Veterinary Immunology and Immunopathology, 25 (1990) 235-247 Elsewer Scmnce Pubhshers B V, Amsterdam - - Prmted m The Netherlands

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Effects of E x o g e n o u s Oestradiol on the N u m b e r and Functional Capacity of Circulating Mononuclear and P o l y m o r p h o n u c l e a r L e u k o c y t e s in the S o w U MAGNUSSON and S EINARSSON

Department of Obstetrics and Gynaecology, Box 7039, College of Vetertnary Medicine, Swedish Unwerslty of Agrwultural Sciences, S-750 07 Uppsala (Sweden) (Accepted 7 December 1989)

ABSTRACT Magnusson, U and Emarsson, S, 1990 Effects of exogenous oestradml on the number and functmnal capacity of circulating mononuclear and polymorphonuclear leukocytes m the sow Vet Immunol Immunopathol , 25 235-247 The effects of exogenous oestradlol-17fl on blood leukocytes were studmd m four ovarlectomlzed gilts The gilts were rejected with oestradml benzoate d~ssolved m arach~dm oil durmg the test pemod and 6 weeks later, during the control pemod, with arachldlc oll alone During both periods, the ~mmune status of the blood was momtored by determining the total numbers of leukocytes, lymphocytes and neutrophlls and the percentage of lmmunoglobuhn (Ig)-bearmg mononuclear cells The serum level of Ig, the number of circulating monocytes with phagocytic activity and the phagocytic functmn of circulating polymorphonuclear leukocytes, estimated by means of chemllummescence, were also determined Durmg the test permd, the total number of lymphocytes and the proportmn of Ig-bearmg mononuclear cells m blood decreased s~gmficantly (P < 0 05 and P < 0 01 respectively) after treatment w~th oestradml benzoate For the polymorphonuclear cells, the time needed to reach the peak value of chemiluminescence was s~gnlflcantly (P < 0 01 ) prolonged after treatment, the phagocytic capacity of polymorphonuclearleukocytes was increased (P < 0 01 ) concurrently None of the changes recorded during the test permd occurred during the control permd The results show that oestradml-17fl depresses some components of the vascular compartment of the porcme ~mmune system, and stimulates other components

INTRODUCTION

In previous studms of the porcine immune system during the pempartum period (Magnusson and Fossum, 1988, 1990 ), we have found considerable variations m the functional capacity and number of circulating ~mmunocompetent cells Statistical relationships between some of the immune parameters mea-

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sured and the plasma levels of oestrogens and cortlsol have also been demonstrated On the basis of these relatmnshlps, we suggested a negative influence of oestrogens on the number of monocytes with phagocytm actlwty and the cellular response to mltogen, but a positive effect on the proportmn of Ig-bearmg mononuclear cells m the clrculatmn However, m these descmptlve studxes, xt has not been possible to determine to what extent other factors operating around the time of partuntmn, other than oestrogens, contribute to or d~mm~sh the observed varmtmns Against this background the present experimental study was undertaken to investigate the effect of oestrogens only, at concentratmns s~mflar to those at partuntmn, on certain immune functmns m the p~g Ovanectomlzed gilts were rejected with oestradml benzoate during a test permd and with placebo during a control permd During both periods, the immune status was momtored by leukocyte, lymphocyte and neutrophfl counts and by determining the proportmn of Ig-bearmg cells The serum level of Ig, the number of monocytes with phagocytm actlwty and the phagocytm functmn of the polymorphonuclear leukocytes (PMNLs) were also determined during both permds Finally, after the test and control permds, the possible stress caused by the bleeding procedure was evaluated The pigs were fitted with a permanent cannula and the cortlsol concentratmns m blood samples collected by vempuncture and cannulatmn were then compared MATERIALSAND METHODS

Animals Four crossbred (Swedish Landrace × Swedish Yorkshire ) gilts, weighing between 111 and 125 kg, were brought to the chmc from commercml farms The gilts were housed m individual pens at the Department of Obstetrics and Gynaecology throughout the study, and were fed according to the Swedish stock standard After going through at least one oestrous cycle, the gilts were anaesthetized by pentothal sodmm (5% 1 v ) and ovarmctomlzed under strictly aseptm conditions Three of the gilts were cannulated according to the method of Rodnguez and Kunavongknt (1983), by inserting a permanent catheter m the jugular veto No chmcal signs of disease were observed m the pigs during the study

Experimental design and blood samphng The study was divided into one test period and one control period During the test period, the gilts were given a single 1 m rejection of 5 mg 17fl-oestradlol benzoate m 2 ml arachldm oll (Ovex B Vet, AB Leo, Malmo, Sweden) Blood samples were drawn on two days before the mjectmn (referred to hereafter collectively as the pretreatment observation), and 1, 2, 3, 5, 8 and 11 days after the rejection

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During the control period, which started 6 weeks after the end of the test period, the gilts were injected 1 m. with 2 ml arachlchc oil (Apoteksbolaget AB, Stockholm, Sweden) as a placebo The scheme for blood sampling was the same as during the test period, except that the samplings on days 5 and 11 were omitted Blood was collected 4-5 h after the morning feeding from the jugular vein m vacutamer tubes with appropriate additives, i.e. EDTA for cell counts and heparm for other essays After the control period, blood samples were taken from the three cannulated pigs 4-5 h after the morning feeding on 6 consecutive days In these blood samples, only the plasma level of cortisol was analysed.

Hormone assays Heparmlzed blood samples for the hormone analyses were immediately centrifuged and the plasma was saved and stored at - 20 ° C until assay. The plasma concentrations of oestradlol-17fl and cortisol were determined by radiolmmunoassays according to the methods validated for pigs by Kunavongkrlt et al (1983) and Nyberg et al (1988) respectively The hormone assays were carried out at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences Blood cell counts The total number of white blood cells (WBC) was counted in a celloscope, and differential WBC counts were carried out using blood smears stained with Glemsa and May-Grunwald solutions The blood analyses were performed according to the standard procedures at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences Isolation of mononuclear cells Mononuclear cells were isolated from heparlmzed blood by centrlfugatlon on Flcoll-Paque (Pharmacla Fine Chemicals, Uppsala, Sweden) according to the method of Boyum (1968), as previously described for porcine blood (Magnusson and Fossum, 1988) Detection of Ig-bearmg cells Ig-bearlng (Ig + ) mononuclear cells were detected by direct lmmunofluorescence using fluoresceln lsothlocyanate (FITC)-conjugated F (ab') 2 fragments purified from hypenmmune rabbit antiserum to porcine Ig (antl-IgFITC) To remove cytophlhc antibodies from the cell surfaces, 3 000 000 mononuclear cells were premcubated in PBS without serum for 30 mm at 37 ° C (Watson, 1976) The cells were then suspended in 50/tl anti-Ig-FITC and incubated for 30 mln at + 4 ° C. Following this treatment, the cells were washed

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three times m P B S with 0 01% human serum albumin ( H S A ) and the proportmn of anti Ig-FITC-pos~t~ve cells was lmmedmtely determined by counting at least 200 mononuclear cells under a fluorescence microscope (Labophot, Nikon, J a p a n )

Serum concentratmn o[ ~mmunoglobuhns The concentration of Ig in serum samples was determined by radial ~mmundfffusmn (RID) In brief, 5 ~1 serum, diluted I 10 m PBS, was put into wells m a 1% agar gel containing 40% rabbit antiserum to pig Ig The RID plates were incubated for 24 h at 37°C and the diameters of the preclpltatmn rings were measured The diameters were read against a standard curve for 4, 2, 1, 0 5, 0 25, 0 125 and 0 0625 mg purified porcine Ig/ml

Ident~f~catmn of monocytes w~th phagocytic actw~ty Monocytes with phagocytic activity (active monocytes) were identified as described earlier (Magnusson and Fossum, 1988) In brief, 4 000 000 mononuclear cells were suspended in 2 ml R P M I 1640 medium, wlth the addition of 2 m M L-glutamme, 0 05 mg/ml gentamycm and 2% Ultroser G (LKB, Bromma, Sweden) The suspension of mononuclear cells was incubated with 5 #1 of 1 96~m fluorescent latex beads (Polysclence I n c , Warrmgton, U S A ) m a slowly rotating mmisorb test tube ( N U N C , D e n m a r k ) for 24 h at 37°C After add1tmn of 75 ,ul 1% Triton X-100 m P B S and 50/~1 propldmm mdlde (1 m g / m l P B S ) , at least 200 cells from each sample were counted m the fluorescence mmroscope Cells with three or more ingested beads were considered as active monocytes The total number of active monocytes/1 of blood was calculated as the proportion of active monocytes multiplied by the total number of mononuclear cells

Measurement of the phagocytic function of polymorphonuclear leukocytes The phagocytic function of P M N L s was assessed by measuring the chemlluminescence (CL) following phagocytosls of opsonized zymosan particles (Zymosan A, Sigma Chemical, St Louis, M O ) The chemiluminescence reactlon, a result of the metabolic burst a s s o o a t e d mainly with the formation of reactive oxygen metabohtes during the phagocytic process, was enhanced by lumlnol (Sigma) and analysed in a L K B 1250 L u m m o m e t e r Chemiluminescence for whole blood was determined according to the method of Tona-Oka et al (1983) and in accordance with the protocol for the L K B 1250 Luminometer, with slight modifications for the porcine system In brmf, for opsomzatlon, 50 mg of zymosan particles were Incubated in a 4-ml solutmn of 75% pooled pig serum in P B S for 30 min at 37°C, washed three times m P B S at 400 ×g and finally resuspended m 4 ml P B S To prepare the sample, the following reagents were added in order to a glass cuvette. 200 #1 0 1 m M lummol, 200 ttl opsonIzed zymosan particles and P B S to a final volume of 900

EFFECT OF OESTRADIOL ON CIRCULATING LEUKOCYTES IN THE SOW

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/~l The background CL was determmedby omitting the zymosan pamcles The samples with zymosan were set up tn tnphcate and the samples without zymosan m duphcate. The assay was started 90 m m after blood samphng by adding 100 pl of hepanmzed blood per cuvette and lmmedmtely placing the cuvettes m the lummometer. The hght emtssmn (mV) from the CL was recorded at intervals of 166 s until the background level of CL was reached after 53 mm The time needed for the PMNLs to reach their peak CL value was recorded (time to peak) The phagocytm capacity per thousand PMNLs was determined by calculating the integral of the CL over 53 mm dlwded by the number of PMNLs added to the assay.

Statistical analysts Stat~stmal analys~s of the data was performed using the GLM procedure of the Statistical Analysis System (SAS, 1985) For each pemod, the pretreatment observatmns were grouped m blocks The variations during each pemod were then analysed separately by applying a statistical model including the effect of ammal (4) and occasion of samphng (7 in the test period and 5 m the control permd respectively ) to the data A total of 28 observatmns during the test permd and 20 during the control permd were available for analysis. However, only 25 and 18 observatmns respectively were complete, 1 e no values were missing All the values presented m the results are least-squares means and standard errors of least-squares means obtained from the analysis of varmnce Correlatmns between the trmts m the test permd were calculated RESULTS

Hormone levels The plasma level of oestradlol-17fl increased dramatmally after rejection of oestradlol benzoate, from 14 pmol/1 before treatment to 2493 pmol/1 on day 1 As shown m Fig 1, the level of oestrachol-17flthen decreased and was low again on day 8 (27 pmol/1). During the control period, the plasma level of oestradiol-17fl remained between 23 2 and 47 8 pmol/1. The plasma level of cortlsol also increased slgmficantly after rejection of oestra&ol benzoate, from 34 1 nmol/1 before treatment to 51 2 nmol/1 on day 3 (P < 0 01 ) From day 3, the concentratton of cortlsol decreased and reached a low level on day 8 (27.0 nmol/1). During the control pemod, the least-squares mean for the plasma level of cortisol fluctuated without slgmficance between 19 0 and 31.8 nmol/1 (Fig. 2) When the pigs were cannulated, the least-squares mean for the plasma level of cortlsol varmd non-slgmficantly between 26.7 and 34 4 nmol/1

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Fig 2 Level of cortlsol m plasma from ovarmctom]zed gilts during test ( H ) (O---O) periods (Least-squares mean and s e m ) P T =pretreatment

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Number o[ blood cells The total number of WBCs decreased slgmficantly (P < 0 01 ) after rejection of oestradlol-17fl, from 16 5 × 109 cells/l blood before treatment to 13 5 × 109 cells/1 on day 3, but returned to the pretreatment value (16 3 × 109 cells/l) on day 5 The total number of WBCs then decreased ( P < 0 05) again to 13 5 × 109 cells/l on day 8 {Fig 3) During the control period, the least-squares mean for the total number of WBCs fluctuated between 14 2 and 15 0 × 109 cells/1 without slgmficance The total number of mononuclear cells was, according to the differential counts, strongly correlated to the total lymphocyte count (r-- 0 98, P < 0 0001 ), hence only the total number of lymphocytes is presented The total number of lymphocytes decreased slgmficantly (P < 0 05 ) after the injection of oestradlol benzoate, from 10 7 × 109 cells/1 before treatment to 8 8 × 109 cells/1 on day 3 (F~g 3) During the control period, the least-squares mean for the number of lym-

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Fig 3 TotalnumberofWBCsmbloodfromovarmctomlzedglltsdurmgtest ( O ~ ) andcontrol ( © - - - O ) permds (upper panel), number of circulating lymphocytes during test ( H ) and control ( O - - - O ) permds (middle panel), and number of circulating neutrophlls durmg test ( D - - ~ ) and control ( [ ] - - - D ) permds (lower panel) (Least-squares mean and s e m ) PT = pretreatment

phocytes varied non-significantly from 8 7 to 9 6 × 109 cells/1 The total number of neutrophlls varied non-slgmficantly at the begmnmg of the test permd (Fig 3), but increased slgmficantly ( P < 0 01) from 3 8>< 109 cells/1 blood on day 3 to 6 2 )< 109 cells/1 blood on day 5 The total number of neutroph,ls then fell to ~ts original level During the control period, the least-squares mean for the number of neutrophlls fluctuated non-significantly between 4 2 and 4 7)< 109 cells/1 (Fig 3 )

Proportion of Ig + mononuclear leukocytes The proportion of Ig + mononuclear cells decreased from 10 2% on the day after the rejection of oestradlol benzoate to 5 8% on the next day (P < 0 01 ) (Fig 4) The proport,on of Ig + cells subsequently increased, reaching 9 0% on day 11 During the control period, the least-squares mean for the proportion of Ig + mononuclear cells vaned non-slgmficantly from 10 2 to 13 7%

Serum levels of ~mmunoglobuhns The least-squares mean for the Ig level in serum vaned non-slgmficantly between 13 5 and 26 1 g/1 during the test period

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Fig 4 Proportmn ( % ) of Ig + cells m the mononuclear cell populatmn m blood obtamed from ovamectomlzed gilts during test ( • - - • ) and control ( • - - - • ) permds (Least-squares mean and s e m ) PT=pretreatment

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Number of actwe monocytes Durmg the test period, changes m the number of active monocytes were not slgmficant (F:g 5) However, the number of active monocytes increased from 0 04 × 109 cells/1 before treatment to 0 18 × 109 cells/12 days after the rejection of oestradlol benzoate, and then decreased to the pretreatment level During the control period, the largest number of active monocytes was measured 3 days after the placebo treatment (0 19 X 109 cells/l), this number being s:gmficantly (P < 0 05) higher than the pretreatment number (0 06 X 109 cells/

1) The phagocytic functmn of polymorphonuclear leukocytes The time needed to reach the peak value of chemiluminescence (time to peak) was slgmficantly (P < 0 0001) prolonged after mjectmn of oestradlol benzoate, : e 626 s before treatment compared w~th 1046 s on day I (Fig. 6) After day 1, the time to peak value decreased gradually, the reduction being sxgmficant ( P < 0 001 ) from day 2 to day 3 At the end of the test permd there

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F:g 6 Time to peak values recorded m the whole-bloodchemiluminescenceassay for blood leukocytes from ovanectom:zed gilts during test ( H ) and control (O---O) periods (upper panel), and phagocytmcapac:ty per thousand PMNLs during test (O---O) and control (O--O ) permds {lowerpanel) (Least-squaresmean and s e m ) PT=pretreatment was no s,gmficant difference m t~me to peak compared to the pretreatment value During the control permd, there was no s:gmficant alteration m the t~me to peak value The phagocytic capacity per t h o u s a n d P M N L s ( c a p a c l t y / P M N L ) mcreased slgmficantly ( P < 0 01) during the test permd, from 13 2 m V × s / cell× 103 before t r e a t m e n t to 17 7 m V × s / c e l l × 103 on day 1 (Fig. 6) No slgmficant d,fferences were detected between the pretreatment value and the values on days 5 and 11 During the control period, there was no slgmficant change m capac,ty/ PMNL The time to peak was positively correlated (r--0 43 ) to the phagocytm cap a c ~ t y / P M N L ( P < 0.05)

Relatmnsh~p between hormone levels and ~mmune parameters Dur,ng the test period, the time to peak value m the CL assay was positively correlated to the levels of both oestradlol- 17fl (r = 0 56; P < 0 001 ) and cortlsol (r= 0 35; P < 0 05) There were no other s,gmficant correlations between hormone levels and :mmune parameters

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DISCUSSION The results from the present experimental study m castrated gilts showed that exogenous oestradlol-17fl reduces the number of circulating lymphocytes and especially the proportmn of Ig-bearmg mononuclear cells. Further, enhanced phagocytic function of polymorphonuclear leukocytes was mdmated during the period of influence of oestradlol-17fl No similar effects were observed after treatment with the drug vehicle The data were stat~stmally analysed within the test and the control periods respectively A case-control study w~th self-pamng was considered inappropriate since the study was d~wded into one test and one control period The peak value of plasma oestradlol-17fl concentration found during the test period (2448 pmol/1) was about twine that m pooled plasma collected from sows just before parturition (Robertson and King, 1974) Hence, the plasma levels of oestradlol-17fl vaned within the physlologmal range m our study The cortlsol concentrations m the blood samples collected through the permanent catheters were at the same level as those m the samples collected by vempuncture A similar finding has been reported by Bald1 et al (1989) Thus, the samphng procedure used during the test and control permds, ~ e vempuncture of pigs housed m mdlwdual pens, did not cause a stress-reduced elevatmn of the plasma cortlsol concentratmn Despite the fact that the pretreatment cort~sol levels m plasma were somewhat h~gher m the test period than m the control permd, there was a slgmficant elevatmn of the cort~sol level up to day 3 of the test period Th~s elevatmn of the cortlsol level was probably reduced by the oestrous behawour that followed mjectmn of oestradml benzoate The existence of such a relatmnshlp between oestrogens, oestrous behawour and cortlsol m the pig has been suggested by Becker et al (1985) Therefore, effects of cortlsol must be considered when the observatmns around day 3 are interpreted The slgmficant decrease m the number of WBCs from the pretreatment value to day 3 of the test permd was mainly due to a decrease m the number of lymphocytes This decrease m lymphocyte number was similar to that observed before partuntmn m the pig (Magnusson and Fossum, 1988), when h~gh plasma levels of oestrogen occur concomitantly with a decreasing number of lymphocytes In consequence, it ~s reasonable to assert that an increasing oestrogen level m plasma does cause a decrease m the number of blood lymphocytes m the sow In addltmn, repeated treatment of castrated cows (Saad and Astrom, 1988) with oestradml benzoate also gives rise to a slgmficant decrease m the proport|on of lymphocytes among WBCs The slgmficant increase m the number of WBCs observed 5 days after the mjectmn of oestradml benzoate was due to an increase m the number of neutrophtls Thxs increase was, however, most probably due to an increased plasma

EFFECT OF OESTRADIOL ON CIRCULATING LEUKOCYTES IN T H E SOW

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level of cortlsol on day 3, as cortlsol is known to induce neutrophlha (Boggs et al, 1964) The most prominent change m the leukocyte population was the drop in the proportion of Ig-bearlng mononuclear cells after injection of oestradml benzoate A statistical correlation between the proportion ofIg + cells and the level of oestrogen observed during the perlpartum period (Magnusson and Fossum 1990) was not found m the present study. However, the graphs presenting the oestrogen levels m blood and the proportion of Ig + cells in the present experimental study show a strong similarity with those describing the peripartum period" high levels of oestrogens are followed by a drop in the proportmn of Ig + cells Hence, oestrogens most hkely reduce the proportion of Ig + cells m the circulation in the pig The concentration of Ig in serum did not vary significantly during the test permd. This finding indicates that the decrease in the serum concentration of gammaglobuhns before parturition in the p~g (Martmsson, 1972, Magnusson and Fossum, 1988) cannot be explained by direct effects of increasing plasma levels of oestrogens at this time The variations m the number of monocytes with phagocytic capacity were similar during the two periods: the number of active monocytes peaked some days after the injection, although the increase was not sigmficant during the test period This similarity could result either from an effect of the placebo (arachidic o11) itself, or from a requirement for a longer time of exposure to oestrogens in order to produce an effect on the number of acttve monocytes For this reason, it is not possible either to support or to reject the suggestmn that the increase in the number of monocytes after parturition in the pig (Magnusson and Fossum, 1988), which has also been observed m women (Buchan et al, 1985), is induced by oestrogens As regards P M N L function, the results from the whole-blood CL assay show that the time to reach the peak CL value m the assay (time to peak) is prolonged under the influence of oestradlol- 17fl The prolonged time to peak could not be explained by altered kinetic conditions for phagocytosis, 1 e a change m the ratio between PMNLs and zymosan particles, since the number of neutrophfls (the overall dominant cell type among the PMNLs of pigs) was unchanged when the time to peak was prolonged (Fig 3) A longer time to peak has previously been reported to reflect a decreased opsonic actlwty m blood (Ton-Oka et al, 1983) Even so, this interpretatmn of the longer time to peak could not be appropriate in the present study since the same preparatmn of pooled p~g serum was used for opsonization throughout the study Instead, the prolonged time to peak may indicate an extended rather than a retarded phagocytic process, since the prolongation observed was concomitant with an increasing phagocytic capacity per thousand PMNLs (capaclty/PMNL) (Fig 6) This interpretation of the prolonged time to peak is supported by the positive correlation between the time to peak and the capacity/PMNL The slg-

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mficant alterations in c a p a c i t y / P M N L generally followed the level of oestradiol-17fl during the test period Hence, these results strengthen the previous suggestion t h a t the increase m the number of P M N L s in the sow at parturition reflects increased oestrogen-mduced activity of th innate immune system (Magnusson and Fossum, 1990) These speculations are further supported by observations in ovanectomlzed mares inoculated m utero with Streptococcus zooep~dem~cus (Washburn et a l , 1982) In these mares, the phagocytic ability of blood P M N L s was enhanced by t r e a t m e n t with oestradml CONCLUSION In this experimental study, the statistical relatmnshlp between oestrogens and some immunological parameters around parturition t h a t has been described previously (Magnusson and Fossum, 1988, 1990) could not be verified The differences m results, which are discussed above, are probably due to the facts that factors other t h a n oestrogen also influence the immune system around parturitmn and t h a t there are temporal differences between high oestrogen levels and measurable effects on the immune system The results of the present study show t h a t oestrogens do cause a reductmn m the number of circulating lymphocytes, and particularly m the proportion of Ig + cells, m the pig The functional studies of the leukocytes suggest t h a t oestrogens enhance the phagocytic function of the o r c u l a t m g P M N L s W h e t h e r these effects of oestrogens on the vascular compartment of the porcine ~mmune system also occur in other compartments, e g the m a m m a r y gland, remains to be studied ACKNOWLEDGEMENTS The authors wish to t h a n k Professor Bror Moreyn and Dr Anna Plym-Forshell for their valuable advice, AgrD Nlls Lundehelm for his help with the statistical analyses, and Ms Catharlna Falkenberg, Ms K e r s t m Llndblad and Ms Helena Andersson for providing techmcal assistance Th~s lnvestlgatmn was supported by the Swedish Council for Forestry and Agricultural Research

REFERENCES Baldy,A, Verga,M, Maffn, M, Canah, E, Chlarawgho,D and Ferran, C, 1989 Effectsof blood samplingprocedures, groupingand adrenal st~mulatlonon stress responses m the growingpig Reprod Nutr Dev ,29 95-103 Becker B A, Ford, J J, Chnstenson, R K, Manak, R C, Hahn, G L and DeShazer, J A, 1985 Cortlsol response of gilts m tether stalls J Atom Scl, 60 264-270 Boggs, D R, Athens, J W, Cartwnght, G E and Wmtrobe, M M, 1964 The effect of adrenal

EFFECT OF OESTRADIOL ON CIRCULATING LEUKOCYTES IN THE SOW

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Effects of exogenous oestradiol on the number and functional capacity of circulating mononuclear and polymorphonuclear leukocytes in the sow.

The effects of exogenous oestradiol-17 beta on blood leukocytes were studied in four ovariectomized gilts. The gilts were injected with oestradiol ben...
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