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Luteinizing Hormone Content in the Pituitary of Laying and Nonlaying Hens1
970
K. L. ARORA AND I. L. KOSIN
Weisbroth, H. S., 1960. The morphological response of gastrulae from three genetic lines of Broad Breasted Bronze turkeys to preincubation storage. M. S. Thesis, Washington State University, Pullman.
Wilson, W. O., U. K. Abbott and H. Abplanalp, 1961. Evaluation of Coturnix (Japanese quail) as pilot animal for poultry. Poultry Sci. 40: 6S1-6S7.
Luteinizing Hormone Content in the Pituitary of Laying and Nonlaying Hens 1
(Received for publication January 31, 1966)
I
T has been reported that the content of gonadotropic hormones in the anterior lobe of pituitary is greater in nonlaying hens than in laying hens when assayed for activity in causing vaginal cornification of rats (Phillips, 1942) or in increasing the weight of chick testes (Nakajo and Imai, 1957; Imai et al., 1964). Also, the gonadotropin content has been reported to be greater in hens having "quiescent" or "regressing" ovarian follicles than in hens having "mature" or "maturing" ovarian follicles when assayed on the mouse uterus (Riley and Fraps, 1942). Since these assays are not specific for either Follicle Stimulating Hormone (FSH) or Luteinizing Hormone (LH) (Evans and Simpson, 1950, a review), it is unknown whether the difference in the gonadotropin content between the laying and the nonlaying hens or between the hens in different ovarian conditions is due to a difference in the content of either FSH or LH, or in the content of both. In this paper, LH content in the anterior lobe of pituitary in laying and nonlaying hens was determined by the rat ovarian ascorbic acid depletion (OAAD) method, which was reported to be a highly specific method for assaying LH (Parlow, 1
Aided by Research Grant from the Ministry of Education of Japan and from Gotoh Hatchery Co., Gifu, Japan.
1958, 1961), although its absolute specificity was criticized recently (Rosemberg et al., 1965). MATERIALS AND METHODS Twenty-six laying and 13 nonlaying White Leghorn hens (18-22 months of age) were the donors of the pituitary glands. The laying hens, which had been laying in regular clutches of 2 to 4 eggs, were killed at three different times after oviposition of the initial or of the second egg of a clutch: (1) 0-3 hr., (2) 5-6 hr., and (3) 8-9 hr. The nonlaying hens, which had not laid for a period of more than 2 weeks and which were molting their wing feathers, were killed at 2:00-4:00 p.m. Their ovarian weight was less than 5.5 g. (mean: 4.0 g., for 13 hens). The anterior lobe of the pituitary was removed and weighed. Two to 6 pituitaries were pooled and homogenized together adding physiological saline solution during cooling in an ice-bath. The homogenates were refrigerated below 4°C. Within 20 hours after making the homogenates, they were assayed for OAAD activity on the immature rats of the Hotzman strain. The OAAD assay was carried out according to the method of Parlow (1961) with some modifications which follow. Like an ordinary design of assay of hormones,
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
KATUHIDE TANAKA, YASUHIRO FUJISAWA AND SHOZO YOSHIOKA Laboratory of Poultry Physiology, Gifu University, Kagamihara, Gifu, Japan
971
PITUITARY LUTEINIZING HORMONE
RESULTS AND DISCUSSION
Table 1 shows the results obtained by the assay of the pituitary from molting nonlaying hens and from laying hens at three different times after oviposition of the initial or of the second egg of a clutch. The weight of the anterior lobe of the pituitary did not differ significantly between groups (P > .05). The ovarian ascorbic acid (OAA) concentration of rats which received pituitaries of the nonlaying hen was signifi* Kindly donated by the Endocrinology Study Section, National Institute of Health.
12,0
„
100
4
X : 0.121
(10)
80 -
60
40 -
20
-J—v*^«S&line
1.0
ao
4.0
FIG. 1. Log dose response relationship for NIH-LH SS (ovine). Numbers in parentheses are the numbers of assay rats. The vertical lines indicate standard errors of mean. X: Index of precision.
cantly less than that of rats which received pituitaries of the laying hen 0-3 hr. or 8-9 hr. after oviposition (Groups 1 and 3) (P < .01), but not significantly different from that of rats which received pituitaries of the laying hen 5-6 hr. after oviposition (Group 2) (P > .05). For the convenience of comparison, LH content in the pituitary was calculated by fitting the OAA values to the regression equation shown in Figure 1. The results indicate that LH content in the pituitary of the nonlaying hen is roughly twice as much as that in the pituitary of the laying hen 0-3 hr. or 8-9 hr. after oviposition. Imai et al. (1964) found by a chick assay that the content of total gonadotropins in the pituitary of molting nonlaying hens was greater than that in the pituitary of laying hens near ovulation. Whether FSH content also differs as does LH content remains to be determined.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
control and test groups of rats were prepared. Injection was made into the saphena vein at 9:30-10:30 a.m. Three hours ± 5 minutes after the injection, both ovaries were removed, weighed and homogenized together during cooling in an ice-bath. Assays of the pituitary from laying hens and from nonlaying hens were run on the same litters of rats. The injected dose was 0.4 ml. pituitary homogenates containing 10 mg. fresh tissue equivalent. Control rats received an injection of the same volume of physiological saline solution. Before doing the assay of the pituitary, NIH-LH SS (ovine)2 was assayed to test the reliability of this OAAD method. The doses used were 1.0, 2.0, and 4.0 (Jig. per rat. The results were statistically analyzed by the method described by Bliss (1952). A linear relationship was observed between the log dose injected and the ovarian ascorbic acid concentration, as shown in Figure 1. The index of precision (X) of this assay was 0.121. This indicates that our modified OAAD method can be used for assaying LH with a satisfactory precision. No parallel assay with the LH standard was carried out simultaneously but LH amounts in the pituitary were obtained by comparing the assay results with the previously determined linear NIH-LH S5 curve.
972
K. TANAKA, Y. FUJISAWA AND S. YOSHIOKA TABLE 1.—Rat ovarian ascorbic acid depletion assay for LH of the anterior lobe of pituitary from molting nonlaying hens and from laying hens at three different times after oviposition of initial or second egg of a clutch Estimated
Source of pituitary
Nonlaying hens3
OAA cone. (Mg./100 mg. ovary)
Weight of ant. pituitary (mg.)
13
7.9±0.39*a**
7
65.7 + 2 . 5 5 V *
2.0
1.6
9
7.7 + 0.31 a
5
77.2 + 3.10 b
1.2
0.9
8
8.0 + 0.32 a
4
63.9 + 2 . 5 4 a
2.2
1.8
9
8.6 + 0 . 5 4 a
6
81.0+1.91 b
0.8
0.7
Control
10
99.2 + 1.00c
LH cone.1 LH content 2 Og./100 mg. Gug./pituitpituitary) ary)
1
Calculated by fitting the values of OAA cone, to the equation shown in Figure 1. Calculated by LH cone, times pituitary weight. Killed at 2:00-4:00 p.m. * Standard error of mean. ** Means followed by the same letter are not significantly different (
2
3
In the laying hen, the content of LH in the pituitary was greater in Group 2 (S-6 hr. after oviposition) than in the other two groups (0-3 hr. and 8-9 hr. after oviposition). This finding confirms a part of the results reported recently by Nelson et al. (1965). As they discussed, the greater content of LH at 5-6 hr. after oviposition may be a result from the neurogenic suppression of LH release from the pituitary, by certain stimuli cause by the presence of egg (irritant) in upper part of oviduct (Huston and Nalbandov, 1953; van Tienhoven, 1953). In this connection, Imai and Nakajo (1961) have reported that the content of total gonadotropins in the pituitary of the laying hen increases after placing a loop of surgical thread in the upper part of oviduct as irritant. The level of LH content in the pituitary of the nonlaying hen is almost the same level as the higher level of the laying hen 5-6 hr. after oviposition. This suggests that the content of LH in the pituitary of the nonlaying hen does not always differ from that in the pituitary of the laying
hen. Although it was demonstrated that LH content in the pituitary fluctuated in the laying hen (Nelson et al, 1965), whether LH content never fluctuates in the pituitary of the nonlaying hen has not been determined yet. REFERENCES Bliss, C. I., 19S2. The Statistics of Bioassay, Academic Press, N.Y. Evans, H. M., and M. E. Simpson, 1950. Physiology of gonadotropins. The Hormones. Vol. 2. Ed. Pincus, G. and K. V. Thimann, Academic Press, N.Y. pp. 351-404. Huston, T. M., and A. V. Nalbandov, 1953. Neurohumoral control of the pituitary in the fowl. Endocrinology, 52: 149-156. Imai, K., and S. Nakajo, 1961. Studies on gonadotropin of the anterior pituitary in domestic fowl. V. Diurnal change of the pituitary content in laying hen. Japanese J. Animal Reprod. 6: 100-102. Imai, K., K. Yamashita and S. Nakajo, 1964. Gonadotropin in the anterior pituitary and in blood plasma of molting hen. Proc. Annual Meetings of W.P.S.A.-Japan, November 1964, pp. 34-36. Nakajo, S., and K. Imai, 1957. Studies on gonadotropin of the anterior pituitary in domestic fowl. II. Gonadotropin content in hens at various
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
Laying hens (1) 0-3 hr. after oviposition (2) S-6 hr. after oviposition (3) 8-9 hr. after oviposition
No. of assay rats
No. of hens
PITUITARY LUTEINIZING HORMONE
potency of unfractioned extracts of poultry pituitaries. Poultry Sci. 2 1 : 161-172. Riley, G. M., and R. M. Fraps, 1942. Relationship of gonad-stimulating activity of female domestic fowl pituitaries to reproductive condition. Endocrinology, 30: 537-541. Rosemberg, E., P. Keller, W. B. Lewis, A. Albert, G. Carl and D. Bennett, 1965. Influence of follicle-stimulating hormone on the estimation of luteinizing hormone in the ventral prostate and ovarian ascorbic acid depletion assays. Endocrinology, 76: 1150-1157. van Tienhoven, A., 1953. Further study of the neurogenic blockage of LH release in the hen. Anat. Rec. 115: 374-375.
Characterization of Strains of Staphylococcus Aureus Isolated from Livers of Commercially Slaughtered Poultry C. GENIGEORGIS AND W. W. SADLER Department of Public Health, School of Veterinary Medicine, University of California, Davis, California (Received for publication February 1, 1966)
INTRODUCTION
S
TAPHYLOCOCCAL food poisoning is presently considered to be the most common type of food-borne disease in this country (Dauer, 1961; Lewis, 1964). The latest summary of food and water-borne disease outbreaks, compiled by the U.S.P.H. Service (1963) for the year 1961, lists staphylococcal food poisoning first, with 46 outbreaks and 1,503 cases, followed by salmonellosis, with 20 outbreaks and 750 cases. Dack (1962) reported that for the years 1956-1961, poultry meat was the third-most-frequent food (after meat and bakery products) implicated in foodpoisoning outbreaks. There is no report available showing to what degree poultry carcasses harbor potentially pathogenic staphylococci immediately after slaughter and before any contamination from the environment. Gunderson et al. (1954) studied the bacteriology of commercial poultry processing by taking surface sam-
pies during the different steps of processing, but did not report the types and numbers of microorganisms found in tissues or inside the carcass before evisceration. Walker and Ay res (1956), in a bacteriological study of organisms associated with commercially dressed poultry, obtained samples aseptically from the body cavities before evisceration, but did not report the incidence of staphylococci. This paper reports some of the characteristics of the staphylococci isolated from poultry livers in a previously reported survey (Sadler et al., 1965). MATERIALS AND METHODS
As previously reported (Sadler et al., 1965), a random sampling of commercially slaughtered poultry was conducted on 67 sampling days in 8 large processing plants to determine the quantitative and qualitative spectrum of pathogens of public health significance indigenous to poultry carcass-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
stages of reproduction. Japanese J. Animal Reprod. 3 : 49-51. Nelson, D. M., H. W. Norton and A. V. Nalbandov, 1965. Changes in hypophysial and plasma LH levels during the laying cycle of the hen. Endocrinology, 77: 889-896. Parlow, A. F., 1958. A rapid bioassay method for LH and factors stimulating LH secretion. Fed. Proc. 17: 402. Parlow, A. F., 1961. Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. Human Pituitary Gonadotropins. Ed. A. Albert, Charles C Thomas Pub., Springfield, 111., pp. 300-312. Phillips, R. E., 1942. Comparative gonadotropic
973
K. L. ARORA AND I. L. KOSIN
Weisbroth, H. S., 1960. The morphological response of gastrulae from three genetic lines of Broad Breasted Bronze turkeys to preincubation storage. M. S. Thesis, Washington State University, Pullman.
Wilson, W. O., U. K. Abbott and H. Abplanalp, 1961. Evaluation of Coturnix (Japanese quail) as pilot animal for poultry. Poultry Sci. 40: 6S1-6S7.
Luteinizing Hormone Content in the Pituitary of Laying and Nonlaying Hens 1
(Received for publication January 31, 1966)
I
T has been reported that the content of gonadotropic hormones in the anterior lobe of pituitary is greater in nonlaying hens than in laying hens when assayed for activity in causing vaginal cornification of rats (Phillips, 1942) or in increasing the weight of chick testes (Nakajo and Imai, 1957; Imai et al., 1964). Also, the gonadotropin content has been reported to be greater in hens having "quiescent" or "regressing" ovarian follicles than in hens having "mature" or "maturing" ovarian follicles when assayed on the mouse uterus (Riley and Fraps, 1942). Since these assays are not specific for either Follicle Stimulating Hormone (FSH) or Luteinizing Hormone (LH) (Evans and Simpson, 1950, a review), it is unknown whether the difference in the gonadotropin content between the laying and the nonlaying hens or between the hens in different ovarian conditions is due to a difference in the content of either FSH or LH, or in the content of both. In this paper, LH content in the anterior lobe of pituitary in laying and nonlaying hens was determined by the rat ovarian ascorbic acid depletion (OAAD) method, which was reported to be a highly specific method for assaying LH (Parlow, 1
Aided by Research Grant from the Ministry of Education of Japan and from Gotoh Hatchery Co., Gifu, Japan.
1958, 1961), although its absolute specificity was criticized recently (Rosemberg et al., 1965). MATERIALS AND METHODS Twenty-six laying and 13 nonlaying White Leghorn hens (18-22 months of age) were the donors of the pituitary glands. The laying hens, which had been laying in regular clutches of 2 to 4 eggs, were killed at three different times after oviposition of the initial or of the second egg of a clutch: (1) 0-3 hr., (2) 5-6 hr., and (3) 8-9 hr. The nonlaying hens, which had not laid for a period of more than 2 weeks and which were molting their wing feathers, were killed at 2:00-4:00 p.m. Their ovarian weight was less than 5.5 g. (mean: 4.0 g., for 13 hens). The anterior lobe of the pituitary was removed and weighed. Two to 6 pituitaries were pooled and homogenized together adding physiological saline solution during cooling in an ice-bath. The homogenates were refrigerated below 4°C. Within 20 hours after making the homogenates, they were assayed for OAAD activity on the immature rats of the Hotzman strain. The OAAD assay was carried out according to the method of Parlow (1961) with some modifications which follow. Like an ordinary design of assay of hormones,
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
KATUHIDE TANAKA, YASUHIRO FUJISAWA AND SHOZO YOSHIOKA Laboratory of Poultry Physiology, Gifu University, Kagamihara, Gifu, Japan
971
PITUITARY LUTEINIZING HORMONE
RESULTS AND DISCUSSION
Table 1 shows the results obtained by the assay of the pituitary from molting nonlaying hens and from laying hens at three different times after oviposition of the initial or of the second egg of a clutch. The weight of the anterior lobe of the pituitary did not differ significantly between groups (P > .05). The ovarian ascorbic acid (OAA) concentration of rats which received pituitaries of the nonlaying hen was signifi* Kindly donated by the Endocrinology Study Section, National Institute of Health.
12,0
„
100
4
X : 0.121
(10)
80 -
60
40 -
20
-J—v*^«S&line
1.0
ao
4.0
FIG. 1. Log dose response relationship for NIH-LH SS (ovine). Numbers in parentheses are the numbers of assay rats. The vertical lines indicate standard errors of mean. X: Index of precision.
cantly less than that of rats which received pituitaries of the laying hen 0-3 hr. or 8-9 hr. after oviposition (Groups 1 and 3) (P < .01), but not significantly different from that of rats which received pituitaries of the laying hen 5-6 hr. after oviposition (Group 2) (P > .05). For the convenience of comparison, LH content in the pituitary was calculated by fitting the OAA values to the regression equation shown in Figure 1. The results indicate that LH content in the pituitary of the nonlaying hen is roughly twice as much as that in the pituitary of the laying hen 0-3 hr. or 8-9 hr. after oviposition. Imai et al. (1964) found by a chick assay that the content of total gonadotropins in the pituitary of molting nonlaying hens was greater than that in the pituitary of laying hens near ovulation. Whether FSH content also differs as does LH content remains to be determined.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
control and test groups of rats were prepared. Injection was made into the saphena vein at 9:30-10:30 a.m. Three hours ± 5 minutes after the injection, both ovaries were removed, weighed and homogenized together during cooling in an ice-bath. Assays of the pituitary from laying hens and from nonlaying hens were run on the same litters of rats. The injected dose was 0.4 ml. pituitary homogenates containing 10 mg. fresh tissue equivalent. Control rats received an injection of the same volume of physiological saline solution. Before doing the assay of the pituitary, NIH-LH SS (ovine)2 was assayed to test the reliability of this OAAD method. The doses used were 1.0, 2.0, and 4.0 (Jig. per rat. The results were statistically analyzed by the method described by Bliss (1952). A linear relationship was observed between the log dose injected and the ovarian ascorbic acid concentration, as shown in Figure 1. The index of precision (X) of this assay was 0.121. This indicates that our modified OAAD method can be used for assaying LH with a satisfactory precision. No parallel assay with the LH standard was carried out simultaneously but LH amounts in the pituitary were obtained by comparing the assay results with the previously determined linear NIH-LH S5 curve.
972
K. TANAKA, Y. FUJISAWA AND S. YOSHIOKA TABLE 1.—Rat ovarian ascorbic acid depletion assay for LH of the anterior lobe of pituitary from molting nonlaying hens and from laying hens at three different times after oviposition of initial or second egg of a clutch Estimated
Source of pituitary
Nonlaying hens3
OAA cone. (Mg./100 mg. ovary)
Weight of ant. pituitary (mg.)
13
7.9±0.39*a**
7
65.7 + 2 . 5 5 V *
2.0
1.6
9
7.7 + 0.31 a
5
77.2 + 3.10 b
1.2
0.9
8
8.0 + 0.32 a
4
63.9 + 2 . 5 4 a
2.2
1.8
9
8.6 + 0 . 5 4 a
6
81.0+1.91 b
0.8
0.7
Control
10
99.2 + 1.00c
LH cone.1 LH content 2 Og./100 mg. Gug./pituitpituitary) ary)
1
Calculated by fitting the values of OAA cone, to the equation shown in Figure 1. Calculated by LH cone, times pituitary weight. Killed at 2:00-4:00 p.m. * Standard error of mean. ** Means followed by the same letter are not significantly different (
2
3
In the laying hen, the content of LH in the pituitary was greater in Group 2 (S-6 hr. after oviposition) than in the other two groups (0-3 hr. and 8-9 hr. after oviposition). This finding confirms a part of the results reported recently by Nelson et al. (1965). As they discussed, the greater content of LH at 5-6 hr. after oviposition may be a result from the neurogenic suppression of LH release from the pituitary, by certain stimuli cause by the presence of egg (irritant) in upper part of oviduct (Huston and Nalbandov, 1953; van Tienhoven, 1953). In this connection, Imai and Nakajo (1961) have reported that the content of total gonadotropins in the pituitary of the laying hen increases after placing a loop of surgical thread in the upper part of oviduct as irritant. The level of LH content in the pituitary of the nonlaying hen is almost the same level as the higher level of the laying hen 5-6 hr. after oviposition. This suggests that the content of LH in the pituitary of the nonlaying hen does not always differ from that in the pituitary of the laying
hen. Although it was demonstrated that LH content in the pituitary fluctuated in the laying hen (Nelson et al, 1965), whether LH content never fluctuates in the pituitary of the nonlaying hen has not been determined yet. REFERENCES Bliss, C. I., 19S2. The Statistics of Bioassay, Academic Press, N.Y. Evans, H. M., and M. E. Simpson, 1950. Physiology of gonadotropins. The Hormones. Vol. 2. Ed. Pincus, G. and K. V. Thimann, Academic Press, N.Y. pp. 351-404. Huston, T. M., and A. V. Nalbandov, 1953. Neurohumoral control of the pituitary in the fowl. Endocrinology, 52: 149-156. Imai, K., and S. Nakajo, 1961. Studies on gonadotropin of the anterior pituitary in domestic fowl. V. Diurnal change of the pituitary content in laying hen. Japanese J. Animal Reprod. 6: 100-102. Imai, K., K. Yamashita and S. Nakajo, 1964. Gonadotropin in the anterior pituitary and in blood plasma of molting hen. Proc. Annual Meetings of W.P.S.A.-Japan, November 1964, pp. 34-36. Nakajo, S., and K. Imai, 1957. Studies on gonadotropin of the anterior pituitary in domestic fowl. II. Gonadotropin content in hens at various
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
Laying hens (1) 0-3 hr. after oviposition (2) S-6 hr. after oviposition (3) 8-9 hr. after oviposition
No. of assay rats
No. of hens
PITUITARY LUTEINIZING HORMONE
potency of unfractioned extracts of poultry pituitaries. Poultry Sci. 2 1 : 161-172. Riley, G. M., and R. M. Fraps, 1942. Relationship of gonad-stimulating activity of female domestic fowl pituitaries to reproductive condition. Endocrinology, 30: 537-541. Rosemberg, E., P. Keller, W. B. Lewis, A. Albert, G. Carl and D. Bennett, 1965. Influence of follicle-stimulating hormone on the estimation of luteinizing hormone in the ventral prostate and ovarian ascorbic acid depletion assays. Endocrinology, 76: 1150-1157. van Tienhoven, A., 1953. Further study of the neurogenic blockage of LH release in the hen. Anat. Rec. 115: 374-375.
Characterization of Strains of Staphylococcus Aureus Isolated from Livers of Commercially Slaughtered Poultry C. GENIGEORGIS AND W. W. SADLER Department of Public Health, School of Veterinary Medicine, University of California, Davis, California (Received for publication February 1, 1966)
INTRODUCTION
S
TAPHYLOCOCCAL food poisoning is presently considered to be the most common type of food-borne disease in this country (Dauer, 1961; Lewis, 1964). The latest summary of food and water-borne disease outbreaks, compiled by the U.S.P.H. Service (1963) for the year 1961, lists staphylococcal food poisoning first, with 46 outbreaks and 1,503 cases, followed by salmonellosis, with 20 outbreaks and 750 cases. Dack (1962) reported that for the years 1956-1961, poultry meat was the third-most-frequent food (after meat and bakery products) implicated in foodpoisoning outbreaks. There is no report available showing to what degree poultry carcasses harbor potentially pathogenic staphylococci immediately after slaughter and before any contamination from the environment. Gunderson et al. (1954) studied the bacteriology of commercial poultry processing by taking surface sam-
pies during the different steps of processing, but did not report the types and numbers of microorganisms found in tissues or inside the carcass before evisceration. Walker and Ay res (1956), in a bacteriological study of organisms associated with commercially dressed poultry, obtained samples aseptically from the body cavities before evisceration, but did not report the incidence of staphylococci. This paper reports some of the characteristics of the staphylococci isolated from poultry livers in a previously reported survey (Sadler et al., 1965). MATERIALS AND METHODS
As previously reported (Sadler et al., 1965), a random sampling of commercially slaughtered poultry was conducted on 67 sampling days in 8 large processing plants to determine the quantitative and qualitative spectrum of pathogens of public health significance indigenous to poultry carcass-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 24, 2015
stages of reproduction. Japanese J. Animal Reprod. 3 : 49-51. Nelson, D. M., H. W. Norton and A. V. Nalbandov, 1965. Changes in hypophysial and plasma LH levels during the laying cycle of the hen. Endocrinology, 77: 889-896. Parlow, A. F., 1958. A rapid bioassay method for LH and factors stimulating LH secretion. Fed. Proc. 17: 402. Parlow, A. F., 1961. Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. Human Pituitary Gonadotropins. Ed. A. Albert, Charles C Thomas Pub., Springfield, 111., pp. 300-312. Phillips, R. E., 1942. Comparative gonadotropic
973