Endotoxin-induced prostaglandin release and corpus luteum function in goats

Animal Reproduction Science, 8 (1985) 109--121

109

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

ENDOTOXIN-INDUCED PROSTAGLANDIN RELEASE AND CORPUS LUTEUM FUNCTION IN GOATS

G U N N A R FREDRIKSSON, H A N S K I N D A H L and LARS-ERIC EDQVIST*

Departments of Obstetrics and Gynaecology and of *ClinicalChemistry, College of Veterinary Medicine, Swedish University of Agricultural Sciences, S-750 07 Uppsala (Sweden) This study was supported by grants from the Swedish Council for Forestry and Agricultural Research. (Accepted 8 May 1984)

ABSTRACT Fredriksson, G., Kindahl, H. and Edqvist, L.E., 1985. Endotoxin-induced prostaglandin release and corpus luteum function in goats. Anim. Reprod. Sci., 8: 109--121. Endotoxin (lipopolysaccharide) from Salmonella typhimurium was administered to non-pregnant and pregnant goats to investigate its capacity to induce synthesis and release of prostaglandin with subsequent alteration of reproductive functions. Prostaglandin release was determined by measurement of 15-keto-13,14-dihydro-PGF2a and the corpus luteum function was monitored by measurement of progesterone. Endotoxin administered into the lumen of the uterus did n o t alter the hormonal patterns. However, when 1.8 ug/kg of the endotoxin was given i.v., a rapid increase of 15-keto-13,14-dihydro-PGF2a and a subsequent decline in progesterone levels were seen. Both non-pregnant and hysterectomized animals shortened their lengths of oestrous cycles. In two out of three pregnant animals the i.v. administration of the endotoxin (0.1 ug/kg) resulted in abortions. When comparing the prostaglandin metabolite response of intact vs. hysterectomized animals, the response was somewhat less in the latter group. When endotoxin was administered in smaller amounts (0.5 ug/kg) to non-pregnant goats it created an immediate increase in prostaglandin metabolite levels, but the subsequent decline in progesterone levels was less pronounced and the length of the oestrous cycle was not altered. From the present study it is obvious that i.v. administered endotoxin can influence the life-span of the corpus luteum, which is probably mediated by induction of synthesis and release of PGF2a.

INTRODUCTION

The injection of a Salmonella enteritidis endotoxin into the uterine artery of the ewe elevates the concentration of PGF2~ in the venous effluent (Roberts et al., 1975). Elevated levels of prostaglandins have also been found after systemic injections of endotoxins (Anderson et al., 1972; 1975a,b; Fletcher and Ramwell, 1977). Prostaglandin F:a acts luteolytically in most domestic species (Horton and Poyser, 1976). The possibil0378-4320/85/$03.30

© 1985 Elsevier Science Publishers B.V.

110 ity then exists that infections by Gram-negative bacteria giving rise to endotoxin production may influence the reproductive functions. It is well known that some Gram-negative infections cause abortion in domestic animals (Roberts, 1971). The aim of the present study was to investigate the ability of an endotoxin isolated from S. typhimurium to induce synthesis and release of prostaglandins and to assess the influence on the reproductive functions, in particular the corpus luteum function in goats. MATERIALS AND METHODS

Animals Six mature Swedish Landrace goats with normal oestrous cyclicity {mean length of cycle 19.8 days, S.E. 0.3 day), one anoestrous goat, two hysterectomized goats and three pregnant goats were used (Table I). They were quartered in groups in pens and fed on 1 kg of good quality hay and 0.2 kg of commercially prepared fodder. The oestrus of the normally cycling does was synchronized, using injections of 10 mg of prostaglandin F2~ (Dinolytic, Upjohn, U.S.A.) given i.m. at 10-day intervals. Testing for oestrus was performed twice daily in the presence of a buck (day 1 = first day of oestrus).

Blood sampling From the onset of oestrus prior to the experiment, 5 ml of blood was collected twice daily. Later the goats were treated differently in accordance with the approach adopted in the study. The pregnant goats were bloodsampled twice weekly from mating until the start of the experiment. After each experiment all goats were blood-sampled 2 or 3 times weekly in order to measure progesterone levels and also to check for eventual return to oestrus. Jugular vein blood samples were withdrawn into heparinized Vacutainer tubes (Becton-Dickinson, U.S.A.) and centrifuged immediately. Plasma was removed and stored at - 2 0 ° C until hormone analysis.

Hormone analysis 15-Ketodihydro-PGF2a (9a,11a
111 TABLE I T r e a t m e n t r e g i m e n s w i t h e n d o t o x i n f r o m Saimone|~a typhfmurium, e n d o c r i n e a n d c l i n i c a l o u t c o m e Goat no.

Route of adminlstration

Reproductive stage

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2

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3

i.u.

4

i.u.

3

i.v.

1

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5

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Results (Inter-oestrous interval given within parentheses)

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NaCI

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1.3

50

Normal length of cycle (19 days)

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2.6

100

Normal length of cycle (20 days)

45

5.2

200

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2.7

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18

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P4<:1 n m o l [ l a f t e r 2 d a y s , o e s t r o s after 3 days (13 days) P4~1 nmolfl after 1 day, oestrus after 4 days (10 days) P4 ~ 1 n m o l ] l a f t e r 1 d a y , b a d status after treatment, weak oestzus after 4 days (10 days) T e m p o r a r y d r o p in P 4 , n e v e r <:1 n m o l f l , n o r m a l l e n g t h o f cycle (20 days) Temporary drop in P4, just one observation ~1 nmol]l, normal length of cycle (19 days) Temporary drop in P4, never <:1 n m o l / l , n o r m a l l e n g t h o f cycle (20 days) P4 a l w a y s l o w , n o signs o f o e s t r u s P4 0 all t h e t i m e , n o s i g n s o f oestros P4~I nmol]l after 1.5 days, oestrus after 5 days Temporary drop in P4, just four observations (1 nmolfl P4 ~ 1 n m o l f l a f t e r 1 d a y , n o signs o f oestl~ls, s p r i n g - s e a s o n , entered anoestrous state P4<:I.5 nnlol]l after 2 days, ~i nmolfl after 5 days, oestros after 6 days Temporary drop in P4, never ~1 nmolfl P4<:1 n m o l ] l a f t e r 1 d a y , n o signs o f o e s t r u s , sp1~ng-season, entered anoestrous state P4 ~ 1 n m o l f l a f t e r 1 . 5 d a y s , a b o r tion after 1 day and signs of oestrus after 2 days P4 ~ 1 n m o l f l a f t e r 3 . 5 d a y s , a b o r tion after 2 days Temporary drop in P4, never ~1 nmolfl pregnancy continued

I n t e r v a l = d a y s s i n c e t r e a t e d l a s t t i m e w i t h e n d o t o x i n , i.u. = i n t ~ a - u t e r i n e , i.v. ffi i n t r a v e n o u s , P4 = progesterone.

Progesterone concentrations were determined by radioimmunoassay (Kindahl et al., 1976). The antibody was raised to an lla-hydroxyprogesterone--hemisuccinate--bovine serum albumin conjugate (Bosu et al., 1976). The antiserum cross-reacted < 1% for progestagens, oestrogens,

112

androgens and corticoids except for deoxycorticosterone (3.8%), pregnenolone {3.8%) and 5a-pregnene~,20-dione (11%). The practical limit of sensitivity was 0.5 nmol/l in analysis of 0.25 ml plasma. The intra-assay coefficient of variation varied between 5.5 and 18.5% for different ranges of the standard curve. The S. typhimurium endotoxin (lipopolysaccharide; LPS) was extracted by the hot phenol-water method from batch-grown cultures of formaldehyde-killed S. typhimurium SH4809, and the LPS was further purified as described previously (Svenson and Lindberg, 1978). The purity and the structure of the LPS were checked by sugar analysis, methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The pyrogenic activity was established in rabbits (Lindberg et al., 1983) In tra-u terine studies On day 9 of the oestrous cycle, 4 goats (nos. 1--4; Table I) were anaesthetized, using 0.22 mg/kg of xylazine chloride and 11 mg/kg of ketamine chloride i.m. The goats were opened surgically by mid-line incision. Goats 1 and 2 received 1 ml of physiological saline injected into the lumen of the uterine horn ipsilateral to the corpus luteum. By the same route, goats nos. 3 and 4 received 50 and 100 pg of the S. typhimurium endotoxin, respectively, which was dissolved in physiological saline to a total volume of 1 ml. Blood sampling was undertaken every hour during the first 3--5 h and then at 3-h intervals until the onset of oestrus. They recovered from surgery without complications. In travenous studies In this study 10 animals were used. They were divided as follows: four normally cycling goats (nos. 1, 3, 5 and 6), one anoestrous goat (no. 7), two hysterectomized goats (nos. 9 and 10) and three pregnant goats (nos. 8, 11 and 12; Table I). Goats 1 and 3 had earlier participated in the intrauterine study. All these animals received endotoxin, injected i.v. into the jugular vein. Goats 5 and 6 were treated again (2 months and 2 weeks later, respectively) with 0.5 pg/kg of endotoxin on day 7 of the oestrous cycle. The anoestrous goat, no. 7, was sampled twice weekly during 3 months and her progesterone value was continuously low, between 0 and 1.7 nmol/1. She was given 3.0 ~g/kg of endotoxin. On day 7 of the oestrous cycle, goats 9 and 10 were hysterectomized using the anaesthesia described previously. During the period of recovery from surgery until administration of endotoxin progesterone levels varied between 11.0 and 24.5 nmol/1, which indicated an undisturbed luteal function. The goats were given 3.3 and 1.8 #g/kg of endotoxin, respectively.

i18

Both animals were treated again 2.5 months later with 0.5 ~g/kg of endotoxin and 15 days thereafter once more with the initial dose. The area under the 15-ketodihydro-PGF2a response curve for the first 5 h following endotoxin injection was calculated for all treatments of non-pregnant and hysterectomized goats using an electronic integrator (Digiplan, Kontron Messger~ite, FRG). The pregnant goats received i.v. 0.1 pg/kg of the S. typhimurium endotoxin at 1.5 months (no. 8) and at 3 months (nos. 11 and 12) of gestation. Goat 8 was treated again 2 weeks post-abortion. The blood sampling applied in all the i.v. studies was an hourly sampling during the first 30--33 h after the administration of endotoxin. It was then continued 4 or 5 times daily for 7--14 days, or until the onset of oestrus. RESULTS

Intra-uterine studies The animals subjected to intra-uterine endotoxin (nos. 3 and 4) or saline (nos. 1 and 2) treatment all responded with small increases in prostaglandin metabolite levels. The duration of the increase was approximately 24 h and the levels varied between 100 and 450 pmol/1 (see Fig. 1, upper and lower panels). In goats 1, 2 and 4, progesterone levels varied between 13 and 24 nmol/l. Goat 3 had low progesterone levels (around 1 nmol/1) at the time of the endotoxin treatment, which she maintained throughout the cycle. By days 16--18 of the oestrous cycles a series of prostaglandin metabolite peaks was seen concomitantly with the drop in progesterone levels. All four goats had onset of oestrus on days 19--21 of the cycle.

Intravenous studies High levels of prostaglandin metabolites were recorded in all goats 0.5--1 h after the intravenous injection of endotoxin. During the first 5 h they reached a magnitude of 1000--5000 pmol/1 (see Fig. 2, upper and middle panels). After the initial pronounced prostaglandin release, lower and decreasing levels of around 200--500 pmol/1 were seen. The initial peak was higher and broader and the return to base line values was more delayed in the goats which had received the higher dose of S. typhimurium endotoxin. Progesterone values declined and reached levels below 1 nmol/1 after 1--2 days. Goats 1, 3 and 5 shortened the length of their oestrous cycle (Table I). Goat 6 received a smaller dose that was later repeated in the same animal and also given to goat 5 (Table I). During the first 12--24 h the progesterone level fell, but never reached levels below 1 nmol/1, except for one observation in goat 5 (Table I). On days 2 and 3 after treatment the progesterone

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concentrations rose again to initial or somewhat lower levels. The length of the oestrous cycle in these goats was normal and they entered oestrus at the expected time (Table I). Three of the goats, nos. 1, 3 and 4, were studied for reproductive performance after the endotoxin studies were finished. They showed oestrous Fig. 2. Peripheral "blood plasma levels o f 15-keto-13,14-dihydro-PGF2~ (e o) and progesterone (o ..... o). Horizontal black bar denotes the time of oestrus. Solid arrow indicated the time o f endotoxin (LPS) injection into the jugular vein. Upper panel -goat 1 (2.7 pg/kg; total dose 100 pg). Middle panel -- goat 6 (0.5 ~g/kg; total dose 18 pg). Lower panel - - g o a t 10, hysterectomized (1.8 pg/kg; total dose 100 #g).

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cyclicity with intervals of 19--21 days. They were mated and approximately 150 days later they gave birth to two kids each. The anoestrous goat (no. 7) responded similarly to the other goats. In the hysterectomized animals (nos. 9 and 10) a peak of prostaglandin metabolites was seen during the first hours after injection of endotoxin (Fig. 2, lower panel). After the initial peak, 1200--2500 pmol/l, lower and decreasing levels of 300--400 nmol/1 were recorded during 20 h until pretreatment concentrations were reached. In both goats 9 and 10, the progesterone levels fell below 1 nmol/1 after 2 and 4.5 days, and they exhibited oestrus after 5 and 6 days, respectively. Progesterone levels after oestrus in these animals rose to normal concentrations and were maintained there for 2.5 months. After the second injection of the lower dose of endotoxin (Table I) an initial, but somewhat smaller peak of prostaglandin metabolite was recorded. Progesterone fell to 1.2 nmol/1 in goat 10 and to 0.6 nmol/1 in goat 9 (below 1 nmol/1 at 4 observations) and then rose again on the 3rd and 4th days after the injection. They exhibited no signs of oestrus this time. E u uJ

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Fig. 3. The d o s e o f e n d o t o x i n is given o n t h e x-axis, t h e r e s p o n s e o f PGF2a o n t h e y-axis. It is e s t i m a t e d as t h e area u n d e r t h e curve o f m e a s u r e d levels in 1 5 - k e t o - 1 3 , 1 4 - d i h y d r o PGF2a during t h e first 5 h a f t e r injection. T h e s y m b o l s are u s e d as f o l l o w s : ~ = i n t a c t animals never t r e a t e d b e f o r e , A = i n t a c t animals t r e a t e d o n c e b e f o r e , o = h y s t e r e c t o m i z e d animals never t r e a t e d before, • = h y s t e r e c t o m i z e d animals t r e a t e d o n c e b e f o r e , • = h y s t e r e c t o m i z e d animals t r e a t e d t w i c e b e f o r e . Line A r e p r e s e n t s t h e i n t a c t animals and line B t h e h y s t e r e c t o m i z e d ones. T h e e q u a t i o n o f t h e line and t h e c o e f f i c i e n t o f correlation is given t o t h e right in t h e diagram.

117

After the third injection the initial prostaglandin metabolite peak resembled the one observed after the first treatment,. Progesterone values fell to below 1 nmol/1 before 24 h. They remained low and did not rise during this period. This last experiment was done in the spring. Regression analyses between the dose of injected endotoxin and the prostaglandin metabolite responses for all treatments of non-pregnant intact goats and hysterectomized goats are given in Fig. 3. The coefficient of correlation was 0.87 for the intact animals and 0.97 for the hysterectomized animals. The response was comparatively lower for the hysterectomized animals. The pregnant goats, nos. 8 and 11, responded with an initial prostaglandin metabolite peak reaching 1500--2500 pmol/1 during the first 5 h (Fig. 4). During the following 5--10 h, levels around 300--700 pmol/1 were recorded. At this time the prostaglandin metabolites measured started to rise to levels of 10000--50000 pmol/1. Prostaglandin levels remained high for almost 2 weeks and then returned to base line levels (50--100 pmol/1). After 1.5 and 3.5 days, respectively, the progesterone level fell below 1 nmol/1, where it remained throughout the rest of the observation period. Abortion occurred after 1 day in goat 8 and after 2 days in goat 11. Goat 8 showed signs of oestrus on days 3 and 4 after treatment. s§

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Goat 12 showed the same initial prostaglandin release pattern as goats 8 and 11 and a similar secondary rise, reaching levels around 4000 pmol/1, but after 2--2.5 days the levels decreased to approximately 200 pmol/1 (Fig. 5). She did not abort. The progesterone values dropped to 2.6 nmol/1, but rose again to initial levels of around 20 nmol/1 when the secondary

118

prostaglandin rise was over on days 2--3. Thereafter she continued to have high progesterone levels. After 150 days o f pregnancy she had two kids. Goat 8, which was treated a second time 15 days after abortion, had an initial prostaglandin metabolite peak of 2 0 0 0 - - 2 5 0 0 pmol/1 during the first 5 h and then decreasing levels of 2 0 0 - - 4 0 0 pmol/1 during the following 24 h. Her progesterone values were < 1 nmol/1 throughout this period. All goats given an injection of more than 1.8 pg/kg of endotoxin exhibited tachycardia, tachyphnoea, fever, general malaise and weakness. In most cases they recovered after about 2 days with the exception of goat 5 which remained weak for several days. The goats which received 0.5 and 0.1 pg/kg of the endotoxin showed much milder and short-lasting clinical symptoms, if any at all. 6000 :

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No reactions were seen, either clinically or in an abnormal hormonal pattern, in the goats which received intra-uterine endotoxin or saline. The slight increase in prostaglandin metabolite levels during, and shortly after, the operation may be due to the surgery. Similar results were obtained in previous attempts to inject physiological saline into the uterine artery of the goat (G. Fredriksson, 1982, unpublished observation). The local uterine environment seems to protect the animal from the injected substance. The administered endotoxin may possibly be absorbed into components in the uterine lumen or into the surface o f the endometrium and not reach prostaglandin-producing cells. In a uterus damaged by infection and with subsequent impaired endothelium, an improved absorption of endotoxin with subsequent clinical effects may well be anticipated. Intravenous administration of endotoxin causes a marked response. In

119

a matter of hours, large amounts of prostaglandins are produced, in sufficient quantities to provoke luteolysis and subsequent manifestation of oestrus in most of the non-pregnant animals and abortion in pregnant animals. The prostaglandin response induced by the endotoxin and the subsequent occurrence of oestrus are dose-dependent (Fig. 3). Thus none of the treated non-pregnant animals given the dose of 0.5 ~g/kg shortened the length of their oestrous cycles. The predominating site of the prostaglandin production is not known. Some of it is probably produced in the uterus, which is an important organ for prostaglandin F ~ synthesis (Horton and Poyser, 1976). However, the production and release of significant amounts of prostaglandins in the hysterectomized goats suggests that organs or cells other than those of the uterus are responsible for most of the synthesis and release. The concentration of progesterone also decreases rapidly in the hysterectomized animals, with subsequent manifestation of oestrus. There is a tendency for the 15-keto-13,14-dihydro-PGF2a response in the hysterectomized animals to be lower than in the intact goats (Fig. 3). It should be pointed out that the dose-response relationships in Fig. 3 apply to animals which had been injected once, twice and even three times. The repeated injections of endotoxin did not seem to change the prostaglandin response markedly, as evidenced by the third endotoxin injection to the hysterectomized animals. The difference in response between the intact and hysterectomized animals is probably explained by the presence or absence of the uterus. It is also obvious that the production of prostaglandins in the other parts of the body is sufficient to influence the corpus luteum function. In this respect the lungs could be an important source of production (Anderson et al., 1975b; Demling et al., 1981). The present results suggest that a Gram-negative infection located any place in the body may affect reproduction, provided that enough endotoxin is resorbed to induce prostaglandin synthesis and release. In cases of uterine infections this might be partially beneficial, since the release of prostaglandin will induce oestrus with a subsequent contraction of the uterus and renewal of uterine cellular elements which may aid the process of recovery. Goats require corpus-luteum-secreted progesterone throughout pregnancy (Meites et al., 1951). An endotoxin-induced prostaglandin synthesis will thus induce abortion. Abortion following endotoxin administration has previously been reported to occur in the pig (Wrathall et al., 1978) and in the rat (Harper and Skarnes, 1972; Skarnes and Harper, 1972). An even smaller dose than the one used in the non-pregnant goats is enough to induce a large initial prostaglandin release, which obviously affects the corpus luteum function, evidenced as a rapid decrease in progesterone levels. It is known that exogenous endotoxin after injection can be localized in the placenta where it causes local damage (Le Bouteiller and Hernandez-Verdun, 1974; Rioux-Darrieulat et al., 1974). Placental lesions may be responsible for the initiation of the secondary rise in prostaglandin metabolite levels and the process of abortion cannot be stopped. The high prosta-

120

glandin metabolite levels seen during the following 2--3 weeks are normal for the post-partum period in the goat (Fredriksson et al., 1984). Goat 12 did not abort. This could be explained by a lower degree of damage of the placenta. Neither did progesterone levels reach sufficiently low levels to allow abortion. The concept that prostaglandins play a role in the patho-physiology of endotoxin shock is well established (Kessler et al., 1973; Philipp-Dormston and Siegert, 1974; Veenendaal et al., 1980; Bottoms et al., 1981; Krausz et al., 1981}. From the present study we conclude that endotoxins do influence the life-span of the corpus luteum, most probably mediated by synthesis and release of PGF2a. In normally cycling animals it alters the length of the oestrous cycle and in pregnant animals it therefore induces abortion or premature delivery. The latter effect is probably more pronounced in those species which are dependent on a functional corpus luteum for maintaining pregnancy, e.g., goats, cattle and pigs. ACKNOWLEDGEMENTS

The authors express their sincere thanks to Dr. Stefan B. Svensson, of the National Bacteriological Laboratory and Karolinska Instituter, Stockholm, for his kind supply of the endotoxin and for his fruitful discussion throughout this study, and to Mr. Samar Basu, Mrs. Kerstin Lindblad, Mrs. Lena Nordberg and Mrs. Ulla Arhed for their skilful technical assistance.

REFERENCES Anderson, F.L., Jubiz, W., Fralios, A.C., Tsagaris, T.J. and Kuida, H., 1972. Plasma prostaglandin levels during endotoxin shock in dogs. Circulation, 45, Suppl. If: 124. Anderson, F.L., Jubiz, W., Tsagaris, T.J. and Kuida, H., 1975a. Endotoxin-induced prostaglandin E and F release in dogs. Am. J. Physiol., 228: 410--414. Anderson, F.L., Tsagaris, T.J., Jubiz, W. and Kuida, H., 1975b. Prostaglandin F and E levels during endotoxin-induced pulmonary hypertension in calves. Am. J. Physiol., 228: 1479--1482. Bosu, W.T.K., Edqvist, L.-E., Lindberg, P., Martinsson, K. and Johansson, E.D.B., 1976. The effect of various dosages of lynesterol on the plasma level of oestrogens and progesterone during the menstrual cycle in the rhesus monkey. Contraception, 13: 677--684. Bottoms, G.D., Fessler, J.F., Roesel, O.F., Moore, A.B, and Frauenfelder, H.C., 1981. Endotoxin-induced hemodynamic changes in ponies: effects of flunixin meglumine. Am. J. Vet. Res., 42: 1514--1518. Demling, R.H., Smith, M., Gunther, R., Flynn, J.T. and Gee, M.H., 1981. Pulmonary injury and prostaglandin production during endotoxemia in conscious sheep. Am. J. Physiol., 240: H348--H353. Fletcher, J.R. and Ramwell, P.W., 1977. Modification, by aspirin and indomethacin, of the haemodynamic and prostaglandin releasing effects of E. coli endotoxin in the dog. Br. J. Pharmacol., 61: 175--181.

121 Fredriksson, G., Kindahl, H. and Edqvist, L.-E., 1984. Periparturient release of prostaglandin F2a in the goat. Zbl. Vet. Med., 31: 386--392. Harper, M.J.K. and Skarnes, R.C., 1972. Inhibition of abortion and fetal death produced by endotoxin or prostaglandin F~a. Prostaglandins, 2: 295--308. Horton, E.W. and Poyser, N.L., 1976. Uterine luteolytic hormone: a physiological role for prostaglandin F2a. Physiol. Rev., 56: 595--651. Kessler, E., Hughes, R.C., Bennett, E.N. and Nadela, S.M., 1973. Evidence for the presence of prostaglandin-like material in the plasma of dogs with endotoxin shock. J. Lab. Clin. Med., 81: 85--94. Kindahl, H., Edqvist, L.-E., GranstrSm, E. and Bane, A., 1976. The release of prostaglandin F~a as reflected by 15-keto-13,14-dihydroprostaglandin F2~ in the peripheral circulation during normal luteolysis in heifers. Prostaglandins, 11 : 871--878. Krausz, M.M., Utsunomiya, T., Feuerstein, G., Wolfe, J.H.N., Shepro, D. and Hechtman, H.B., 1981. Prostacyclin reversal of lethal endotoxemia in dogs. J. Clin. Invest., 67: 1118--1125. Le Bouteiller, Ph. and Hernandez-Verdun, D., 1974. Action des endotoxines bact~riennes sur l'ultrastructure du placenta de rat. Pathol. Biol., 22: 29--38. Lindberg, A.A., Sheldon, G.E. and Svenson, S.B., 1983. Induction of endotoxin tolerance with nonpyrogenic O-antigenic oligosaccharide--protein conjugates. Infect. Immun., 41 : 888--895. Meites, J., Webster, H.D., Young, F.W., Thorp, F., Jr. and Hatch, R.N., 1951. Effects of corpora lutea removal and replacement with progesterone on pregnancy in goats. J. Anita. Sci., 10: 411--416. Philipp-Dormston, W.K. and Siegert, R., 1974. Plasma prostaglandins of the E and F series in rabbits during fever induced by Newcastle disease virus, E. coli-endotoxin, or endogenous pyrogen. Z. Naturforsch. Tell C, 29: 773--776. Rioux-Darrieulat, F., Parant, M. and Chedid, L., 1974. Effets l~tal et abortif des endotoxines bact~riennes. Ann. Biol., 9--10: 481--500. Roberts, J.S., Barcikowski, B., Wilson, L., Skarnes, R.C. and McCracken, J.A., 1975. Hormonal and related factors affecting the release of prostaglandin F2a from the uterus. J. Steroid Biochem., 6 : 1 0 9 1 - - 1 0 9 7 . Roberts, S.J., 1971. Veterinary Obstetrics and Genital Diseases. Ithaca, New York (publ. by author), pp. 107--116, 135--137,155--160. Skarnes, R.C. and Harper, M.J.K., 1972. Relationship between endotoxin-induced abortion and the synthesis of prostaglandin F. Prostaglandins, 1 : 191--203. Svenson, S.B. and Lindberg, A.A., 1978. Immunochemistry of Salmonella O-antigens: Preparations of an octasaccharide-bovine serum albumin immunogen representative of Salmonella serogroup B O-antigen and characterization of the antibody response. J. Immunol., 120: 1750--1757. Veenendaal, G.H., Woutersen-Van Nijnanten, F.M.A., Van Duin, C.Th.M. and Van Miert, A.S.J.P.A.M., 1980. Role of circulating prostaglandins in the genesis of pyrogen (endotoxin)-induced ruminal stasis in conscious goats. J. Vet. Pharmacol. Therap., 3 : 59---68. Wrathall, A.E., Wray, C., Bailey, J. and Wells, D.E., 1978. Experimentally induced bacterial endotoxaemia and abortion in pigs. Br. Vet. J., 134: 225--230.