherwise, these birds were handled exactly as those in Group 1. RESULTS The pH values in the oviducts of Group 1 are plotted in Figure 1. The horizontal positions of most points are approximate only, i.e. each column of points represents measurements taken at random throughout the corresponding area. The electrode was positioned precisely only at the infundibulum-magnum and uterovaginal junctions. The pH values are highly variable. In most of the regions the values spanned nearly one pH unit; only in the utero-vaginal junction were they relatively constant. In spite of their variability, however, the plotted points describe a curve, starting with an average pH of 7.742 in the infundibulum and falling steadily to a minimum of 7.17 in the pos2
It should be noted that since pH values are logarithms, arithmetic averages (plotted in Figures 1 and 2 and quoted in the text) are geometric rather than arithmetic means of the hydrogen-ion concentrations. However, the use of average pH values is justified by the relatively homogeneous variance they yield.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
FIG. 1. Group 1: pH values of mucosal surface plotted against region of oviduct. Except at junctions, placement of electrode within regions shown was approximate only.
terior magnum. The curve then climbs sharply to a maximum of 7.87 in the utero-vaginal junction; thereafter it declines slightly, to 7.70 in the vagina. Of 82 individual measurements, only four (one in the posterior magnum and three in the isthmus) were below pH 7.0. The results obtained with the birds in Group 2 were similar to those of Group 1. With more points measured, a steady drop in pH was observed from the anterior infundibulum through the anterior isthmus (Figure 2). At the posterior isthmus the pH values increased again, rising to an abrupt maximum of 7.81 on the vaginal side of the utero-vaginal junction. Considering differences in care with which the electrode was positioned, and the variability of individual values, the two curves are in essential agreement in all regions except the uterus, where the average pH in Group 2 birds was about 0.25 units lower than in birds of Group 1. Utero-vaginal junctions of Group 2 birds were measured in three carefully defined but closely spaced regions: squarely within, and on either margin of the sphincter. Typical uterine pH values were found on the uterine margin and within the sphincter, but on the vaginal margin (which includes the glandular region—see below) they were sharply elevated. The electrode had been placed in the utero-vaginal junctions of Group 1 birds without anticipating this sharp pH difference; consequently, the high values obtained probably resulted from fortuitous positioning of the electrode on the vaginal margin in each of these birds. In the vagina proper the pH was a little lower in both groups, averaging 7.71 with a range of 7.20 to 8.30. Additional measurements via the cloaca, in 13 of the same birds prior to anesthesia yielded an identical average and a similar range of values.
5
P H OF THE OVIDUCT
r«r 1 r The pooled error variances of Groups 1 and 2 were respectively 0.249 and 0.232, leading to fiducial limits (P = 0.05) for individual oviduct areas in the two groups varying from +0.14 to +0.29 pH units, depending on the number of observations made in that area. Unfortunately, these data did not allow for separate estimation of variance due to bird individuality. An attempt was made to assess this potential source of variabili t y through calculation of correlation coefficients between areas in eight of the birds of Group 2 for which pH measurements were available in each of five areas. FIG. 2. Group 2: pH values of mucosal surface These coefficients were all nonsignificant plotted against region of oviduct. The rectangles and negative nearly as often as positive, drawn above the oviduct diagram show the extent except for the one between the adjacent of regions represented by the points plotted directly isthmus and mid-uterine areas; this was above. The two regions on either side of the uterovaginal junction were actually closer to the junction significant at the 5% level (r = 0.77). than shown in the diagram. Thus, in these birds, at least, little or no variability appeared to be attributable to DISCUSSION bird individuality. The pH values in the oviduct reported Likewise, little of the variability aphere are of interest considering reported peared to be attributable to the secretory pH optima for cock spermatozoa. Alstage of the oviduct, at least insofar as this could be estimated by the positions of though these optima are not sharp, they descending ova. Positions of ova in rela- apparently lie between 7.0 and 7.5 for in tion to the point of pH measurement are vitro life span (Ogasawara, 1957). Lardy indicated in Figure 2. To be sure, most of and Phillips (1943) found an optimum pH values obtained distal to an approaching of 7.25 for oxidative metabolism, and Wilovum were clustered in upper regions of cox and Shaffner (1957) reported the optithe pH distributions, but there were mum for maintenance of fertilizing capacseveral exceptions; otherwise the points ity, when the semen was stored several appeared to be distributed very nearly at hours in a phosphate-saline diluent, to be between 7.03 and 7.27. These optima are random. The pH measurements made 30 to 45 all lower than the pH observed here in any minutes after death of the hens dropped part of the oviduct except the posterior an average of 0.7 units. The drop was more magnum and anterior isthmus. Sperm are extreme at the ends of the oviduct than in stored in the oviduct in the lumina of the middle; the average pH in different tubular glands in a narrow region on the regions ranged from 6.5 to 7.0. This rapid vaginal margin of the sphincter in the shift in pH after death doubtless accounts utero-vaginal junction (Bobr, 1962; Bobr for the low values previously reported by et al., 1962). Yet this region had the highest average pH in the entire oviduct. HowBuckner and Martin (1929). • • bord-shelled egq in ultras • - ovum ol poinl
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
InlirKjbulum
6
F. X. OGASAWARA, H. P. VAN KREY AND F. W. LORENZ
SUMMARY
Determinations of pH were made in oviducts of anesthetized laying hens, using an electrode probe assembly. Individual values, measured on the mucosal surface, were nearly all alkaline; average values were highest in the infundibulum and
utero-vaginal junction and lowest in the posterior magnum and anterior isthmus. REFERENCES Buckner, G. D., and J. H. Martin, 1929. The hydrogen ion concentration of the reproductive organs of the White Leghorn chicken. Am. J. Physiol. 89: 164-169. Bobr, L. W., 1962. Oviducal distribution of spermatozoa and fertility of the domestic fowl. Thesis, Univ. of Calif. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1962. The role of the uterovaginal junction in storage of cock spermatozoa. Poultry Sci. 41: 1628. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Lardy, H. A., and P. H. Phillips, 1943. Effects of pH and certain electrolytes on the metabolism of ejaculated spermatozoa. Am. J. Physiol. 138: 741-746. Ogasawara, F. X., 1957. Oxidative metabolism of fowl spermatozoa as influenced by extracts of the hen's oviduct. Thesis, Univ. of Calif. Olsen, M. W., and B. H. Neher, 1948. The site of fertilization in the domestic fowl. J. Exp. Zool. 109:35S-366. Wilcox, F. H., and C. S. Shaffner, 1957. The effect of differences in salt and hydrogen ion concentration on the fertilizing ability of avian sperm. J. Appl. Physiol. 11:429-434.
Alkyl Mercury-Treated Seed in Food Grain MERCURY IN TISSUES AND EGGS FROM HENS FED WITH GRAIN CONTAINING METHYL MERCURY DICYANDIAMIDE Department
STIG TEJNING 1 AND RAGNAR VESTERBERG of Industrial Hygiene, National Institute of Public Health, Stockholm,
Sweden
(Received for publication December 3, 1962)
I
N Sweden the disinfectants most commonly used for seed treatment are liquids—"Panogen" (methyl mercury dicyandiamide) and "Betoxin F " (ethyl mercuric chloride)—containing 0.8 percent "Present address: Department of Occupational Medicine, University Hospital, Lund, Sweden.
mercury as active substance. Dust disinfectants are rarely used nowadays. About 85 percent of seed corn is treated with "Panogen." For approximately 100 kg. of wheat, 200 ml. of this disinfectant are used, which implies that 1 g. of seed contains about 16 p.g., and each grain about 0.5 j/.g. of mercury.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
ever, the pH of the inner surface of the oviduct in this region is not necessarily identical with that deep in the sperm glands. Also, it should not be expected that particular optimum conditions in vivo are necessarily identical with corresponding in vitro optima unless all conditions are made identical. It is of interest in this connection that the pH of the inf undibulum, the site of fertilization (Olsen and Neher, 1948), was also high. The authors recently were privileged to read an unpublished manuscript by C. M. Winget and C. A. Mephan, in which cyclic shifts of considerable magnitude in uterine pH were reported. Very likely, similar cyclic shifts throughout the oviduct may account, in part at least, for the variability in pH reported here.
A
MATERIALS AND METHODS
Two groups of 12 Single Comb White Leghorn hens from the department flock were used. All hens were in active egg production and had ovulated during the previous 30 hours. Sodium pentobarbital (diluted with three parts 0.9% NaCl) was injected intravenously until the wink reflex was suppressed. The hens were then laparotomized, taking precautions to minimize hemorrhage. A long, thin electrode assembly fTurkman Prr.hP Aggpmhlyr No. 39176) was inserted through incisions approximately 5 mm. long in. the oviduct and positioned at various places on the inner wall by gentle palpation; pH was read on a Beckman pH meter, Model G. Hard shelled eggs in the uterus were expelled manually prior to inserting the 1 This investigation was supported in part by funds from National Science Foundation grant G9837, and in part by a Public Health Service training grant 2G 646 from the Institute of General Medical Sciences, Public Health Service.
electrode; eggs higher in the oviduct were left undisturbed. Four incisions were necessary to explore the entire oviduct. A laboratory thermometer was inserted through each incision immediately after removal of the probe. Temperatures never fell below 35°C. and usually were closer to 39°C. The data for the first group of birds were obtained over a period of several weeks. Measurements of pH were made in 9 locations throughout the oviducts, all within 15 minutes after laparotomy. Because of this time limitation, and in a few instances because of obstructing eggs, not all locations were measured in all birds. In several birds an additional estimate of vaginal pH was made, prior to anesthesia, by inserting the probe into the vagina which had been everted according to the method ordinarily used for artificial insemination (Burrows and Quinn, 1936). All birds survived the above manipulations. Immediately thereafter they were killed with additional sodium pentobarbital. The oviducts were removed, placed between saline-wetted paper towels and left undisturbed for at least 30 minutes. The pH readings were then repeated, with the pH meter readjusted to the lower temperatures (approximately 25°C), to which the tissues had fallen. The variability in the results, described below, led to selection of a second group of birds all of which were laying at high rates. Determinations of pH on this group were made within a period of 2 days, and pH was measured at 14 points. Extra care was
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
S PART of an investigation of the role - the environment in survival of inseminated spermatozoa, information was obtained on pH of the oviduct lumen. A previous study (Buckner and Martin, 1929) with homogenized tissue segments of oviducts of recently killed hens yielded average pH values of 5.8 to 6.4. In the present investigation pH was determined in oviduct lumina of anesthetized hens. These values were compared with values obtained in the same oviducts shortly after excision.
4
F. X. OGASAWARA, H. P. VAN KREY AND F. W. LORENZ
lintundibulum [ junction
mnnnum
i REGION
isthmus
i
litems
| vgqinn junction
i
OF OVIDUCT
taken to place the electrode as precisely as possible at each point within the oviduct, and the positions of any ova present were recorded. Otherwise, these birds were handled exactly as those in Group 1. RESULTS The pH values in the oviducts of Group 1 are plotted in Figure 1. The horizontal positions of most points are approximate only, i.e. each column of points represents measurements taken at random throughout the corresponding area. The electrode was positioned precisely only at the infundibulum-magnum and uterovaginal junctions. The pH values are highly variable. In most of the regions the values spanned nearly one pH unit; only in the utero-vaginal junction were they relatively constant. In spite of their variability, however, the plotted points describe a curve, starting with an average pH of 7.742 in the infundibulum and falling steadily to a minimum of 7.17 in the pos2
It should be noted that since pH values are logarithms, arithmetic averages (plotted in Figures 1 and 2 and quoted in the text) are geometric rather than arithmetic means of the hydrogen-ion concentrations. However, the use of average pH values is justified by the relatively homogeneous variance they yield.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
FIG. 1. Group 1: pH values of mucosal surface plotted against region of oviduct. Except at junctions, placement of electrode within regions shown was approximate only.
terior magnum. The curve then climbs sharply to a maximum of 7.87 in the utero-vaginal junction; thereafter it declines slightly, to 7.70 in the vagina. Of 82 individual measurements, only four (one in the posterior magnum and three in the isthmus) were below pH 7.0. The results obtained with the birds in Group 2 were similar to those of Group 1. With more points measured, a steady drop in pH was observed from the anterior infundibulum through the anterior isthmus (Figure 2). At the posterior isthmus the pH values increased again, rising to an abrupt maximum of 7.81 on the vaginal side of the utero-vaginal junction. Considering differences in care with which the electrode was positioned, and the variability of individual values, the two curves are in essential agreement in all regions except the uterus, where the average pH in Group 2 birds was about 0.25 units lower than in birds of Group 1. Utero-vaginal junctions of Group 2 birds were measured in three carefully defined but closely spaced regions: squarely within, and on either margin of the sphincter. Typical uterine pH values were found on the uterine margin and within the sphincter, but on the vaginal margin (which includes the glandular region—see below) they were sharply elevated. The electrode had been placed in the utero-vaginal junctions of Group 1 birds without anticipating this sharp pH difference; consequently, the high values obtained probably resulted from fortuitous positioning of the electrode on the vaginal margin in each of these birds. In the vagina proper the pH was a little lower in both groups, averaging 7.71 with a range of 7.20 to 8.30. Additional measurements via the cloaca, in 13 of the same birds prior to anesthesia yielded an identical average and a similar range of values.
5
P H OF THE OVIDUCT
r«r 1 r The pooled error variances of Groups 1 and 2 were respectively 0.249 and 0.232, leading to fiducial limits (P = 0.05) for individual oviduct areas in the two groups varying from +0.14 to +0.29 pH units, depending on the number of observations made in that area. Unfortunately, these data did not allow for separate estimation of variance due to bird individuality. An attempt was made to assess this potential source of variabili t y through calculation of correlation coefficients between areas in eight of the birds of Group 2 for which pH measurements were available in each of five areas. FIG. 2. Group 2: pH values of mucosal surface These coefficients were all nonsignificant plotted against region of oviduct. The rectangles and negative nearly as often as positive, drawn above the oviduct diagram show the extent except for the one between the adjacent of regions represented by the points plotted directly isthmus and mid-uterine areas; this was above. The two regions on either side of the uterovaginal junction were actually closer to the junction significant at the 5% level (r = 0.77). than shown in the diagram. Thus, in these birds, at least, little or no variability appeared to be attributable to DISCUSSION bird individuality. The pH values in the oviduct reported Likewise, little of the variability aphere are of interest considering reported peared to be attributable to the secretory pH optima for cock spermatozoa. Alstage of the oviduct, at least insofar as this could be estimated by the positions of though these optima are not sharp, they descending ova. Positions of ova in rela- apparently lie between 7.0 and 7.5 for in tion to the point of pH measurement are vitro life span (Ogasawara, 1957). Lardy indicated in Figure 2. To be sure, most of and Phillips (1943) found an optimum pH values obtained distal to an approaching of 7.25 for oxidative metabolism, and Wilovum were clustered in upper regions of cox and Shaffner (1957) reported the optithe pH distributions, but there were mum for maintenance of fertilizing capacseveral exceptions; otherwise the points ity, when the semen was stored several appeared to be distributed very nearly at hours in a phosphate-saline diluent, to be between 7.03 and 7.27. These optima are random. The pH measurements made 30 to 45 all lower than the pH observed here in any minutes after death of the hens dropped part of the oviduct except the posterior an average of 0.7 units. The drop was more magnum and anterior isthmus. Sperm are extreme at the ends of the oviduct than in stored in the oviduct in the lumina of the middle; the average pH in different tubular glands in a narrow region on the regions ranged from 6.5 to 7.0. This rapid vaginal margin of the sphincter in the shift in pH after death doubtless accounts utero-vaginal junction (Bobr, 1962; Bobr for the low values previously reported by et al., 1962). Yet this region had the highest average pH in the entire oviduct. HowBuckner and Martin (1929). • • bord-shelled egq in ultras • - ovum ol poinl
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
InlirKjbulum
6
F. X. OGASAWARA, H. P. VAN KREY AND F. W. LORENZ
SUMMARY
Determinations of pH were made in oviducts of anesthetized laying hens, using an electrode probe assembly. Individual values, measured on the mucosal surface, were nearly all alkaline; average values were highest in the infundibulum and
utero-vaginal junction and lowest in the posterior magnum and anterior isthmus. REFERENCES Buckner, G. D., and J. H. Martin, 1929. The hydrogen ion concentration of the reproductive organs of the White Leghorn chicken. Am. J. Physiol. 89: 164-169. Bobr, L. W., 1962. Oviducal distribution of spermatozoa and fertility of the domestic fowl. Thesis, Univ. of Calif. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1962. The role of the uterovaginal junction in storage of cock spermatozoa. Poultry Sci. 41: 1628. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Lardy, H. A., and P. H. Phillips, 1943. Effects of pH and certain electrolytes on the metabolism of ejaculated spermatozoa. Am. J. Physiol. 138: 741-746. Ogasawara, F. X., 1957. Oxidative metabolism of fowl spermatozoa as influenced by extracts of the hen's oviduct. Thesis, Univ. of Calif. Olsen, M. W., and B. H. Neher, 1948. The site of fertilization in the domestic fowl. J. Exp. Zool. 109:35S-366. Wilcox, F. H., and C. S. Shaffner, 1957. The effect of differences in salt and hydrogen ion concentration on the fertilizing ability of avian sperm. J. Appl. Physiol. 11:429-434.
Alkyl Mercury-Treated Seed in Food Grain MERCURY IN TISSUES AND EGGS FROM HENS FED WITH GRAIN CONTAINING METHYL MERCURY DICYANDIAMIDE Department
STIG TEJNING 1 AND RAGNAR VESTERBERG of Industrial Hygiene, National Institute of Public Health, Stockholm,
Sweden
(Received for publication December 3, 1962)
I
N Sweden the disinfectants most commonly used for seed treatment are liquids—"Panogen" (methyl mercury dicyandiamide) and "Betoxin F " (ethyl mercuric chloride)—containing 0.8 percent "Present address: Department of Occupational Medicine, University Hospital, Lund, Sweden.
mercury as active substance. Dust disinfectants are rarely used nowadays. About 85 percent of seed corn is treated with "Panogen." For approximately 100 kg. of wheat, 200 ml. of this disinfectant are used, which implies that 1 g. of seed contains about 16 p.g., and each grain about 0.5 j/.g. of mercury.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 11, 2015
ever, the pH of the inner surface of the oviduct in this region is not necessarily identical with that deep in the sperm glands. Also, it should not be expected that particular optimum conditions in vivo are necessarily identical with corresponding in vitro optima unless all conditions are made identical. It is of interest in this connection that the pH of the inf undibulum, the site of fertilization (Olsen and Neher, 1948), was also high. The authors recently were privileged to read an unpublished manuscript by C. M. Winget and C. A. Mephan, in which cyclic shifts of considerable magnitude in uterine pH were reported. Very likely, similar cyclic shifts throughout the oviduct may account, in part at least, for the variability in pH reported here.