Departments of Clinical Biochemistry and Clinical Radiology, Royal Veterinary College, Stockholm, and Department of Obstetrics and Gynaecology, University of Uppsala, Sweden, the Laboratory for Comparative Orthopaedics, the Hospital for Special Surgery, New York, N. Y. The
the
BLOOD PLASMA LEVELS OF PROGESTERONE AND OESTRADIOL IN THE DOG THE DURING OESTROUS CYCLE AND PREGNANCY
By L.-E.
Edqvist,
E. D. B.
Johansson,
H. Kasstr\l=o"\m,
S.-E. Olsson and M. Richkind ABSTRACT Plasma oestradiol and progesterone levels were studied in German Shepherd and Greyhound bitches during the normal oestrous cycle, pregnancy and at parturition. The mean oestradiol level increased from approximately 7 to 13 pg/ml during the fifth week before onset of oestrus. Oestradiol stayed at this level until pro oestrus which was characterized by a steady increase to a mean peak level of about 30 pg/ml 5\p=n-\6days before oestrus. At the start of oestrus the level was approximately 12 pg/ml. A level between 10\p=n-\15pg/ml was kept during the following 10 weeks whether or not the bitch was pregnant and no change occurred at parturition. The mean progesterone level was found to be very low, around or under 1 ng/ml, until 1\p=n-\4days after the oestradiol peak after which the level gradually increased to around 10 ng/ml at the start of oestrus. During oestrus and metoestrus or pregnancy the pattern was inconsistent. In some bitches the level gradually increased during 3\p=n-\4weeks and reached a peak value of 30\p=n-\50ng/ml. The level then gradually decreased during 5\p=n-\6 weeks. During the first 5 weeks of the same period progesterone in other bitches fluctuated between similar maximum levels and extremely low levels. After the fifth week the pattern was the same for all bitches. In the pregnant bitches there was a significant drop of the progesterone level at parturition. The data suggest that the dog is not an ideal test animal for steroids synthetized for use in man.
and
dog has been used as an experimental animal in metabolic studies and toxicity testings of synthetic steroid hormones made for use as oral contra¬ ceptives in women during recent years; data on the pattern of sex steroid hormones during the normal oestrous cycle and pregnancy have become avail¬ able (Christie et al. 1971; Bell et al. 1971; Jones et al. I913a,b,c; Smith 8c McDonald 1974). The dog has apparently a sex steroid pattern which differs from that of most other species of laboratory or domestic animals and man. The aim of the present study was to determine peripheral plasma levels of both progesterone1) and oestrogens and their sequential changes during the normal canine oestrous cycle, pregnancy and at parturition in the same in¬ The
dividuals. MATERIAL AND METHODS
Pure-bred female German Shepherds and racing Greyhounds were used (Table 1). The German Shepherds were between 2 and 5 years of age and the Greyhounds between 4 and 6 years of age. All animals were kept in pens and runs and were given a commercial diet and water ad libitum. They were regularly dewormed and inoculated against distemper and hepatitis. Pregnant bitches were given an additional adequate supply of calories, minerals and vitamins during the last trimester of preg¬ nancy.
The females were checked daily for presence of blood stained mucus in the vulva, which was used as the criterion for beginning of pro-oestrus. Blood samples were collected from dogs in the morning before feeding once or twice weekly during the anoestrous period. Starting with the first day of pro-oestrus, blood samples were taken daily or every second day until the female accepted a male. Sampling was then resumed twice a week for 8 weeks. During the ninth week after acceptance, daily blood samples were taken until parturition or in the case of unmated females, for 7 to 10 days, whereafter samples twice a week were taken for another 2 to 4 weeks. Five to eight ml of blood were collected from the cephalic vein in heparinized tubes. After centrifugation the plasma was removed and stored at below -20°C until analyzed. Analyzing of oestradiol and progesterone was done in the following manner; plasma levels of oestradiol in all dogs, plasma levels of progesterone in the Greyhounds (except dog No. 10, which was analyzed only during the peripartal period). Plasma levels of progesterone were measured by radioimmunoassay (Thorneycroft Se Stone 1972). Plasma levels of oestrogens were measured by radioimmunoassay using the same procedure and reagents as described by Edqvist 8- Johansson (1972). Antiserum to an oestradiol-17/? 17-succinyl-bovine serum albumin was a gift from National Institute of Health, Bethesda, USA, and was used in a dilution of 1:50 000 in a phos-
following abbreviations and trivial names are Oestradiol-17^: l,3,5(10)-oestratriene-3,17/?-diol.
') The
Oestrone: 3-hydroxy-l,3,5(10)-oestratrien-17-one. Progesterone: 4-pregnene-3,20-dione. standard error of the standard deviation.
sem: s:
mean.
used:
Table 1. Clinical data on the
Dog No.
Breed
pregnancy
German
58
Shepherd 2
German
58
Shepherd 3
German
Shepherd 4
German
German
6
German
not
7 S 9
Shepherd Greyhound Greyhound Greyhound
10
Greyhound
(days)
12 12
no
distinct
peak 3 males 1 male 5 females
mated not
Greyhound
males females males females
pregnant 62
Shepherd
11
4 3 3 2
(days)
mated not
Shepherd 5
Live
Time inter¬ val between
Time interval between pro-oestrus oestradiol peak and 1st day and 1st day Stillborn of acceptance of acceptance
Litter at birth
Duration of
(days) 1
dogs.
10
2
13
4
14
7
14 14 17
3 5 2
pregnant mated
pregnant
not mated not mated
65 60
4 3 5 2 5 1
males 2 males females
16
males
females males female
1 male 1 female
11
phate buffer, pH 7.4, containing 0.1 "lo gelatin. The degree of cross-reaction was identical to other antisera prepared against oestradiol-17/5? conjugated at position 17 (Boilert ci al. 1973; Lindberg et al. 1974). The reliability of the oestradiol radio¬ immunoassay was tested by two methods from pooled dog plasma. The rapid radio¬ immunoassay procedure and the radioimmunoassay after separation on Sephadex LH-20 columns were compared. A high degree of correlation between the two methods was found whether or not purification of the plasma extract was done. The sensitivity
of the assay system used was 2 pg as read off the standard curve. The coefficient of variation in the range 2-5 pg/ml was 16.3 per cent calculated from 30 duplicate estimations. One ml plasma was extracted with 5 ml diethyl ether by vigorous shaking for 1 min. The plasma blank was negligible and the diethyl ether blank was corrected for by adding 5 ml of diethyl ether to each point of the standard curve. The standard samples as well as the unknown were simultaneously evaporated to dryness at 37°C. All values obtained during the present study were uncorrected for packed cell volume and well above the limit of detection of the assay system.
RESULTS
The time interval between start of pro-oestrus and first day of acceptance (= onset of oestrus) is given in Table 1. Pro-oestrus was considered to start at the first appearance of blood stained mucus in the vulva. Pro-oestrus started 12.8 (s 2.6) days before onset of oestrus. All pregnant dogs delivered normal sized litters without obstetrical assistance and after a gestation of normal length (Table 1). The five stillborn puppies were all of full term size and were not macerated. The mortality rate in this study was the same as normally found in the kennel. The mean peripheral plasma level of oestradiol in 11 bitches during late anoestrus, pro-oestrus and until the first day of acceptance is given in Fig. 1. The mean oestradiol level increased from 7 to 13 pg/ml from the fifth to the fourth week before first day of acceptance (Fig. 1). Thereafter the level remained fairly constant to about the start of pro-oestrus. During the pro-oestrous period the mean oestradiol level increased continu¬ ously and the highest mean values of approximately 30 pg/ml were seen on days 6 and 5 before start of oestrus (Fig. 1). During this period column separa¬ tion on Sephadex LH-20 revealed an oestradiol/oestrone ratio of about 2.5. The time interval between the oestradiol peak and oestrus in each individual dog is given in Table 1. Thereafter the mean level decreased to the initial pro-oestrous levels. On the first day of acceptance (= day 0) a mean value of 12 pg/ml was found. The time interval between the oestradiol peak and first day of acceptance was found to vary between 2 and 7 days (Table 1). The individual peripheral plasma levels of progesterone in four Greyhounds (Nos. 7, 8, 9 and 11) are also given in Fig. 1. No marked change of the levels was seen prior to 5 days before oestrus. The levels obtained up to this point were mostly below 1 ng/ml. During late pro-oestrus the progesterone levels in the four bitches increased gradually. In three of the Greyhounds this in¬ crease was found to start on the day after the oestradiol peak in each bitch, while one bitch (No. 11) showed no change of the progesterone level until 4 days after the oestradiol peak. The mean peripheral plasma levels of oestradiol in pregnant as well as in non-pregnant bitches during the 8 weeks after onset of oestrus are shown in Fig. 2. It was found that the mean levels were about the same in both preg¬ nant and non-pregnant bitches. The mean peripheral plasma level of oestradiol was between 10 and 15 pg/ml in both these two groups. During the luteal column phase separation on Sephadex LH-20 revealed an oestradiol/oestrone ratio of about 1. Fig. 3 shows the individual peripheral plasma levels of oestra¬ diol before and after the delivery in the six pregnant bitches. Also in Fig. 3 the oestradiol values for non-pregnant bitches are shown during the time inter¬ val 57-70 days after oestrus. All the pregnant bitches gave birth during this =
Fig. 1. Peripheral plasma levels of oestradiol (X ± SEM)for all bitches and individual peri¬ pheral plasma levels of progesterone in four Greyhound bitches for the five weeks proceeding oestrus. Arrows indicate individual oestradiol peak value.
period (Table 1). One non-pregnant dog (No. 6) showed signs of approaching oestrus during this time interval and is therefore excluded from the material drawn in Fig. 3. The peripheral plasma levels of oestradiol were found to be similar in both pregnant and non-pregnant bitches and no significant change occurred when comparing the mean oestradiol levels before and after the delivery (Students i-test). No statistically significant difference was found when comparing the mean oestradiol measurements of individual pregnant and non-pregnant Greyhounds and German Shepherds from oestrus and 60 days onward. Neither was there any statistically significant difference found when all oestradiol measurements were compared between breeds (Student's
Fig.
2.
levels of oestradiol (X + sem) for pregnant bitches (broken lines) and non-pregnant bitches (shadowed area) during the 8 weeks following onset of oestrus. Individual peripheral plasma levels of progesterone in two pregnant and two non-pregnant Greyhound bitches for 11 weeks following oestrus.
Peripheral plasma
30
a.
w
Parturition
20
10-
O
-6
Days
-A -2 0 +2 +4 +6 before and after parturition
Fig.
58
Days
62 64 66 68 70 after onset of oestrus
60
3.
Peripheral plasma levels of oestradiol in six pregnant bitches between one week before and after parturition. For comparative reasons the oestradiol levels in four non-pregnant bitches are also given during the 9th and 10th week after onset of oestrus, which was the time of parturition in the pregnant bitches. The mean value for Greyhounds was 12.9 (sem 0.53) pg/ml and for German Shepherds 11.6 (sem 0.60) pg/ml. The peripheral plasma levels of progesterone in two pregnant (Nos. 9 and 11) and two non-pregnant bitches (Nos. 7 and 8) are given in Fig. 2. Two
/-test).
=
=
bitches, the
pregnant (No. 11) and one non-pregnant (No. 8) showed about pattern with continuously increasing values of progesterone from to about 3 to 4 weeks thereafter, at which time maximum values one
same
oestrus
of approximately 30 and 50 ng/ml were seen. The levels then gradually decreased and the non-pregnant bitch (No. 8) showed values below 0.5 ng/ml from the latter part of the 9th week after oestrus and onwards. The pregnant bitch No. 11 showed progesterone values of 2.5-5 ng/ml during the 9th week after oestrus. After the delivery on day 65 the level diminished rapidly to values below 0.5 ng/ml. The other two bitches, one pregnant (No. 9) and one non-pregnant (No. 7), showed a very inconsistent pattern of pro¬ gesterone levels with low concentrations 2 and 3 days after oestrus followed by an increase and thereafter low levels again during the 4th week (No. 7) and during the 5th week (No. 9). After the 5th week the pattern for these two dogs followed the same pattern as that of dogs Nos. 9 and 11. In the pregnant bitch No. 9 the progesterone levels fell rapidly after parturition on day 65, while the non-pregnant bitch No. 7 showed relatively higher values during the 10th and 11th week after oestrus compared with the other two dogs
(Fig. 2). In Fig. 4
given the peripheral plasma levels of oestradiol and proges¬ and after parturition in three bitches (Nos. 9, 10 and 11). The terone before progesterone level dropped at parturition and after the delivery very low levels were encountered. The peripheral plasma levels of oestradiol showed no significant change during this period. are
4-4
Dog
nr
.,
..
9 10 Parturition
Parturition
Parturition
6i cu ¬
£ U 30
ÌL 2
-o cû
20 3 10 o
-5 -3
-1 A
Days
3
-3
-1.1
before and after
parturition
Fig. 4. Peripheral plasma levels of oestradiol (solid line) and progesterone (broken line) in three individual Greyhound bitches during the immediate pre- and post-partum period.
DISCUSSION
The
length of the pro-oestrous period found in the present investigation was 12.8 (s 2.6; range 8-17) days. This interval is considerably longer than the interval of 4.6 (range 2-8) days reported by Christie et al. (1971). However, these investigators considered oestrus to start before the bitch fully accepted the male (Christie 8c Bell 1971). Smith 8c McDonald (1974), who calculated the length of the pro-oestrous period using the same criterion as in the present investigation found pro-oestrus to last for 9.1 (s 1.0) days. =
=
Metoestrus commences with the end of the receptive period and lasts for about 60 days. Thereafter the animal enters the anoestrous period which lasts for about 7 months until start of next pro-oestrus. If the bitch is mated and pregnant the pregnancy usually lasts for about 60 days (Christie 8c Bell 1971). The peripheral plasma levels of oestradiol during the pro-oestrous peak recorded in the present study compares well with the levels described by Jones et al (I913a,b) and Hadley (1973). The highest individual oestradiol peak value in the present investigation was 100 pg/ml. Due to the variation of the peak values found in the present study it is reasonable to assume that the oestradiol peak in the dog is of short duration. Daily blood samples might therefore not be frequent enough to catch the maximum peak value. Smith 8c McDonald (1974) found serum LH levels to start to increase ap¬ proximately 3 days prior to oestrus, with a peak on the day before first day of acceptance. The same investigators found an increase in serum progesterone soon after the increase in LH. Jones et al. (1973e) suggested that LH release in the dog is stimulated by an increase in oestradiol secretion during prooestrus. This observation is supported by the present study, in which the pro¬ gesterone levels in the four dogs investigated started to increase on the day after the oestradiol peak in three bitches, and 4 days after the oestradiol peak in one bitch. Jones et al (1913b) found peripheral plasma levels of oestradiol to fall to a minimum of 9.2 pg/ml in early pregnancy (days 1 to 6 of oestrus). The level then rose and remained fairly constant at 13.8 pg/ml until day 50 when it declined to 7.7 pg/ml on day 60 of pregnancy. The authors also concluded that the levels of oestradiol found during metoestrus were somewhat greater than those of the non-pregnant dog during metoestrus (Jones et al. 1973e). The present study revealed a rather constant mean level of oestradiol of about 10-15 pg/ml throughout pregnancy. No decrease of the level was found for the pregnant bitches during early pregnancy. Furthermore, no marked dif¬ ference was found when comparing the oestradiol levels in pregnant and nonpregnant dogs during metoestrus and the peripartal period. Johnson et al. (1972) investigated peripheral plasma levels of oestradiol during pregnancy and puerperium in four bitches and found no marked change of the levels before as compared to after the delivery.
levels of progesterone during metoestrus or preg¬ nancy showed an inconsistent pattern in two of the four dogs investigated. The regular pattern of two of the dogs, one non-pregnant (No. 8) and one pregnant (No. 11) are in agreement with previous reports (Christie et al. 1971; Jones et al. 1973a,b; Jöchle et al. 1973; Smith Sc McDonald 1974). The levels of progesterone of these two dogs are in accordance with findings by Smith 8c McDonald (1974) and Jöchle et al. (1973) but considerably higher than those of Jones et al. (I973a,b) and Hadley (1973), who recorded levels about half of that found in the present study. Christie et al. (1971) examined 13 nonpregnant bitches during metoestrus and found a considerable degree of in¬ dividual variation; for example, the authors recorded a five fold difference between the highest and lowest progesterone concentration. However, all of the 13 dogs they investigated followed a relatively regular pattern. The other two dogs, one non-pregnant (No. 7) and one pregnant (No. 9), showed a very irregular pattern. Progesterone levels ranged from undetectable levels up to 45 ng/ml. This irregular pattern has previously been described in non-pregnant animals during metoestrus by Jöchle et al. (1973). Parkes et al. (1972) reported on pregnant bitches in which progesterone plasma levels were found below 1 ng/ml. Due to the relatively small number of bitches investigated and the extreme irregular pattern of progesterone levels in two of them no comparison of the level in pregnant and non-pregnant dogs can be made. In the pregnant dog parturition was accompanied by a marked drop of the peri¬ pheral plasma levels of progesterone. This was also found by Smith Se McDonald (1974). The levels in the non-pregnant dogs gradually diminished showing no marked drop. As pointed out previously in the present study and in another (Johnson et al. 1972) no marked change was found in the oestradiol levels from the immediate pre-partum period to the immediate post-partum period. The relatively low levels of oestrogens during the reproductive cycle in the dog is intriguing when compared with the extremely high sensitivity of the dog for exogenously administered oestrogens (Gustafsson 8c Beling 1969). One possible explanation is that the dog lacks a specific binding protein in plasma. Preliminary studies indicate a very low concentration of such a protein (Kasström et al, in press). This would make all circulating oestrogens avail¬ able to the target organs and thus to rapid metabolism. Plager et al. (1963) investigated the cortisol binding property of dog-plasma. These authors en¬ countered no change of the cortisol binding properties in four dogs treated with oestrogens. Human cortisol binding protein (transcortin) increased its concentration after oestrogen treatment. Following the finding of mammary nodules in Beagle dogs and monkeys during treatment with ethynerone the Food and Drug Administration (FDA) in the United States made it mandatory to study any steroidal or non-steroidal potential contraceptive for 7 years in the Beagle dog and 10 years in monkeys.
The
peripheral plasma
Three dose levels have been required, one or two times, 10 and 25 times the human dose per kilogram (Finkel 8c Berliner 1973). Chlormadinone acetate and megestrol acetate were found to be highly tumourigenic in the mammary glands of the Beagle beginning in the second or third year of drug administration, respectively. In monkeys no tumours oc¬ curred. Chlomadinone acetate and megestrol acetate are progesterone deriva¬ tives while 19 nor-testosterone derivatives as norethindrone or d-norgestrel have up to date shown no tumourigenic potential. The extrapolation of the findings in the Beagle dogs to women has been heavily criticized, mainly due to the lack of knowledge of the reproductive physiology of the Beagle dog and also the lack of information regarding the potency and metabolic fate of the administered sex steroids in the dog. Great differences in the hormone levels and sensitivity between the Beagle bitch and women are known for some substances. For instance chlormadinone acetate, which in women is slightly more progestational per unit weight than norethindrone, is found to be 225 times more potent than norethindrone in the Beagle dog (Hill et al. 1970). When comparing the data obtained in the present study to similar data in women it can be concluded that a great difference exists in the ovarian steroid function between these two species. This adds more evidence to suggest that the dog is less suitable as an experimental animal for the testing of syn¬ thetic steroids for use in man.
ACKNOWLEDGMENTS This
investigation was supported by
the
John
M. Olin Foundation and Ford Foundation.
REFERENCES Bell E.
T., Christie D. W. & Younglai E. V.: J. Endocr. 51 (1971) 225. Edqvist L.-E., Johansson E. D. B., Lindberg P. Se Martinsson K.: Steroids
Boilert B.,
(1973)
22
891.
Christie D. W. Se Bell E. T.: J. small Anim. Pract. 12 (1971) 383. Christie D. W., Bell E. T., Horili C E. & Palmer R. F.: Acta endocr.
(1971) 543. Edqvist L.-E.
(Kbh.)
68
Se Johansson E. D. B.: Acta endocr. (Kbh.) 71 (1972) 716. Finkel M. J. 8c Berliner V. R.: The Extrapolation of Experimental Findings (Animal to Man); the Dilemma of the Systemically Administered Contraceptives. 62nd Annual Meeting, International Academy of Pathology (1973). Gustafsson P. O. Se Beling C. G.: Endocrinology 85 (1969) 481. Hadley J. C: Vet. Ree. 93 (1973) 77. Hill R., Averkin E., Brown W.. Gagne W. E. Se Segre E.: Contraception 2 (1970) 381. Johnson J. ., Davis J. O. Se Hotchkiss J.: Endocrinology 90 (1972) 322. Jones G. E., Boyns A. R., Cameron E. H. D.. Bell E. T.. Christie D. W. Se Parkes M. F.: J. Endocr. 57 (1973«) 331.
Jones G. E., Boyns A. R., Cameron E. H. D., Bell E. T., Christie D. W. Se Parkes M. F.: J. Reprod. Fértil. 35 (19736) 187. Jones G. E., Boyns A. R., Cameron E. H. D., Beli E. T., Christie D. W. & Parkes M. F.: Acta endocr (Kbh.) 72 (1973c) 573. Jöchle W., Tomlinson R. V. 8e Andersen A. C: Prostaglandins 3 (1973) 209. Lindberg B. S., Lindberg P., Martinsson K. Se Johansson E. D. B.: Acta obstet, gynec. scand. Suppl. 32 (1974) 5. Parkes M. F., Bell E. T. Se Christie D. W.: Brit. vet. J. 128 (1972) XV. Plager J. E., Knopp R., Slaunwhite N. R. 8- Sandberg . .: Endocrinology 73 (1963) 353. Smith M. S. Se McDonald L. E.: Endocrinology 94 (1974) 404. Thomcycroft 1. H. Se Stone S. G: Contraception 5 (1972) 129.
Received
on
March 23rd, 1974.
the
BLOOD PLASMA LEVELS OF PROGESTERONE AND OESTRADIOL IN THE DOG THE DURING OESTROUS CYCLE AND PREGNANCY
By L.-E.
Edqvist,
E. D. B.
Johansson,
H. Kasstr\l=o"\m,
S.-E. Olsson and M. Richkind ABSTRACT Plasma oestradiol and progesterone levels were studied in German Shepherd and Greyhound bitches during the normal oestrous cycle, pregnancy and at parturition. The mean oestradiol level increased from approximately 7 to 13 pg/ml during the fifth week before onset of oestrus. Oestradiol stayed at this level until pro oestrus which was characterized by a steady increase to a mean peak level of about 30 pg/ml 5\p=n-\6days before oestrus. At the start of oestrus the level was approximately 12 pg/ml. A level between 10\p=n-\15pg/ml was kept during the following 10 weeks whether or not the bitch was pregnant and no change occurred at parturition. The mean progesterone level was found to be very low, around or under 1 ng/ml, until 1\p=n-\4days after the oestradiol peak after which the level gradually increased to around 10 ng/ml at the start of oestrus. During oestrus and metoestrus or pregnancy the pattern was inconsistent. In some bitches the level gradually increased during 3\p=n-\4weeks and reached a peak value of 30\p=n-\50ng/ml. The level then gradually decreased during 5\p=n-\6 weeks. During the first 5 weeks of the same period progesterone in other bitches fluctuated between similar maximum levels and extremely low levels. After the fifth week the pattern was the same for all bitches. In the pregnant bitches there was a significant drop of the progesterone level at parturition. The data suggest that the dog is not an ideal test animal for steroids synthetized for use in man.
and
dog has been used as an experimental animal in metabolic studies and toxicity testings of synthetic steroid hormones made for use as oral contra¬ ceptives in women during recent years; data on the pattern of sex steroid hormones during the normal oestrous cycle and pregnancy have become avail¬ able (Christie et al. 1971; Bell et al. 1971; Jones et al. I913a,b,c; Smith 8c McDonald 1974). The dog has apparently a sex steroid pattern which differs from that of most other species of laboratory or domestic animals and man. The aim of the present study was to determine peripheral plasma levels of both progesterone1) and oestrogens and their sequential changes during the normal canine oestrous cycle, pregnancy and at parturition in the same in¬ The
dividuals. MATERIAL AND METHODS
Pure-bred female German Shepherds and racing Greyhounds were used (Table 1). The German Shepherds were between 2 and 5 years of age and the Greyhounds between 4 and 6 years of age. All animals were kept in pens and runs and were given a commercial diet and water ad libitum. They were regularly dewormed and inoculated against distemper and hepatitis. Pregnant bitches were given an additional adequate supply of calories, minerals and vitamins during the last trimester of preg¬ nancy.
The females were checked daily for presence of blood stained mucus in the vulva, which was used as the criterion for beginning of pro-oestrus. Blood samples were collected from dogs in the morning before feeding once or twice weekly during the anoestrous period. Starting with the first day of pro-oestrus, blood samples were taken daily or every second day until the female accepted a male. Sampling was then resumed twice a week for 8 weeks. During the ninth week after acceptance, daily blood samples were taken until parturition or in the case of unmated females, for 7 to 10 days, whereafter samples twice a week were taken for another 2 to 4 weeks. Five to eight ml of blood were collected from the cephalic vein in heparinized tubes. After centrifugation the plasma was removed and stored at below -20°C until analyzed. Analyzing of oestradiol and progesterone was done in the following manner; plasma levels of oestradiol in all dogs, plasma levels of progesterone in the Greyhounds (except dog No. 10, which was analyzed only during the peripartal period). Plasma levels of progesterone were measured by radioimmunoassay (Thorneycroft Se Stone 1972). Plasma levels of oestrogens were measured by radioimmunoassay using the same procedure and reagents as described by Edqvist 8- Johansson (1972). Antiserum to an oestradiol-17/? 17-succinyl-bovine serum albumin was a gift from National Institute of Health, Bethesda, USA, and was used in a dilution of 1:50 000 in a phos-
following abbreviations and trivial names are Oestradiol-17^: l,3,5(10)-oestratriene-3,17/?-diol.
') The
Oestrone: 3-hydroxy-l,3,5(10)-oestratrien-17-one. Progesterone: 4-pregnene-3,20-dione. standard error of the standard deviation.
sem: s:
mean.
used:
Table 1. Clinical data on the
Dog No.
Breed
pregnancy
German
58
Shepherd 2
German
58
Shepherd 3
German
Shepherd 4
German
German
6
German
not
7 S 9
Shepherd Greyhound Greyhound Greyhound
10
Greyhound
(days)
12 12
no
distinct
peak 3 males 1 male 5 females
mated not
Greyhound
males females males females
pregnant 62
Shepherd
11
4 3 3 2
(days)
mated not
Shepherd 5
Live
Time inter¬ val between
Time interval between pro-oestrus oestradiol peak and 1st day and 1st day Stillborn of acceptance of acceptance
Litter at birth
Duration of
(days) 1
dogs.
10
2
13
4
14
7
14 14 17
3 5 2
pregnant mated
pregnant
not mated not mated
65 60
4 3 5 2 5 1
males 2 males females
16
males
females males female
1 male 1 female
11
phate buffer, pH 7.4, containing 0.1 "lo gelatin. The degree of cross-reaction was identical to other antisera prepared against oestradiol-17/5? conjugated at position 17 (Boilert ci al. 1973; Lindberg et al. 1974). The reliability of the oestradiol radio¬ immunoassay was tested by two methods from pooled dog plasma. The rapid radio¬ immunoassay procedure and the radioimmunoassay after separation on Sephadex LH-20 columns were compared. A high degree of correlation between the two methods was found whether or not purification of the plasma extract was done. The sensitivity
of the assay system used was 2 pg as read off the standard curve. The coefficient of variation in the range 2-5 pg/ml was 16.3 per cent calculated from 30 duplicate estimations. One ml plasma was extracted with 5 ml diethyl ether by vigorous shaking for 1 min. The plasma blank was negligible and the diethyl ether blank was corrected for by adding 5 ml of diethyl ether to each point of the standard curve. The standard samples as well as the unknown were simultaneously evaporated to dryness at 37°C. All values obtained during the present study were uncorrected for packed cell volume and well above the limit of detection of the assay system.
RESULTS
The time interval between start of pro-oestrus and first day of acceptance (= onset of oestrus) is given in Table 1. Pro-oestrus was considered to start at the first appearance of blood stained mucus in the vulva. Pro-oestrus started 12.8 (s 2.6) days before onset of oestrus. All pregnant dogs delivered normal sized litters without obstetrical assistance and after a gestation of normal length (Table 1). The five stillborn puppies were all of full term size and were not macerated. The mortality rate in this study was the same as normally found in the kennel. The mean peripheral plasma level of oestradiol in 11 bitches during late anoestrus, pro-oestrus and until the first day of acceptance is given in Fig. 1. The mean oestradiol level increased from 7 to 13 pg/ml from the fifth to the fourth week before first day of acceptance (Fig. 1). Thereafter the level remained fairly constant to about the start of pro-oestrus. During the pro-oestrous period the mean oestradiol level increased continu¬ ously and the highest mean values of approximately 30 pg/ml were seen on days 6 and 5 before start of oestrus (Fig. 1). During this period column separa¬ tion on Sephadex LH-20 revealed an oestradiol/oestrone ratio of about 2.5. The time interval between the oestradiol peak and oestrus in each individual dog is given in Table 1. Thereafter the mean level decreased to the initial pro-oestrous levels. On the first day of acceptance (= day 0) a mean value of 12 pg/ml was found. The time interval between the oestradiol peak and first day of acceptance was found to vary between 2 and 7 days (Table 1). The individual peripheral plasma levels of progesterone in four Greyhounds (Nos. 7, 8, 9 and 11) are also given in Fig. 1. No marked change of the levels was seen prior to 5 days before oestrus. The levels obtained up to this point were mostly below 1 ng/ml. During late pro-oestrus the progesterone levels in the four bitches increased gradually. In three of the Greyhounds this in¬ crease was found to start on the day after the oestradiol peak in each bitch, while one bitch (No. 11) showed no change of the progesterone level until 4 days after the oestradiol peak. The mean peripheral plasma levels of oestradiol in pregnant as well as in non-pregnant bitches during the 8 weeks after onset of oestrus are shown in Fig. 2. It was found that the mean levels were about the same in both preg¬ nant and non-pregnant bitches. The mean peripheral plasma level of oestradiol was between 10 and 15 pg/ml in both these two groups. During the luteal column phase separation on Sephadex LH-20 revealed an oestradiol/oestrone ratio of about 1. Fig. 3 shows the individual peripheral plasma levels of oestra¬ diol before and after the delivery in the six pregnant bitches. Also in Fig. 3 the oestradiol values for non-pregnant bitches are shown during the time inter¬ val 57-70 days after oestrus. All the pregnant bitches gave birth during this =
Fig. 1. Peripheral plasma levels of oestradiol (X ± SEM)for all bitches and individual peri¬ pheral plasma levels of progesterone in four Greyhound bitches for the five weeks proceeding oestrus. Arrows indicate individual oestradiol peak value.
period (Table 1). One non-pregnant dog (No. 6) showed signs of approaching oestrus during this time interval and is therefore excluded from the material drawn in Fig. 3. The peripheral plasma levels of oestradiol were found to be similar in both pregnant and non-pregnant bitches and no significant change occurred when comparing the mean oestradiol levels before and after the delivery (Students i-test). No statistically significant difference was found when comparing the mean oestradiol measurements of individual pregnant and non-pregnant Greyhounds and German Shepherds from oestrus and 60 days onward. Neither was there any statistically significant difference found when all oestradiol measurements were compared between breeds (Student's
Fig.
2.
levels of oestradiol (X + sem) for pregnant bitches (broken lines) and non-pregnant bitches (shadowed area) during the 8 weeks following onset of oestrus. Individual peripheral plasma levels of progesterone in two pregnant and two non-pregnant Greyhound bitches for 11 weeks following oestrus.
Peripheral plasma
30
a.
w
Parturition
20
10-
O
-6
Days
-A -2 0 +2 +4 +6 before and after parturition
Fig.
58
Days
62 64 66 68 70 after onset of oestrus
60
3.
Peripheral plasma levels of oestradiol in six pregnant bitches between one week before and after parturition. For comparative reasons the oestradiol levels in four non-pregnant bitches are also given during the 9th and 10th week after onset of oestrus, which was the time of parturition in the pregnant bitches. The mean value for Greyhounds was 12.9 (sem 0.53) pg/ml and for German Shepherds 11.6 (sem 0.60) pg/ml. The peripheral plasma levels of progesterone in two pregnant (Nos. 9 and 11) and two non-pregnant bitches (Nos. 7 and 8) are given in Fig. 2. Two
/-test).
=
=
bitches, the
pregnant (No. 11) and one non-pregnant (No. 8) showed about pattern with continuously increasing values of progesterone from to about 3 to 4 weeks thereafter, at which time maximum values one
same
oestrus
of approximately 30 and 50 ng/ml were seen. The levels then gradually decreased and the non-pregnant bitch (No. 8) showed values below 0.5 ng/ml from the latter part of the 9th week after oestrus and onwards. The pregnant bitch No. 11 showed progesterone values of 2.5-5 ng/ml during the 9th week after oestrus. After the delivery on day 65 the level diminished rapidly to values below 0.5 ng/ml. The other two bitches, one pregnant (No. 9) and one non-pregnant (No. 7), showed a very inconsistent pattern of pro¬ gesterone levels with low concentrations 2 and 3 days after oestrus followed by an increase and thereafter low levels again during the 4th week (No. 7) and during the 5th week (No. 9). After the 5th week the pattern for these two dogs followed the same pattern as that of dogs Nos. 9 and 11. In the pregnant bitch No. 9 the progesterone levels fell rapidly after parturition on day 65, while the non-pregnant bitch No. 7 showed relatively higher values during the 10th and 11th week after oestrus compared with the other two dogs
(Fig. 2). In Fig. 4
given the peripheral plasma levels of oestradiol and proges¬ and after parturition in three bitches (Nos. 9, 10 and 11). The terone before progesterone level dropped at parturition and after the delivery very low levels were encountered. The peripheral plasma levels of oestradiol showed no significant change during this period. are
4-4
Dog
nr
.,
..
9 10 Parturition
Parturition
Parturition
6i cu ¬
£ U 30
ÌL 2
-o cû
20 3 10 o
-5 -3
-1 A
Days
3
-3
-1.1
before and after
parturition
Fig. 4. Peripheral plasma levels of oestradiol (solid line) and progesterone (broken line) in three individual Greyhound bitches during the immediate pre- and post-partum period.
DISCUSSION
The
length of the pro-oestrous period found in the present investigation was 12.8 (s 2.6; range 8-17) days. This interval is considerably longer than the interval of 4.6 (range 2-8) days reported by Christie et al. (1971). However, these investigators considered oestrus to start before the bitch fully accepted the male (Christie 8c Bell 1971). Smith 8c McDonald (1974), who calculated the length of the pro-oestrous period using the same criterion as in the present investigation found pro-oestrus to last for 9.1 (s 1.0) days. =
=
Metoestrus commences with the end of the receptive period and lasts for about 60 days. Thereafter the animal enters the anoestrous period which lasts for about 7 months until start of next pro-oestrus. If the bitch is mated and pregnant the pregnancy usually lasts for about 60 days (Christie 8c Bell 1971). The peripheral plasma levels of oestradiol during the pro-oestrous peak recorded in the present study compares well with the levels described by Jones et al (I913a,b) and Hadley (1973). The highest individual oestradiol peak value in the present investigation was 100 pg/ml. Due to the variation of the peak values found in the present study it is reasonable to assume that the oestradiol peak in the dog is of short duration. Daily blood samples might therefore not be frequent enough to catch the maximum peak value. Smith 8c McDonald (1974) found serum LH levels to start to increase ap¬ proximately 3 days prior to oestrus, with a peak on the day before first day of acceptance. The same investigators found an increase in serum progesterone soon after the increase in LH. Jones et al. (1973e) suggested that LH release in the dog is stimulated by an increase in oestradiol secretion during prooestrus. This observation is supported by the present study, in which the pro¬ gesterone levels in the four dogs investigated started to increase on the day after the oestradiol peak in three bitches, and 4 days after the oestradiol peak in one bitch. Jones et al (1913b) found peripheral plasma levels of oestradiol to fall to a minimum of 9.2 pg/ml in early pregnancy (days 1 to 6 of oestrus). The level then rose and remained fairly constant at 13.8 pg/ml until day 50 when it declined to 7.7 pg/ml on day 60 of pregnancy. The authors also concluded that the levels of oestradiol found during metoestrus were somewhat greater than those of the non-pregnant dog during metoestrus (Jones et al. 1973e). The present study revealed a rather constant mean level of oestradiol of about 10-15 pg/ml throughout pregnancy. No decrease of the level was found for the pregnant bitches during early pregnancy. Furthermore, no marked dif¬ ference was found when comparing the oestradiol levels in pregnant and nonpregnant dogs during metoestrus and the peripartal period. Johnson et al. (1972) investigated peripheral plasma levels of oestradiol during pregnancy and puerperium in four bitches and found no marked change of the levels before as compared to after the delivery.
levels of progesterone during metoestrus or preg¬ nancy showed an inconsistent pattern in two of the four dogs investigated. The regular pattern of two of the dogs, one non-pregnant (No. 8) and one pregnant (No. 11) are in agreement with previous reports (Christie et al. 1971; Jones et al. 1973a,b; Jöchle et al. 1973; Smith Sc McDonald 1974). The levels of progesterone of these two dogs are in accordance with findings by Smith 8c McDonald (1974) and Jöchle et al. (1973) but considerably higher than those of Jones et al. (I973a,b) and Hadley (1973), who recorded levels about half of that found in the present study. Christie et al. (1971) examined 13 nonpregnant bitches during metoestrus and found a considerable degree of in¬ dividual variation; for example, the authors recorded a five fold difference between the highest and lowest progesterone concentration. However, all of the 13 dogs they investigated followed a relatively regular pattern. The other two dogs, one non-pregnant (No. 7) and one pregnant (No. 9), showed a very irregular pattern. Progesterone levels ranged from undetectable levels up to 45 ng/ml. This irregular pattern has previously been described in non-pregnant animals during metoestrus by Jöchle et al. (1973). Parkes et al. (1972) reported on pregnant bitches in which progesterone plasma levels were found below 1 ng/ml. Due to the relatively small number of bitches investigated and the extreme irregular pattern of progesterone levels in two of them no comparison of the level in pregnant and non-pregnant dogs can be made. In the pregnant dog parturition was accompanied by a marked drop of the peri¬ pheral plasma levels of progesterone. This was also found by Smith Se McDonald (1974). The levels in the non-pregnant dogs gradually diminished showing no marked drop. As pointed out previously in the present study and in another (Johnson et al. 1972) no marked change was found in the oestradiol levels from the immediate pre-partum period to the immediate post-partum period. The relatively low levels of oestrogens during the reproductive cycle in the dog is intriguing when compared with the extremely high sensitivity of the dog for exogenously administered oestrogens (Gustafsson 8c Beling 1969). One possible explanation is that the dog lacks a specific binding protein in plasma. Preliminary studies indicate a very low concentration of such a protein (Kasström et al, in press). This would make all circulating oestrogens avail¬ able to the target organs and thus to rapid metabolism. Plager et al. (1963) investigated the cortisol binding property of dog-plasma. These authors en¬ countered no change of the cortisol binding properties in four dogs treated with oestrogens. Human cortisol binding protein (transcortin) increased its concentration after oestrogen treatment. Following the finding of mammary nodules in Beagle dogs and monkeys during treatment with ethynerone the Food and Drug Administration (FDA) in the United States made it mandatory to study any steroidal or non-steroidal potential contraceptive for 7 years in the Beagle dog and 10 years in monkeys.
The
peripheral plasma
Three dose levels have been required, one or two times, 10 and 25 times the human dose per kilogram (Finkel 8c Berliner 1973). Chlormadinone acetate and megestrol acetate were found to be highly tumourigenic in the mammary glands of the Beagle beginning in the second or third year of drug administration, respectively. In monkeys no tumours oc¬ curred. Chlomadinone acetate and megestrol acetate are progesterone deriva¬ tives while 19 nor-testosterone derivatives as norethindrone or d-norgestrel have up to date shown no tumourigenic potential. The extrapolation of the findings in the Beagle dogs to women has been heavily criticized, mainly due to the lack of knowledge of the reproductive physiology of the Beagle dog and also the lack of information regarding the potency and metabolic fate of the administered sex steroids in the dog. Great differences in the hormone levels and sensitivity between the Beagle bitch and women are known for some substances. For instance chlormadinone acetate, which in women is slightly more progestational per unit weight than norethindrone, is found to be 225 times more potent than norethindrone in the Beagle dog (Hill et al. 1970). When comparing the data obtained in the present study to similar data in women it can be concluded that a great difference exists in the ovarian steroid function between these two species. This adds more evidence to suggest that the dog is less suitable as an experimental animal for the testing of syn¬ thetic steroids for use in man.
ACKNOWLEDGMENTS This
investigation was supported by
the
John
M. Olin Foundation and Ford Foundation.
REFERENCES Bell E.
T., Christie D. W. & Younglai E. V.: J. Endocr. 51 (1971) 225. Edqvist L.-E., Johansson E. D. B., Lindberg P. Se Martinsson K.: Steroids
Boilert B.,
(1973)
22
891.
Christie D. W. Se Bell E. T.: J. small Anim. Pract. 12 (1971) 383. Christie D. W., Bell E. T., Horili C E. & Palmer R. F.: Acta endocr.
(1971) 543. Edqvist L.-E.
(Kbh.)
68
Se Johansson E. D. B.: Acta endocr. (Kbh.) 71 (1972) 716. Finkel M. J. 8c Berliner V. R.: The Extrapolation of Experimental Findings (Animal to Man); the Dilemma of the Systemically Administered Contraceptives. 62nd Annual Meeting, International Academy of Pathology (1973). Gustafsson P. O. Se Beling C. G.: Endocrinology 85 (1969) 481. Hadley J. C: Vet. Ree. 93 (1973) 77. Hill R., Averkin E., Brown W.. Gagne W. E. Se Segre E.: Contraception 2 (1970) 381. Johnson J. ., Davis J. O. Se Hotchkiss J.: Endocrinology 90 (1972) 322. Jones G. E., Boyns A. R., Cameron E. H. D.. Bell E. T.. Christie D. W. Se Parkes M. F.: J. Endocr. 57 (1973«) 331.
Jones G. E., Boyns A. R., Cameron E. H. D., Bell E. T., Christie D. W. Se Parkes M. F.: J. Reprod. Fértil. 35 (19736) 187. Jones G. E., Boyns A. R., Cameron E. H. D., Beli E. T., Christie D. W. & Parkes M. F.: Acta endocr (Kbh.) 72 (1973c) 573. Jöchle W., Tomlinson R. V. 8e Andersen A. C: Prostaglandins 3 (1973) 209. Lindberg B. S., Lindberg P., Martinsson K. Se Johansson E. D. B.: Acta obstet, gynec. scand. Suppl. 32 (1974) 5. Parkes M. F., Bell E. T. Se Christie D. W.: Brit. vet. J. 128 (1972) XV. Plager J. E., Knopp R., Slaunwhite N. R. 8- Sandberg . .: Endocrinology 73 (1963) 353. Smith M. S. Se McDonald L. E.: Endocrinology 94 (1974) 404. Thomcycroft 1. H. Se Stone S. G: Contraception 5 (1972) 129.
Received
on
March 23rd, 1974.
