GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Hormonal California
32, 163-166 (1977)
Control of Delayed Development in the Leaf-Nosed Bat, Macrotus ca/ifornic&
III. Changes
in Plasma
Progesterone
during
Pregnancy
JOHN M. BURNS AND RONALD G.EASLEY Department
of
Biological Sciences, Texas Tech University, Lubbock, Texas 79409 Accepted January 28, 1977
Plasma samples were collected from 95 h4. californicus at 9 different intervals during the bat’s 9 month gestation period. Radioimmunoassay for progesterone concentration of these samples revealed 2 peaks; the first occurring in the first trimester of pregnancy (22 rig/ml), followed by a 3 month period in which progesterone concentrations were decreased to an average of 7 @ml. A second peak in hormone levels occurred in mid-May (32 &ml) followed by a sharp decline in progesterone in the early June, preparturition samples. The first progesterone peak corresponds to the time of placentation followed by reduced hormone concentration during the retarded embryonic growth phase. The second peak in progesterone concentrations coincides with the accelerated growth rate of the embryo and perhaps represents the additional hormonal stimulus needed for completion of fetal development.
The California leaf-nosed bat, Mucrotus et al. (1972) found that animals which were at a constant 27” and 12 hr (formerly known as M. maintained Davis and Baker, 1974) has photoperiod and supplied with more than been shown to exhibit a rather curious re- adequate food still required 9 months for productive pattern which Bradshaw (1962) the development of the young. This study termed “delayed development.” Copulaalso pointed out that although the pattern of tion occurs in September or early October, maternal thyroid hormone (thyroxine) sefollowed by immediate fertilization and im- cretion during pregnancy was different plantation (Bleier, 1975a). The embryo de- from that reported for animals with no revelops at a very slow rate. For example, 5 productive delay, exogenous thyroxine had months are required to attain the primitive no influence on the rate of fetal developstreak stage of development (Bradshaw, ment. 1962). The young (usually one weighing ap The present study was undertaken to quantify the plasma progesterone pattern proximately 7 g) are born the following June. A similar delay pattern has also been which occurs during pregnancy in this animal and compare this pattern with those of reported in the frugivorous bat, Artibeus other mammals which have no delay or difjumuicensis (Fleming, 1971). Information which might explain a hor- ferent types of delay mechanisms in an atmonal regulation of such a mammalian re- tempt to elucidate a possible control mechproductive scheme is indeed scarce. Burns anism for delayed development. californicus waterhousii,
* This project was supported by a grant from the Ti%?XMTeck~ Uaiverity hl&tw for ReWWsk and by NSF grant No. GB-40654.
MATERIALS
AND METHODS
T%e at&n& used ttkis study were coHecte&bom abandoned mine sites in southern Arizona. Shortly 163
Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0016-6480
164
BURNS
AND
EASLEY
Beckman LS-150 scintillation counter using 10 ml of scintillation cocktail (Beckman flouralloy TLA mixed in toluene and 10% biosolv, BBS-3 solution). All plasma samples were run within a 4 week period and standard curves were run with each batch of samples to allow for interassay variation. Statistical analysis was performed using a complete random design analysis of variance (F test). The data were further analyzed using program DMDO7V developed by the Department of Bio-medical Sciences of the University of California, Los Angeles using an IBM 3600 computer.
after capture, the bats were sacrificed by decapitation and the blood was collected in heparinized tubes. The blood samples were immediately placed in ice for transport to Texas Tech. The plasma was collected by centrifugation at 4” and stored at -20” until the time of progesterone assay. Plasma samples of 2-3 bats were pooled so that 1 ml of plasma could be used for assay. A total of 95 animals was used in the study. Plasma progesterone was extracted 3 times with fresh diethyl ether (Squibb anesthesic) and the extracts were pooled and evaporated to dryness with nitrogen. Approximately 800 cpm of progesterone (Progesterone [I .2-3H(N)], New England Nuclear) were added to each sample so that percentage recovery could be calculated. A progesterone fraction was isolated by Celite microcolumns and the concentration of hormone was determined by the radioimmunoassay of Abraham et al. (1971) using progesterone antibody (S-49) purchased from Harbor General Hospital, Torrence, Calif. The specificity of the antibody for steroids in this fraction was essentially that reported by Abraham et al. (1971). A I: 1000 dilution of the antibody in 0.1 M phosphate buffer (pH 7), bound 72.8% of labeled progesterone after 4 hr of incubation at 4”. The within and between assay variance was evaluated by calculation of the coefficient of variation (CV) for duplicate samples using the formulas described by Abraham et a/. (1971). In 20 duplication determinations from two separate assays the CV was 10%. Bound and unbound progesterone were separated using dextran-charcoal mixture and centrifuging at 2000 rpm at 4”. Percentage bound progesterone was determined by counting the radioactivity in a
RESULTS
The results of plasma progesterone analysis from bats in various stages of pregnancy are presented in Fig. 1. The August sample represents nonpregnant animals and the progesterone values appear as “zero” since no hormone could be detected by the assay. A peak in progesterone was recorded in the December sample, followed by much lower values for the next three months. Plasma progesterone values began to show significant increases (P < 0.05) in the April sample and continued to increase until the early May sample. In fact, the progesterone concentration for the May sample is a 500% increase over the hormone values recorded during the retarded
13 t
9
t 6 14
t
I
I. AUP
t
9
t
1 OCt
No*
08s
JOtl DATE
Fob Of
Mar
APr
Mov
JtJn
SAMPLE
FIG. 1. Progesterone concentrations from bats at various intervals in the 9 month gestation period. Values represent means IT 2 standard errors. Numerals indicate the numbers of samples run for each group.
PREGNANCY
embryonic growth phase of JanuaryMarch. The sample taken in early June represents blood from animals within a few days prior to parturition; in fact, about one-half of the animals collected at this time had delivered their young and were lactating. No progesterone values of these nonpregnant animals are presented.
IN
BAT
165
crease would be sheer speculation on our part but probably supports the belief that the hormone is needed for implantation (Mead, 1975). The lower hormone values recorded for the January-March period coincide with the retarded growth rate of the embryo, while the second peak in progesterone concentration corresponds to the period in which the embryo and placenta develop at DISCUSSION an accelerated rate. Once again, the elecThe pattern of progesterone concen- tron micrographs of Bleier (1975b) correlate nicely with the reported changes in progestrations reported here shows a difference from animals which undergo no reproducterone values. Bleier found that the corpus tive delay mechanism. In the rabbit, for luteum appeared to be more metabolically example, progesterone values increase active in November and December, inacsteadily until the midpoint of pregnancy tive during January through March, and and then decline to 5 rig/ml on the day of quite active from late March until the terparturition (Challis et al., 1973). In animals mination of gestation. which are known to exhibit delayed implanProgesterone values have not been retation, blood progesterone values stay ported for other chiroptera during pregslightly above nonpregnant concentrations nancy, however, our values are within the until shortly before the time of implantarange of those presented for other mamtion, at which time they began to increase, mals. Edqvist et al. (1975) reported progesfollowed by a decline during the latter half terone values in pregnant dogs which of the postimplantation period (Mead and ranged from 10 rig/ml at the onset of pregnancy to 50 rig/ml during the third week of Eik-Nes, 1969). The progesterone pattern in the mink in- gestation. Robertson and King (1974) recreases from the time of copulation to a ported much lower values in the pregnant peak of approximately 105 rig/ml at 20 days pig ranging from 12 to 5 rig/ml. Finally, prior to parturition followed by a gradual Hartmann et al. (1973) found peak progesdecline as the pregnancy is terminated terone values in pregnant ewes of 28 rig/ml. (Moller, 1973). In the stoat, a biphasic The progesterone pattern which we report here is very similar to an earlier study progesterone pattern has been reported (Gulamhusein and Thawley, 1974). How- showing a biphasic plasma estrogen pattern in M. californicus during pregnancy (Bums ever, the first peak in hormone concentration corresponds to the time of copulation and Wallace, 1975). The estrogen and progesterone values show a spurt around the and decreases quite rapidly and remains perhaps to facilitate less than 5 rig/ml during the period of de- time of implantation the implantation process and the initiation layed implantation. of the chorioallantoic The pattern for M. californicus is also of the development however, is not biphasic and differs from the mustelids in placenta. Placentation, that the first phase starts to increase just complete until late March or early April prior to copulation and then continues to (Bodley, 1974) during which time both estrogen and progesterone values again show increase for a 3 month period at which time an increase. implantation is complete and placentation The possible endogenous source of prois well underway (Bodley, 1974, and Bleier, 1975b). The cause of this progesterone in- gesterone can only be speculated at this
166
BURNS
AND
time. However, the ovary seems to be the major site of synthesis. In separate experiments (unpublished data), we found that ovariectomy at any stage of gestation resulted in abortion of the fetus. Sham operations, on the other hand, resulted in abortions in less than 10% of the animals. We feel that the progesterone secretion reported here supports our earlier work, as well as that of other investigators (see references) working with the reproductive physiology of M. californicus and that this species exemplifies a reproductive pattern which is perhaps quite different from delayed implantation. ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. John Capeheart, Mr. Mike Owen and William Wallace for their help in collecting animals used in this study. Thanks are also extended to Dr. Francis Rose for his assistance in the preparation of the manuscript.
EASLEY nwterhousii. II. Radioimmunoassay of plasma estrone and estradiol 17-p during pregnancy. GetI. Comp.
Abraham, G. E., Swerdloff, R. S., Tulchinsky, D.. Hopper, K., and Odell, W. 0. (1971). Radioimmunoassay of plasma progesterone. J. C/in. Endocrinol. 32, 619-624. Bleier, W. J. (1975a). Early embryology and implantation in the California leaf-nosed bat, Mucrotus californicus.
Anat.
Rec.
182, 237-254.
Bleier, W. J. (1975b). Fine structure of implantation and corpus luteum in the California leaf-nosed bat, Mucrotus californicus. PhD dissertation, Texas Tech. Bodley, H. D. (1974). The development of the chorioallantoic placental barrier in the bat Macrotus
waterhousii.
Anat.
Rec.
178, 313-325.
Bradshaw, G. V. R. (1962). Reproductive cycle of the California leaf-nosed bat, Macrotus californicus. Science
136, 645.
Burns, J. M.. and Wallace, W. E. (1975). Hormonal control of delayed development in Macrotus
25, 529-533.
Camp.
Endocrinol.
18, 54-58.
Challis, J. R. CL.. Davies, I. J.. and Ryan, K. (1973). The concentrations of progesterone, estrone and estradiol 17-p in the plasma of pregnant rabbits. Endocrinolog>~ 93, 971-976. Davis, B. L.. and Baker, R. J. (1974). Morphometerics, evolution, and cytotaxonomy of mainland bats of the genus Macrotus (Chiroptera: Phyllostomatidae). System. Zoo. 23, 239-269. Edqvist, L. E., Johansson, E. D. B., Kasstrom, H., Olsson, S. E., and Richkind, M. (1975). Blood plasma levels of progesterone and oestradiol in the dog during the oestrus cycle and pregnancy. Acta
Endocrinol.
78, 554-564.
Fleming, T. H. (1971). Artibeus jamaicensis: delayed embryonic development in a neotropical bat. Science
171, 402-404.
Gulamhusein, A. P., and Thawley, A. R. (1974). Plasma progesterone levels in the stoat. J. Reprod.
REFERENCES
Endocrinol.
Burns. J. M.. Baker. R. J., and Bleier. W. J. (1972). Hormonal control of delayed development in Macrotus Mwterhousii. I. Changes in plasma thyroxine during pregnancy and lactation. Gen.
Fert.
36, 405-408.
Hartmann. P. E., Trevethan. P., and Shelton, J. N. (1973). Progesterone and oestrogen and the initiation of lactation in ewes. J. Endocrine/. 59, 249-259. Mead, R. A., and Eik-Nes. K. B. (1969). Seasonal variation in plasma levels of progesterone in western forms of the spotted skunk. J. Reprod. Fert.
Suppl.
6, 397-403.
Mead, R. A. (1975). Effects of hypophysectomy on blastocyst survival, progesterone secretion and nidation in the spotted skunk. Biol. Reprod. 12, 526-533. Moller. 0. D. (1973). The progesterone concentrations in the peripheral plasma of the mink (Mustella vison) during pregnancy. .I. Endocrinol. 56, 121-132. Robertson. H. A.. and King, G. J. (1974). Plasma concentrations of progesterone, oestrone, oestradiol 17-p and of oestrone sulphate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fert. 40, 133-141.
AND
COMPARATIVE
ENDOCRINOLOGY
Hormonal California
32, 163-166 (1977)
Control of Delayed Development in the Leaf-Nosed Bat, Macrotus ca/ifornic&
III. Changes
in Plasma
Progesterone
during
Pregnancy
JOHN M. BURNS AND RONALD G.EASLEY Department
of
Biological Sciences, Texas Tech University, Lubbock, Texas 79409 Accepted January 28, 1977
Plasma samples were collected from 95 h4. californicus at 9 different intervals during the bat’s 9 month gestation period. Radioimmunoassay for progesterone concentration of these samples revealed 2 peaks; the first occurring in the first trimester of pregnancy (22 rig/ml), followed by a 3 month period in which progesterone concentrations were decreased to an average of 7 @ml. A second peak in hormone levels occurred in mid-May (32 &ml) followed by a sharp decline in progesterone in the early June, preparturition samples. The first progesterone peak corresponds to the time of placentation followed by reduced hormone concentration during the retarded embryonic growth phase. The second peak in progesterone concentrations coincides with the accelerated growth rate of the embryo and perhaps represents the additional hormonal stimulus needed for completion of fetal development.
The California leaf-nosed bat, Mucrotus et al. (1972) found that animals which were at a constant 27” and 12 hr (formerly known as M. maintained Davis and Baker, 1974) has photoperiod and supplied with more than been shown to exhibit a rather curious re- adequate food still required 9 months for productive pattern which Bradshaw (1962) the development of the young. This study termed “delayed development.” Copulaalso pointed out that although the pattern of tion occurs in September or early October, maternal thyroid hormone (thyroxine) sefollowed by immediate fertilization and im- cretion during pregnancy was different plantation (Bleier, 1975a). The embryo de- from that reported for animals with no revelops at a very slow rate. For example, 5 productive delay, exogenous thyroxine had months are required to attain the primitive no influence on the rate of fetal developstreak stage of development (Bradshaw, ment. 1962). The young (usually one weighing ap The present study was undertaken to quantify the plasma progesterone pattern proximately 7 g) are born the following June. A similar delay pattern has also been which occurs during pregnancy in this animal and compare this pattern with those of reported in the frugivorous bat, Artibeus other mammals which have no delay or difjumuicensis (Fleming, 1971). Information which might explain a hor- ferent types of delay mechanisms in an atmonal regulation of such a mammalian re- tempt to elucidate a possible control mechproductive scheme is indeed scarce. Burns anism for delayed development. californicus waterhousii,
* This project was supported by a grant from the Ti%?XMTeck~ Uaiverity hl&tw for ReWWsk and by NSF grant No. GB-40654.
MATERIALS
AND METHODS
T%e at&n& used ttkis study were coHecte&bom abandoned mine sites in southern Arizona. Shortly 163
Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0016-6480
164
BURNS
AND
EASLEY
Beckman LS-150 scintillation counter using 10 ml of scintillation cocktail (Beckman flouralloy TLA mixed in toluene and 10% biosolv, BBS-3 solution). All plasma samples were run within a 4 week period and standard curves were run with each batch of samples to allow for interassay variation. Statistical analysis was performed using a complete random design analysis of variance (F test). The data were further analyzed using program DMDO7V developed by the Department of Bio-medical Sciences of the University of California, Los Angeles using an IBM 3600 computer.
after capture, the bats were sacrificed by decapitation and the blood was collected in heparinized tubes. The blood samples were immediately placed in ice for transport to Texas Tech. The plasma was collected by centrifugation at 4” and stored at -20” until the time of progesterone assay. Plasma samples of 2-3 bats were pooled so that 1 ml of plasma could be used for assay. A total of 95 animals was used in the study. Plasma progesterone was extracted 3 times with fresh diethyl ether (Squibb anesthesic) and the extracts were pooled and evaporated to dryness with nitrogen. Approximately 800 cpm of progesterone (Progesterone [I .2-3H(N)], New England Nuclear) were added to each sample so that percentage recovery could be calculated. A progesterone fraction was isolated by Celite microcolumns and the concentration of hormone was determined by the radioimmunoassay of Abraham et al. (1971) using progesterone antibody (S-49) purchased from Harbor General Hospital, Torrence, Calif. The specificity of the antibody for steroids in this fraction was essentially that reported by Abraham et al. (1971). A I: 1000 dilution of the antibody in 0.1 M phosphate buffer (pH 7), bound 72.8% of labeled progesterone after 4 hr of incubation at 4”. The within and between assay variance was evaluated by calculation of the coefficient of variation (CV) for duplicate samples using the formulas described by Abraham et a/. (1971). In 20 duplication determinations from two separate assays the CV was 10%. Bound and unbound progesterone were separated using dextran-charcoal mixture and centrifuging at 2000 rpm at 4”. Percentage bound progesterone was determined by counting the radioactivity in a
RESULTS
The results of plasma progesterone analysis from bats in various stages of pregnancy are presented in Fig. 1. The August sample represents nonpregnant animals and the progesterone values appear as “zero” since no hormone could be detected by the assay. A peak in progesterone was recorded in the December sample, followed by much lower values for the next three months. Plasma progesterone values began to show significant increases (P < 0.05) in the April sample and continued to increase until the early May sample. In fact, the progesterone concentration for the May sample is a 500% increase over the hormone values recorded during the retarded
13 t
9
t 6 14
t
I
I. AUP
t
9
t
1 OCt
No*
08s
JOtl DATE
Fob Of
Mar
APr
Mov
JtJn
SAMPLE
FIG. 1. Progesterone concentrations from bats at various intervals in the 9 month gestation period. Values represent means IT 2 standard errors. Numerals indicate the numbers of samples run for each group.
PREGNANCY
embryonic growth phase of JanuaryMarch. The sample taken in early June represents blood from animals within a few days prior to parturition; in fact, about one-half of the animals collected at this time had delivered their young and were lactating. No progesterone values of these nonpregnant animals are presented.
IN
BAT
165
crease would be sheer speculation on our part but probably supports the belief that the hormone is needed for implantation (Mead, 1975). The lower hormone values recorded for the January-March period coincide with the retarded growth rate of the embryo, while the second peak in progesterone concentration corresponds to the period in which the embryo and placenta develop at DISCUSSION an accelerated rate. Once again, the elecThe pattern of progesterone concen- tron micrographs of Bleier (1975b) correlate nicely with the reported changes in progestrations reported here shows a difference from animals which undergo no reproducterone values. Bleier found that the corpus tive delay mechanism. In the rabbit, for luteum appeared to be more metabolically example, progesterone values increase active in November and December, inacsteadily until the midpoint of pregnancy tive during January through March, and and then decline to 5 rig/ml on the day of quite active from late March until the terparturition (Challis et al., 1973). In animals mination of gestation. which are known to exhibit delayed implanProgesterone values have not been retation, blood progesterone values stay ported for other chiroptera during pregslightly above nonpregnant concentrations nancy, however, our values are within the until shortly before the time of implantarange of those presented for other mamtion, at which time they began to increase, mals. Edqvist et al. (1975) reported progesfollowed by a decline during the latter half terone values in pregnant dogs which of the postimplantation period (Mead and ranged from 10 rig/ml at the onset of pregnancy to 50 rig/ml during the third week of Eik-Nes, 1969). The progesterone pattern in the mink in- gestation. Robertson and King (1974) recreases from the time of copulation to a ported much lower values in the pregnant peak of approximately 105 rig/ml at 20 days pig ranging from 12 to 5 rig/ml. Finally, prior to parturition followed by a gradual Hartmann et al. (1973) found peak progesdecline as the pregnancy is terminated terone values in pregnant ewes of 28 rig/ml. (Moller, 1973). In the stoat, a biphasic The progesterone pattern which we report here is very similar to an earlier study progesterone pattern has been reported (Gulamhusein and Thawley, 1974). How- showing a biphasic plasma estrogen pattern in M. californicus during pregnancy (Bums ever, the first peak in hormone concentration corresponds to the time of copulation and Wallace, 1975). The estrogen and progesterone values show a spurt around the and decreases quite rapidly and remains perhaps to facilitate less than 5 rig/ml during the period of de- time of implantation the implantation process and the initiation layed implantation. of the chorioallantoic The pattern for M. californicus is also of the development however, is not biphasic and differs from the mustelids in placenta. Placentation, that the first phase starts to increase just complete until late March or early April prior to copulation and then continues to (Bodley, 1974) during which time both estrogen and progesterone values again show increase for a 3 month period at which time an increase. implantation is complete and placentation The possible endogenous source of prois well underway (Bodley, 1974, and Bleier, 1975b). The cause of this progesterone in- gesterone can only be speculated at this
166
BURNS
AND
time. However, the ovary seems to be the major site of synthesis. In separate experiments (unpublished data), we found that ovariectomy at any stage of gestation resulted in abortion of the fetus. Sham operations, on the other hand, resulted in abortions in less than 10% of the animals. We feel that the progesterone secretion reported here supports our earlier work, as well as that of other investigators (see references) working with the reproductive physiology of M. californicus and that this species exemplifies a reproductive pattern which is perhaps quite different from delayed implantation. ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. John Capeheart, Mr. Mike Owen and William Wallace for their help in collecting animals used in this study. Thanks are also extended to Dr. Francis Rose for his assistance in the preparation of the manuscript.
EASLEY nwterhousii. II. Radioimmunoassay of plasma estrone and estradiol 17-p during pregnancy. GetI. Comp.
Abraham, G. E., Swerdloff, R. S., Tulchinsky, D.. Hopper, K., and Odell, W. 0. (1971). Radioimmunoassay of plasma progesterone. J. C/in. Endocrinol. 32, 619-624. Bleier, W. J. (1975a). Early embryology and implantation in the California leaf-nosed bat, Mucrotus californicus.
Anat.
Rec.
182, 237-254.
Bleier, W. J. (1975b). Fine structure of implantation and corpus luteum in the California leaf-nosed bat, Mucrotus californicus. PhD dissertation, Texas Tech. Bodley, H. D. (1974). The development of the chorioallantoic placental barrier in the bat Macrotus
waterhousii.
Anat.
Rec.
178, 313-325.
Bradshaw, G. V. R. (1962). Reproductive cycle of the California leaf-nosed bat, Macrotus californicus. Science
136, 645.
Burns, J. M.. and Wallace, W. E. (1975). Hormonal control of delayed development in Macrotus
25, 529-533.
Camp.
Endocrinol.
18, 54-58.
Challis, J. R. CL.. Davies, I. J.. and Ryan, K. (1973). The concentrations of progesterone, estrone and estradiol 17-p in the plasma of pregnant rabbits. Endocrinolog>~ 93, 971-976. Davis, B. L.. and Baker, R. J. (1974). Morphometerics, evolution, and cytotaxonomy of mainland bats of the genus Macrotus (Chiroptera: Phyllostomatidae). System. Zoo. 23, 239-269. Edqvist, L. E., Johansson, E. D. B., Kasstrom, H., Olsson, S. E., and Richkind, M. (1975). Blood plasma levels of progesterone and oestradiol in the dog during the oestrus cycle and pregnancy. Acta
Endocrinol.
78, 554-564.
Fleming, T. H. (1971). Artibeus jamaicensis: delayed embryonic development in a neotropical bat. Science
171, 402-404.
Gulamhusein, A. P., and Thawley, A. R. (1974). Plasma progesterone levels in the stoat. J. Reprod.
REFERENCES
Endocrinol.
Burns. J. M.. Baker. R. J., and Bleier. W. J. (1972). Hormonal control of delayed development in Macrotus Mwterhousii. I. Changes in plasma thyroxine during pregnancy and lactation. Gen.
Fert.
36, 405-408.
Hartmann. P. E., Trevethan. P., and Shelton, J. N. (1973). Progesterone and oestrogen and the initiation of lactation in ewes. J. Endocrine/. 59, 249-259. Mead, R. A., and Eik-Nes. K. B. (1969). Seasonal variation in plasma levels of progesterone in western forms of the spotted skunk. J. Reprod. Fert.
Suppl.
6, 397-403.
Mead, R. A. (1975). Effects of hypophysectomy on blastocyst survival, progesterone secretion and nidation in the spotted skunk. Biol. Reprod. 12, 526-533. Moller. 0. D. (1973). The progesterone concentrations in the peripheral plasma of the mink (Mustella vison) during pregnancy. .I. Endocrinol. 56, 121-132. Robertson. H. A.. and King, G. J. (1974). Plasma concentrations of progesterone, oestrone, oestradiol 17-p and of oestrone sulphate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fert. 40, 133-141.
