- Email: [email protected]
Circulating concentrations of porcine relaxin in cows: Evaluation of vehicles and routes of administration
THERIOGENOLOGY
CIRCULATING CONCENTRATIONS OF PORCINE RELAKIN IN COWS: EVALUATION OF VEHICLES AND ROUTES OF ADMINISTRATION D.L. Paccamonti,lL S.T. Chang? W. Dubois,zb C.M. Barrosy M. Drost.,’ C. J. Wilcox3 and M. J. Field?” Department of Large Animal Clinical Sciences ‘School of Veterinary Medicine ‘Department of Animal Science 3Department of Dairy Science University of Florida, Gainesville, FL 32611 Received for publication: Accepted:
October 30, 1990 April 15, 1991
ABSTRACT The effectiveness of various delivery vehicles and routes of administration in providing a sustained concentration of circulating relaxin in the cow was evaluated. Porcine relaxin (1 mg) was administered to cows during estrus as follows: 1) intramuscularly in 5% beeswax in sesame oil, 2) subcutaneously in beeswax/oil, 3) intracervically in K-Y gel, 4) intramuscularly in saline, and 5) intravenously in saline. Blood samples were taken over a 48-h period after administration, and plasma concentrations of relaxin were determined by radioimmunoassay. The half-life of relaxin from intravenous administration was determined to be 16.6 min. The beeswax/oil carrier diminished the release of relaxin, with plasma concentrations consistently lower than those seen with relaxin in a saline carrier. Relaxin, administered intramuscularly in saline, reached a peak concentration of 12.6 f 1.2 n@nl (Mean f SEM) at 1.1 f 0.2 h compared with only 2.8 f 1.3 ng/ml at 1.8 f 0.5 h when administered intramuscularly in oil. In addition, use of the beeswax/oil carrier unexpectedly acted like an adjuvant, resulting in the development of antibodies against porcine relaxin. After relaxin was deposited in the cervix, systemic concentrations of the hormone were marginally detectable (0.4 f 0.2 @ml). Key words: relaxin, half-life, antibodies, cow
Acknowledgments Florida Agricultural Experiment Station Journal Series No.R-01485. ‘Present address: Department of Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge. bhesent address: Department of Math and Natural Sciences, D’ Yourville College, Buffalo, NY. Present address: Department of Pharmacology, Universidade E&dual Paulista, Sao Paulo, Brazil (supported by CNPq fellowship). *Reprint requests. Supported by grant US-1 160-86C from BARD, The United States-Israel Binational Agricultural Research and Development Fund.
JUNE 1991 VOL. 36 NO. 6
1131
THERIOGENOLOGY INTRODUCTION Relaxin is a peptide hormone that plays an important role in pregnancy and parturition (l-5). Relaxin is apparently necessary for normal parturition and delivery of young in pigs (2), mice (3,4) and rats (4.5). The periparturient cow responds to the administration of porcine relaxin by dilatation of the cervix (6) and enlargement of the pelvic opening (7). Relaxin has ken shown to successfully induce parturition alone (7,8) or in combination with corticosteroids or prostaglandins (9), and to reduce the incidence of retained placenta (9). Carrier vehicles and routes of administration of relaxin used in these studies included KY gel/cervical (7,8) and saline/intramuscular (7-9). Other studies with the cow have included relaxin administered in beeswax/intramuscularly (10.1 l), saline/intramuscularly (10) and water/subcutaneously and intracervically (12). The vehicle in which relaxin is suspended or absorbed was shown to have an effect on its potency when administered to laboratory animals. Investigation of effects on enlargement of the mouse pubic ligament found that relaxin in a suspension of beeswax/oil produced a 65fold increase in potency compared with a solution of relaxin in saline (13). The suspension also reduced the minimum effective dose to l/150 of the saline dose (14). In the guinea pig, a single injection of relaxin in a beeswax/oil carrier was found to be 10 times more effective than a single dose in saline for prolonging the relaxation of the pubic symphysis (14). If a delivery vehicle was found to have produced similar effects in the cow, a more effective use of relaxin might be attained. This study was undertaken to determine the effectiveness of different delivery vehicles and routes of administration in providing a sustained concentration of circulating relaxin in the cow. MATERIALS AND METHODS Porcine Relaxin Ovaries were harvested from mid- to late-gestation sows, transported to the laboratory in liquid nitrogen and stored at -20°C. Relaxin was purified from an acidacetone extract of the ovaries by fractionation with Sephadex G-50 chromatography, followed by CM-cellulose chromatography (15,16). Reverse-phase, high performance liquid chromatography (C4, 5 u column, RP-304)” with a linear gradient of acetonitrile showed no evidence of the presence of proteins other than relaxin in the CM-celluloseisolated product. Amino acid analysis showed CM-cellulose purified relaxin to be 88.3% relaxin by weight. This material was also biologically active since 100 ng inhibited the spontaneous contractions of the uterus from an estrogen-primed prepubertal mouse in vitro (15,17).
“Bio-Rad, Richmond, CA.
1132
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY Relaxin Assay Bovine plasma was assayed for porcine relaxin using a double antibody homologous porcine relaxin radioimmunoassay (RIA). Development of the primary antibody (UF-1M) to porcine relaxin was reported previously (18). Relaxin was iodinated according to a modification (19) of the technique described by Bolton et al. (20). Each assay tube received 200 pl of Tris hydmxymethylaminomethan (Tris)-HCl buffered saline (60 mM, pH 7.6) with 0.2% normal rabbit serum (NRS) and 0.2% bovine serum albumin (BSA). In each assay a standard curve was generated in triplicate, with porcine relaxin in amounts of 5.0, 2.5, 1.25, 0.625, 0.312,0.156,0.078, 0.039, and 0.019 rig/tube. Next 10 to 100 pl bovine plasma samples were assayed in duplicate. To each assay tube was then added 6,000 to 8,000 cpm of [luI&relaxin in 100 ~160 mM Tris-HCl buffer. Then 100 pl of rabbit antiserum to relaxin, at a 1:40,000 working dilution (which binds 35% to 45% of total cpm at zero dose) was added to each assay tube. After incubating for 22 h at 4” C, 100 pl of goat anti-rabbit gamma globulin,’ diluted 1:2.5, was added. After an additional 22 h incubation at 4”C, 1 ml of 60 mM Tris-HCl without NRS or BSA was added. The tubes were centrifuged at 3500 rpm for 35 min. The supematant was then decanted and the pellet counted. Sensitivity of the assay, determined as two standard deviations above the zero dose level, was 0.015 f 0.002 ngkube (mean f SEM). The mean slope of the standard curves for 18 assays was - 1.032 f 0.042. Estimated dose at 20,50 and 80% binding was 997 f 77,227 f 17, and 59 f 5 pgkube, respectively. Interand intra-assay coefficients of variation were 12.9 and 6.6%, respectively. Detection of Antibodies to Porcine Relaxin in the Cow To determine if cows receiving multiple injections of porcine relaxin developed antibodies to the relaxin, ammonium sulfate precipitation of gamma globulin was used. To 100 pl bovine plasma, 200 ~160 mM Tris-HCl buffer with 0.2% NRS and 0.2% BSA, and 100 pl [‘UI]relaxin (6.000-8,000 cpm) were added and incubated at 4°C for 22 h. Saturated ammonium sulfate (200 ul) was then added and allowed to incubate at 4°C for 5 to 8 h. One milliliter of 33% ammonium sulfate in 60 mM Tris-HCl buffer without NRS or BSA was added just prior to centrifugation. Centrifugation, decanting and counting were performed as in the relaxin RIA. Samples were assayed in duplicate at dilutions of l:l, 1:5, l:lO, l:lOO, and 1:lOOO; the percentage of [lYI]relaxin in precipitates was determined. Experiment 1: Relaxin Administration
in Beeswax in Oil
Five nonpregnant Angus cows in diestrus were estrus synchronized with PGFkE.
‘Calbiochem, La Jolla, CA. Qnalyse,
Upjohn Co., Kalamazoo, MI.
JUNE 1991 VOL. 36 NO. 6
1133
THERIOGENOLOGY
Seventy-two hours after the PGFz, injection, each of four cows was treated with 1 mg porcine relaxin via one of the following methods: 1) in 1 ml K-Y gelh :PBS (l:l, v:v), intracervically (i.cx.); 2) in 1 ml 5% beeswax in sesame oil, intramuscularly; 3) in 1 ml 5% beeswax/oil, subcutaneously; 4) in 1 ml saline intravenously As a control, the fifth cow was treated with 1 ml beeswax/oil carrier without relaxin, intramuscularly (Figure 1). Intracetvical placement was performed using a 55 cm infusion pipette, depositing the relaxin midcervix (-4.5 cm) while stabilizing the cervix per rectum. Blood was collected into heparinized containers via the jugular vein at -10, -5,0,2,4,8,10,15,20,25, and 30 min and at 1,2,4,8,12,24,36, and 48 h after relaxin administration. Samples were transported on ice, centrifuged and plasma was stored at -WC. The entire procedure, including estrus synchronization, was repeated at 2-wk intervals for a total of five times. The same five cows were used each time but treatments were assigned so each cow received each treatment only once (Figure 1). Experiment 2: Relaxin Administration
in Saline
The procedure described in Experiment 1 was repeated with five nonpregnant Hereford cows to compare saline with beeswax oil as a carrier for relaxin. One milligram porcine relaxin was administered: 1) in 1 ml saline, intravenously; 2) in 1 ml saline, intramuscularly; or 3) in 1 ml 5% beeswax in sesame oil, intramuscularly. Blood samples were drawn at the same intervals and treated in the same manner as in the previous experiment (Figure 1). In experiment 2, five cows were used three times each (Figure 1). Statistical Analyses Data were analyzed using the Nonlinear Model (21). Analysis was performed on post-treatment values of relaxin, corrected for assay background by subtracting 90% of the minimum concentration of relaxin (-10 and -5 min sample) observed for a given cow, receiving a given treatment, from each value recorded for that cow during that period. The analysis consisted of fitting by the method of least squares analysis of variance a model that best described the response over time for each cow receiving a given treatment, and then, using that model, solving for peak concentration of relaxin, time of the peak, and area under the curve. Analysis of variance then was performed on the estimated parameters to test for treatment differences. The linear nature of control data precluded the use of a nonlinear curve for this treatment. The responses for all other treatments were assumed to be bell shaped and skewed to the right. These data also were analyzed using the General Linear Model (21) utilizing least squares multiple regression analysis of variance. Least squares polynomial regression equations for response over time and tests of heterogeneity of regression between treatments were obtained.
hJohnson & Johnson Co., New Brunswick, NJ.
1134
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY
JUNE 1991 VOL. 35 NO. 6
1135
THERIOGENOLOGY RESULTS As time increased within 48 h post treatment, the plasma concentration of relaxin decreased, approaching the detection limit of the assay. Control cows injected with carrier alone did not have detectable relaxin in the plasma. For analysis of data, when the concentration of relaxin in a sample was found to be below the detection limit of the assay, which was 0.152 ng/ml, a value of 0.152 was assigned to that sample. Figure 2 represents the curves for the intracervical, intramuscular, and subcutaneous data from Experiment 1. Data analyzed using the General Linear Model supported the findings from the Nonlinear method. Tests of heterogeneity of regression of the fifth order polynomials indicated that highly significant differences existed between treatments (i.e., curves were not parallel). Concentrations of relaxin in cows after intracervical administration were characterized by a flattened curve reaching a peak concentration of 0.4 nglml at 2.7 h (Table 1, Figure 2). Intramuscular and subcutaneous administration of relaxin in beeswax/oil resulted in a slow release of relaxin into the systemic circulation, with a peak concentration of 1.0 and 1.8 @ml not seen until 8.3 and 10.8 h postinjection (Table 1, Figure 2). The decline in relaxin concentration also was delayed in these cases, occurring over the next 24 h and returning to baseline concentrations by 36 h post treatment. Predicted maximum value of relaxin, time of maximum value and area under the curve were tested with a conservative one-way analysis of variance; thus, the probability values listed may be smaller than those reported in Table 1. Much variation existed between individual animals in Experiment 1. This appears, in part, to reflect variations in absorption and delivery within the carriers used. To induce homogeneity of error, logs of these parameters were used in the ANOVA. The failure of relaxin administered in oil intramuscularly to attain a high concentration in plasma led to Experiment 2: a comparison of an intramuscular injection of relaxin in beeswax oil with relaxin in saline. In addition, relaxin was administered in saline intravenously, The curves for the intramuscular in oil, intramuscular in saline, and intravenous treatments from Experiment 2 are shown in Figures 3 and 4. Administration of relaxin in oil intramuscularly resulted in a peak concentration of 2.8 ng/ml 1.8 h after treatment (Table 1, Figure 3). The concentration of relaxin in plasma returned to baseline by 24 h post injection. However, as in Experiment 1, cows injected with relaxin in oil intramuscularly varied considerably in their response. The peak concentration seen with the intramuscular route using saline as the carrier is more than four times greater than with the beeswax/oil (P
1136
JUNE 1991 VOL. 35 NO. 6
Table 1.
Treatment
Peak concentrations of relaxin, times of peak concentration and areas under the curve following a single injection of 1 mg porcine relaxin.
Peak concentration (ng/ml)
Time peak concentration (h)
Area under curve
Exueriment 1 intracervical K-Y gel
0.4 f 0.2’
2.7 f 1.0”
5.5 f 2.4’
intramuscular beeswax/oil
1.0 f 0.3b
8.3 f 2.v
30.0 f 10.6”
subcutaneous beeswax/oil
1.8 f 0.5’
10.8 f 1.9’
30.5 f 6.8”
Exueriment 2 intramuscular beeswax/oil
2.8 f 1.3*
1.8 f 0.5
26.0 f 3.4’
12.6 f 1.2’
1.1 f 0.2
75.5 f 4.6’
intramuscular saline intravenous saline
__
__
71.7 f 5.3’
Figures within a column within an experiment with different superscripts differ Experiment 1: a vs c P~0.05; b vs c PcO.10; d vs e PcO.03; d vs f P~0.01. Experiment 2: g vs h PcO.005; i vs j P~O.001. The half-life of relaxin was calculated using the equation tm = [(ln 2)/b]* (22), and was determined to be 16.6 min from the intravenous injections (Figure 4). The half-life of porcine relaxin administered intravenously in the ewe was reported to be 8 min (23). Neither identity of the cow nor period significantly affected the concentration of relaxin over time (P S.10) in either experiment. The plasma concentration of progesterone (1.08 f 0.16 q/ml, Mean f SEM) in the periestrous cows at the time of relaxin administration also did not affect the concentration of relaxin in either Experiment 1 or 2 as determined by ANOVA (P ~0.10). Data from Periods 3 to 5 in Experiment 1 and Period 3 in Experiment 2, as indicated by circled treatments in Figure 1 (i.e., 245, Period 5, subcutaneously beeswax) were not included in the statistical analyses of these
JUNE 1991 VOL. 35 NO. 6
1137
0
12
Time (hours)
24
36
................Intracervically -Intramuscularly -----Subcutaneously
Relaxin
1
48
(K-Y gel) (oil) (oil)
Figure 2. Regression curves of plasma concentrations of relaxin in cows administered 1 mg relaxin in 1) K-Y gel intracervically, 2) 5% beeswax in sesame oil intramuscularly, 3) 5% beeswax in sesame oil subcutaneously.
0
4
8
12
1 mg Porcine
EXPERIMENT
6
18
24
(saline)
-intramuscularly
Time (hours)
12
(oil)
-----intramuscularly
Relaxin
Figure 3. Regression curves of plasma concentrations of relaxin in cows administered 1 mg of relaxin intramuscularly in a) 5% beeswax in sesame oil or b) saline.
0
1 mg Porcine
EXPERIMENT 2
0
2
Time &ours)
6
388 v 3103
300
0 q
270
0
cow
a
Figure 4. Regression curve of plasma concentrations of relaxin in cows administered 1 mg relaxin intravenously in 1 ml saline.
0
10
20
30
40
50
EXPERIMENT 2 1 mg Porcine Relaxin
THERIOGENOLOGY data. Samples collected at these times had aberrantly high concentrations of relaxin that are believed to be a function of endogenous antibody production influencing the relaxin assay. Numerous sets of samples were found to contain elevated concentrations of relaxin, even in the -10 and -5 min pretreatment samples. In Experiment 1, Period 3, one cow (336) had apparent pretreatment relaxin concentrations of 11 to 15 @ml. In Period 4, this same animal treated intracervically had apparent pretreatment concentrations of relaxin of 27 to 29 ng/ml and post treatment values that ranged from 19 to 32 ng/ml over the 48-h period. Concentrations of relaxin of this magnitude normally were seen only within the fist 30 min following intravenous administration. In Period 5, plasma samples from Cow 336, (this time serving as a control receiving only the beeswax/oil carrier) had an apparent pretreatment concentration of relaxin of 1 r&ml, which decreased over the next 24 h. However, at 36 h, the apparent concentration of relaxin rose to 31 nglml. This animal showed a pattern similar to Cow 839, which also had high pretreatment concentrations of 13 @ml in Period 4 and 37 to 40 r&ml in Period 5. Once an animal began to show elevated pretreatment concentrations of relaxin (11.0 rig/ml), its pretreatment concentrations remained elevated in subsequent periods. While only one animal (Cow 336) in Period 3 had high pretreatment concentrations, there were two animals (Cows 336 and 839) in Period 4, and four animals (Cows 245,336,449 and 839) in Period 5 that had elevated pretreatment concentrations of relaxin (Figure 1). This pattern of values with increased frequency of exposure to relaxin treatment suggested the production of antibodies in the cow to the injected porcine relaxin. The presence of [‘2SI]relaxin in the ammonium sulfate precipitates of plasma samples taken prior to treatment indicated the presence of antibodies to porcine relaxin. Plasma samples that, at 1:lO dilution, were able to precipitate 15% or more of the added [rzI]relaxin were considered to contain antibodies to relaxin at a concentration high enough to interfere with the relaxin assay (Figure 5). Data from periods in which cows showed antibody production were not used in the statistical analyses of Experiments 1 and 2. Once a cow developed an antibody response, she continued to show the response in subsequent periods. In addition, with repeated exposures, additional cows demonstrated the antibody response. DISCUSSION This study investigated the effect of different routes of administration of relaxin, and the use of a beeswax/oil carrier to prolong high concentrations of exogenous relaxin in the cow. The beeswax/oil carrier resulted in variation in plasma concentrations of relaxin among cows, which may reflect a lack of uniform distribution of relaxin throughout the carrier and/or differences in absorption of relaxin from the injection site. The use of saline as an alternative carrier resulted in greater similarity of plasma concentrations of relaxin among cows. The rate of release of relaxin from beeswax/oil was diminished, and the maximum concentrations attained were low in relation to those seen after administration of relaxin in saline. In addition, there was no difference between
JUNE 1991 VOL. 36 NO. 6
1141
-.
1 \ \
l/10
\
cow
\
COW
\ \
zg -_ 2
1 1/1000
\ \ \
--._
--_
‘..
\
... .
l/l
.
i
.
,
\
1
\
\
.
\
.
\
‘..
\
l/10
\
cow
.
\
\
\
\ \
.
..
f
..
--
.. *.
* ?
l/1006
-.
sulfate from plasma
l/100
\
270
-.
.* *4
-
--
-0
-.4
..
-------N
\
.. ...
636
Relaxin
cow
---_
*
Anti-porcine
Plasma Concentration
l/100
\
4224
336
Titer of Bovine Plasma
Figure 5. Percentage of El*%]relaxin precipitated by ammonium collected prior to relaxin administration.
Otl/l
20.
40.
60.
c zii looK
F 3!
loo 1
THERIOGENOLOGY the plasma concentrations of relaxin administered intramuscularly in saline or beeswax/oil at 24 h post treatment. Although potentiation is assessed by biological response, and is not solely dependent upon concentrations in the blood, it is not likely that the beeswax/oil carrier would result in potentiation or prolongation of relaxin in the cow in view of these results. We cannot explain the differences seen in the magnitude of circulating porcine relaxin (21 ng/ml) in the studies of Musah et al. (73) compared with those of our study (0.4 ng/ml after intracervical and 12.6 q/ml after intramuscular in saline administration). Caldwell et al. (25) reported a concentration of 7.5 ng/ml 2-h after intramuscular administration of 1 mg porcine relaxin in saline to beef heifers. Although samples were obtained only at 2-h intervals (23, the appearance of the curve depicting the concentration of relaxin in plasma over time was similar to that obtained in our present study. The carrier used for the intracervical deposition and the method and site of deposition are the same in this study as in those of Musah et al. (7,24). It has been demonstrated in humans that the hormone vasopressin injected into the cervical tissue will produce systemic cardiovascular effects (26). However, the authors are unaware of information available on the systemic uptake of protein hormones, other than relaxin, deposited in the cervical lumen. An additional trial conducted to compare concentrations of Circulating relaxin after mid-cervical and uterine body administration revealed similar low systemic uptake ( C.M. Barros, D.L. Paccamonti, and M.J. Fields, unpublished data). One difference between the studies of Musah et al. (724) and this study is the use of nonpregnant versus pregnant cows. The copious mucus produced during estrus which flows toward the vulva may result in the flushing of material placed in the cervix. During pregnancy, the cervical plug could serve to trap substances placed in the cervix. Also, differences in the endocrine state of the pregnant vs nonpregnant cow may have affected the uptake of relaxin from the cervix into the systemic circulation. Musah et al. (7,24) used late-term pregnant beef heifers, animals which would be under a longer period of increasing estrogen influence. The animals used in the present study were estrus induced with PGF, and were under a much shorter period of estrogenic influence. An attempt was made to control the variability of the stage of the estrous cycle by estrus synchronization, but no attempt was made to compare pregnant and nonpregnant cows. However, when four late pregnant Hereford cows received relaxin intracervically, systemic concentrations were similarly low (M. Ombionyo and M.J. Fields, unpublished data). When cows received relaxin repeatedly at 2-wk intervals, antibody production was observed. However, the production of antibody to porcine relaxin did not occur until after the animal was injected intramuscularly with relaxin in 5% beeswax/oil. In Experiment 1, cows whose plasma sample precipitated the highest percentage of [‘~I]relaxin had also been treated with a subcutaneous injection of relaxin in 5% beeswax/oil. In several instances cows developed antibodies to porcine relaxin after only two injections of relaxin, one of which was in oil intramuscularly. Plasma samples from each of those cows, when treated with relaxin in beeswax/oil intramuscularly when examined, demonstrated the presence of antibodies against porcine relaxin. Apparently, the beeswax in the sesame oil carrier acted as an adjuvant, in effect immunizing the cows against
JUNE 1991 VOL. 36 NO. 6
1143
THERIOGENOLOGY relaxin, rather than potentiating or prolonging the effects of relaxin, as seen in mice (13) and guinea pigs (14). This possibility of immunizing cows against relaxin may provide a means for studying the role of relaxin in pregnancy and parturition in this species. The clinical significance of these findings should not be overemphasized since cows would seldom receive repeated injections of relaxin, particularly at such short intervals. In light of the findings in this study, it is also highly unlikely that a beeswax/oil carrier would be used. Production of antibodies to porcine relaxin would not be expected under field conditions, particularly in view of the lack of antibody production when the carriers K-Y gel or saline were utilized. Nonetheless, a preparation of bovine relaxin could be an ideal alternative for clinical uses,such as the induction of parturition in the cow REFERENCES 1.
Downing, S.J. and Sherwood, O.D. The physiological role of relaxin in the pregnant rat. II. The influence of relaxin on uterine contractile activity. Endocrinology l&:1206-1214 (1985).
2.
Nara, B.S., Welk, EA., Rutherford, J.E., Sherwood, O.D., and Fist, N.L. Effect of relaxin on parturition and frequency of live births in pigs. J. Reprod. Fertil. &%359-365 (1982).
3.
Steinetz, B.G., Beach, V.L. and Kroc, R.L. The influence of progesterone, relaxin and estrogen on some structural and functional changes in the pre-parturient mouse. Endocrinology a:271-280 (1957).
4.
Kroc, R.L., Steinetz, B.G. and Beach, V.L. The effects of estrogens, progestagens, and relaxin in pregnant and nonpregnant laboratory rodents. Ann. N.Y. Acad. Sci. =942-980 (1959).
5.
Downing, S.J. and Sherwood, O.D. The physiological role of relaxin in the pregnant rat. I. The influence of relaxin on parturition. Endocrinology 116: 1200- 1205 (1985).
6.
Perezgrovas, R. and Anderson, L.L. Effect of porcine relaxin on cervical dilatation, pelvic area and parturition in beef heifers. Biol. Reprod. %765-776 (1982).
7.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Pelvic development as affected by relaxin in three genetically selected frame sizes of beef heifers. Biol. Reprod. %:363-369 (1986).
8.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Relaxin on induction of parturition in beef heifers. Endocrinology m:1476-1482 (1986).
9.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Induction of parturition, progesterone secretion, and delivery of placenta in beef heifers given
1144
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY relaxin with cloprostenol or dexamethasone. Biol. Reprod. 37:797-803 (1987). 10.
Hall, V.A., Moore, C.L. and Dracy, A.E. Preliminary report on dilating the cervix of a non-pregnant cow. Proc. SD. Acad. Sci. 37:53-63 (1958).
11.
Eggee, C.J. and Dracy, A.E. Histological study of effects of relaxin on the bovine cervix. J. Dairy Sci. 49:1053-1057 (1966).
12.
Graham, E.F. Some Mechanical and Hormonal Methods of Dilating the Bovine Cervix. MS. Thesis, South Dakota State College, Brookings 1952.
13.
Kliman, B. and Greep, R.O. The enhancement of relax&induced pubic ligament in mice. Endocrinology Q~586-595 (1958).
14.
Steinetz, B.G., Beach, V.L. and Rroc, R.L. The physiology of relaxin in laboratory animals. In: Lloyd, C.W. (ed) Recent Progress in the Endocrinology of Reproduction. Academic Press, New York, 1959, pp. 389-427.
15.
Fields, M.J., Fields, P.A., Castro-Hemandez, A. and Larkin, L.H. Evidence for relaxin in corpora lutea of late pregnant cows. Endocrinology m869-876 (1980).
16.
Fields, M.J., Roberts, R. and Fields, P.A. Gctadecylsilica and carboxymethyl cellulose isolation of bovine and porcine relaxin. Ann. N.Y. Acad. Sci. m:36-46 (1982).
17.
Larkin, L.H., Fields, P.A. and Pardo, R. Mouse uterus bioassay for relaxin. In: Bryant-Greenwood, G.D., Niall, H.D. and Greenwood, F.C. (eds), Relaxin. Elsevier North Holland, New York, 1981, pp. 321-330.
18.
Fields, P.A. and Fields, M.J. Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig. Biol. Reprod. 21169-1179 (1985).
19.
McMurtry, J.P., Kwok, S.C.M. and Bryant-Greenwood, G.D. Target tissues for relaxin identified in vitro with ‘ZSI-labelled porcine relaxin. J. Reprod. Fertil. $$209-216 (1978).
20.
Bolton, A.E. and Hunter, W.M. The labelling of proteins to high specific radioactivities by conjugation to a ‘2SI-containing acylating agent, B&hem. J. 133:529-539 (1973).
21.
Statistical Analysis System, SAS User’s Guide. Statistical Analysis System Institute, Inc., Car-y, NC, 1985.
22.
Baggot, J.D. Principles of pharmacokinetics. In: Principles of Drug Disposition in Domestic Animals: The Basis of Veterinary Clinical Pharmacology. W.B. Saunders Co., Philadelphia, 1977, pp. 158-162.
JUNE 1991 VOL. 36 NO. 6
growth of the
1146
THERIOGENOLOGY 23.
Bryant-Greenwood, G.D. Radioimmunoassay of relaxin. In: Abraham, G.E. (ed) A Handbook of Radioimmunoassay. Marcel Dekker. New York, 1977, pp. 243-273.
24.
Musah, A.I., Schwabe, C. and Anderson, L.L. Acute decrease in progesterone and increase in estrogen secretion caused by relaxin during late pregnancy in beef heifers. Endocrinology -120:317-324 (1987).
25.
Caldwell, R.W., Bellows, R.A., Hall, J.A. and Anthony, R.V. Administration of pig relaxin to beef heifers 4 or 7 days pre partum. J. Reprod. Fertil. 90:165-174 (1990).
26.
Rundqvist, E., Allen, D. and Larsson, G. Comparison between lysine vasopressin and a long-acting analogue (Na-triglycyl-lysine vasopressin) used as local hemostatic agents for conization. Acta Obstet. Gynecol. Stand. 67:301-305 (1988).
1146
JUNE 1991 VOL. 36 NO. 6
CIRCULATING CONCENTRATIONS OF PORCINE RELAKIN IN COWS: EVALUATION OF VEHICLES AND ROUTES OF ADMINISTRATION D.L. Paccamonti,lL S.T. Chang? W. Dubois,zb C.M. Barrosy M. Drost.,’ C. J. Wilcox3 and M. J. Field?” Department of Large Animal Clinical Sciences ‘School of Veterinary Medicine ‘Department of Animal Science 3Department of Dairy Science University of Florida, Gainesville, FL 32611 Received for publication: Accepted:
October 30, 1990 April 15, 1991
ABSTRACT The effectiveness of various delivery vehicles and routes of administration in providing a sustained concentration of circulating relaxin in the cow was evaluated. Porcine relaxin (1 mg) was administered to cows during estrus as follows: 1) intramuscularly in 5% beeswax in sesame oil, 2) subcutaneously in beeswax/oil, 3) intracervically in K-Y gel, 4) intramuscularly in saline, and 5) intravenously in saline. Blood samples were taken over a 48-h period after administration, and plasma concentrations of relaxin were determined by radioimmunoassay. The half-life of relaxin from intravenous administration was determined to be 16.6 min. The beeswax/oil carrier diminished the release of relaxin, with plasma concentrations consistently lower than those seen with relaxin in a saline carrier. Relaxin, administered intramuscularly in saline, reached a peak concentration of 12.6 f 1.2 n@nl (Mean f SEM) at 1.1 f 0.2 h compared with only 2.8 f 1.3 ng/ml at 1.8 f 0.5 h when administered intramuscularly in oil. In addition, use of the beeswax/oil carrier unexpectedly acted like an adjuvant, resulting in the development of antibodies against porcine relaxin. After relaxin was deposited in the cervix, systemic concentrations of the hormone were marginally detectable (0.4 f 0.2 @ml). Key words: relaxin, half-life, antibodies, cow
Acknowledgments Florida Agricultural Experiment Station Journal Series No.R-01485. ‘Present address: Department of Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge. bhesent address: Department of Math and Natural Sciences, D’ Yourville College, Buffalo, NY. Present address: Department of Pharmacology, Universidade E&dual Paulista, Sao Paulo, Brazil (supported by CNPq fellowship). *Reprint requests. Supported by grant US-1 160-86C from BARD, The United States-Israel Binational Agricultural Research and Development Fund.
JUNE 1991 VOL. 36 NO. 6
1131
THERIOGENOLOGY INTRODUCTION Relaxin is a peptide hormone that plays an important role in pregnancy and parturition (l-5). Relaxin is apparently necessary for normal parturition and delivery of young in pigs (2), mice (3,4) and rats (4.5). The periparturient cow responds to the administration of porcine relaxin by dilatation of the cervix (6) and enlargement of the pelvic opening (7). Relaxin has ken shown to successfully induce parturition alone (7,8) or in combination with corticosteroids or prostaglandins (9), and to reduce the incidence of retained placenta (9). Carrier vehicles and routes of administration of relaxin used in these studies included KY gel/cervical (7,8) and saline/intramuscular (7-9). Other studies with the cow have included relaxin administered in beeswax/intramuscularly (10.1 l), saline/intramuscularly (10) and water/subcutaneously and intracervically (12). The vehicle in which relaxin is suspended or absorbed was shown to have an effect on its potency when administered to laboratory animals. Investigation of effects on enlargement of the mouse pubic ligament found that relaxin in a suspension of beeswax/oil produced a 65fold increase in potency compared with a solution of relaxin in saline (13). The suspension also reduced the minimum effective dose to l/150 of the saline dose (14). In the guinea pig, a single injection of relaxin in a beeswax/oil carrier was found to be 10 times more effective than a single dose in saline for prolonging the relaxation of the pubic symphysis (14). If a delivery vehicle was found to have produced similar effects in the cow, a more effective use of relaxin might be attained. This study was undertaken to determine the effectiveness of different delivery vehicles and routes of administration in providing a sustained concentration of circulating relaxin in the cow. MATERIALS AND METHODS Porcine Relaxin Ovaries were harvested from mid- to late-gestation sows, transported to the laboratory in liquid nitrogen and stored at -20°C. Relaxin was purified from an acidacetone extract of the ovaries by fractionation with Sephadex G-50 chromatography, followed by CM-cellulose chromatography (15,16). Reverse-phase, high performance liquid chromatography (C4, 5 u column, RP-304)” with a linear gradient of acetonitrile showed no evidence of the presence of proteins other than relaxin in the CM-celluloseisolated product. Amino acid analysis showed CM-cellulose purified relaxin to be 88.3% relaxin by weight. This material was also biologically active since 100 ng inhibited the spontaneous contractions of the uterus from an estrogen-primed prepubertal mouse in vitro (15,17).
“Bio-Rad, Richmond, CA.
1132
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY Relaxin Assay Bovine plasma was assayed for porcine relaxin using a double antibody homologous porcine relaxin radioimmunoassay (RIA). Development of the primary antibody (UF-1M) to porcine relaxin was reported previously (18). Relaxin was iodinated according to a modification (19) of the technique described by Bolton et al. (20). Each assay tube received 200 pl of Tris hydmxymethylaminomethan (Tris)-HCl buffered saline (60 mM, pH 7.6) with 0.2% normal rabbit serum (NRS) and 0.2% bovine serum albumin (BSA). In each assay a standard curve was generated in triplicate, with porcine relaxin in amounts of 5.0, 2.5, 1.25, 0.625, 0.312,0.156,0.078, 0.039, and 0.019 rig/tube. Next 10 to 100 pl bovine plasma samples were assayed in duplicate. To each assay tube was then added 6,000 to 8,000 cpm of [luI&relaxin in 100 ~160 mM Tris-HCl buffer. Then 100 pl of rabbit antiserum to relaxin, at a 1:40,000 working dilution (which binds 35% to 45% of total cpm at zero dose) was added to each assay tube. After incubating for 22 h at 4” C, 100 pl of goat anti-rabbit gamma globulin,’ diluted 1:2.5, was added. After an additional 22 h incubation at 4”C, 1 ml of 60 mM Tris-HCl without NRS or BSA was added. The tubes were centrifuged at 3500 rpm for 35 min. The supematant was then decanted and the pellet counted. Sensitivity of the assay, determined as two standard deviations above the zero dose level, was 0.015 f 0.002 ngkube (mean f SEM). The mean slope of the standard curves for 18 assays was - 1.032 f 0.042. Estimated dose at 20,50 and 80% binding was 997 f 77,227 f 17, and 59 f 5 pgkube, respectively. Interand intra-assay coefficients of variation were 12.9 and 6.6%, respectively. Detection of Antibodies to Porcine Relaxin in the Cow To determine if cows receiving multiple injections of porcine relaxin developed antibodies to the relaxin, ammonium sulfate precipitation of gamma globulin was used. To 100 pl bovine plasma, 200 ~160 mM Tris-HCl buffer with 0.2% NRS and 0.2% BSA, and 100 pl [‘UI]relaxin (6.000-8,000 cpm) were added and incubated at 4°C for 22 h. Saturated ammonium sulfate (200 ul) was then added and allowed to incubate at 4°C for 5 to 8 h. One milliliter of 33% ammonium sulfate in 60 mM Tris-HCl buffer without NRS or BSA was added just prior to centrifugation. Centrifugation, decanting and counting were performed as in the relaxin RIA. Samples were assayed in duplicate at dilutions of l:l, 1:5, l:lO, l:lOO, and 1:lOOO; the percentage of [lYI]relaxin in precipitates was determined. Experiment 1: Relaxin Administration
in Beeswax in Oil
Five nonpregnant Angus cows in diestrus were estrus synchronized with PGFkE.
‘Calbiochem, La Jolla, CA. Qnalyse,
Upjohn Co., Kalamazoo, MI.
JUNE 1991 VOL. 36 NO. 6
1133
THERIOGENOLOGY
Seventy-two hours after the PGFz, injection, each of four cows was treated with 1 mg porcine relaxin via one of the following methods: 1) in 1 ml K-Y gelh :PBS (l:l, v:v), intracervically (i.cx.); 2) in 1 ml 5% beeswax in sesame oil, intramuscularly; 3) in 1 ml 5% beeswax/oil, subcutaneously; 4) in 1 ml saline intravenously As a control, the fifth cow was treated with 1 ml beeswax/oil carrier without relaxin, intramuscularly (Figure 1). Intracetvical placement was performed using a 55 cm infusion pipette, depositing the relaxin midcervix (-4.5 cm) while stabilizing the cervix per rectum. Blood was collected into heparinized containers via the jugular vein at -10, -5,0,2,4,8,10,15,20,25, and 30 min and at 1,2,4,8,12,24,36, and 48 h after relaxin administration. Samples were transported on ice, centrifuged and plasma was stored at -WC. The entire procedure, including estrus synchronization, was repeated at 2-wk intervals for a total of five times. The same five cows were used each time but treatments were assigned so each cow received each treatment only once (Figure 1). Experiment 2: Relaxin Administration
in Saline
The procedure described in Experiment 1 was repeated with five nonpregnant Hereford cows to compare saline with beeswax oil as a carrier for relaxin. One milligram porcine relaxin was administered: 1) in 1 ml saline, intravenously; 2) in 1 ml saline, intramuscularly; or 3) in 1 ml 5% beeswax in sesame oil, intramuscularly. Blood samples were drawn at the same intervals and treated in the same manner as in the previous experiment (Figure 1). In experiment 2, five cows were used three times each (Figure 1). Statistical Analyses Data were analyzed using the Nonlinear Model (21). Analysis was performed on post-treatment values of relaxin, corrected for assay background by subtracting 90% of the minimum concentration of relaxin (-10 and -5 min sample) observed for a given cow, receiving a given treatment, from each value recorded for that cow during that period. The analysis consisted of fitting by the method of least squares analysis of variance a model that best described the response over time for each cow receiving a given treatment, and then, using that model, solving for peak concentration of relaxin, time of the peak, and area under the curve. Analysis of variance then was performed on the estimated parameters to test for treatment differences. The linear nature of control data precluded the use of a nonlinear curve for this treatment. The responses for all other treatments were assumed to be bell shaped and skewed to the right. These data also were analyzed using the General Linear Model (21) utilizing least squares multiple regression analysis of variance. Least squares polynomial regression equations for response over time and tests of heterogeneity of regression between treatments were obtained.
hJohnson & Johnson Co., New Brunswick, NJ.
1134
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY
JUNE 1991 VOL. 35 NO. 6
1135
THERIOGENOLOGY RESULTS As time increased within 48 h post treatment, the plasma concentration of relaxin decreased, approaching the detection limit of the assay. Control cows injected with carrier alone did not have detectable relaxin in the plasma. For analysis of data, when the concentration of relaxin in a sample was found to be below the detection limit of the assay, which was 0.152 ng/ml, a value of 0.152 was assigned to that sample. Figure 2 represents the curves for the intracervical, intramuscular, and subcutaneous data from Experiment 1. Data analyzed using the General Linear Model supported the findings from the Nonlinear method. Tests of heterogeneity of regression of the fifth order polynomials indicated that highly significant differences existed between treatments (i.e., curves were not parallel). Concentrations of relaxin in cows after intracervical administration were characterized by a flattened curve reaching a peak concentration of 0.4 nglml at 2.7 h (Table 1, Figure 2). Intramuscular and subcutaneous administration of relaxin in beeswax/oil resulted in a slow release of relaxin into the systemic circulation, with a peak concentration of 1.0 and 1.8 @ml not seen until 8.3 and 10.8 h postinjection (Table 1, Figure 2). The decline in relaxin concentration also was delayed in these cases, occurring over the next 24 h and returning to baseline concentrations by 36 h post treatment. Predicted maximum value of relaxin, time of maximum value and area under the curve were tested with a conservative one-way analysis of variance; thus, the probability values listed may be smaller than those reported in Table 1. Much variation existed between individual animals in Experiment 1. This appears, in part, to reflect variations in absorption and delivery within the carriers used. To induce homogeneity of error, logs of these parameters were used in the ANOVA. The failure of relaxin administered in oil intramuscularly to attain a high concentration in plasma led to Experiment 2: a comparison of an intramuscular injection of relaxin in beeswax oil with relaxin in saline. In addition, relaxin was administered in saline intravenously, The curves for the intramuscular in oil, intramuscular in saline, and intravenous treatments from Experiment 2 are shown in Figures 3 and 4. Administration of relaxin in oil intramuscularly resulted in a peak concentration of 2.8 ng/ml 1.8 h after treatment (Table 1, Figure 3). The concentration of relaxin in plasma returned to baseline by 24 h post injection. However, as in Experiment 1, cows injected with relaxin in oil intramuscularly varied considerably in their response. The peak concentration seen with the intramuscular route using saline as the carrier is more than four times greater than with the beeswax/oil (P
1136
JUNE 1991 VOL. 35 NO. 6
Table 1.
Treatment
Peak concentrations of relaxin, times of peak concentration and areas under the curve following a single injection of 1 mg porcine relaxin.
Peak concentration (ng/ml)
Time peak concentration (h)
Area under curve
Exueriment 1 intracervical K-Y gel
0.4 f 0.2’
2.7 f 1.0”
5.5 f 2.4’
intramuscular beeswax/oil
1.0 f 0.3b
8.3 f 2.v
30.0 f 10.6”
subcutaneous beeswax/oil
1.8 f 0.5’
10.8 f 1.9’
30.5 f 6.8”
Exueriment 2 intramuscular beeswax/oil
2.8 f 1.3*
1.8 f 0.5
26.0 f 3.4’
12.6 f 1.2’
1.1 f 0.2
75.5 f 4.6’
intramuscular saline intravenous saline
__
__
71.7 f 5.3’
Figures within a column within an experiment with different superscripts differ Experiment 1: a vs c P~0.05; b vs c PcO.10; d vs e PcO.03; d vs f P~0.01. Experiment 2: g vs h PcO.005; i vs j P~O.001. The half-life of relaxin was calculated using the equation tm = [(ln 2)/b]* (22), and was determined to be 16.6 min from the intravenous injections (Figure 4). The half-life of porcine relaxin administered intravenously in the ewe was reported to be 8 min (23). Neither identity of the cow nor period significantly affected the concentration of relaxin over time (P S.10) in either experiment. The plasma concentration of progesterone (1.08 f 0.16 q/ml, Mean f SEM) in the periestrous cows at the time of relaxin administration also did not affect the concentration of relaxin in either Experiment 1 or 2 as determined by ANOVA (P ~0.10). Data from Periods 3 to 5 in Experiment 1 and Period 3 in Experiment 2, as indicated by circled treatments in Figure 1 (i.e., 245, Period 5, subcutaneously beeswax) were not included in the statistical analyses of these
JUNE 1991 VOL. 35 NO. 6
1137
0
12
Time (hours)
24
36
................Intracervically -Intramuscularly -----Subcutaneously
Relaxin
1
48
(K-Y gel) (oil) (oil)
Figure 2. Regression curves of plasma concentrations of relaxin in cows administered 1 mg relaxin in 1) K-Y gel intracervically, 2) 5% beeswax in sesame oil intramuscularly, 3) 5% beeswax in sesame oil subcutaneously.
0
4
8
12
1 mg Porcine
EXPERIMENT
6
18
24
(saline)
-intramuscularly
Time (hours)
12
(oil)
-----intramuscularly
Relaxin
Figure 3. Regression curves of plasma concentrations of relaxin in cows administered 1 mg of relaxin intramuscularly in a) 5% beeswax in sesame oil or b) saline.
0
1 mg Porcine
EXPERIMENT 2
0
2
Time &ours)
6
388 v 3103
300
0 q
270
0
cow
a
Figure 4. Regression curve of plasma concentrations of relaxin in cows administered 1 mg relaxin intravenously in 1 ml saline.
0
10
20
30
40
50
EXPERIMENT 2 1 mg Porcine Relaxin
THERIOGENOLOGY data. Samples collected at these times had aberrantly high concentrations of relaxin that are believed to be a function of endogenous antibody production influencing the relaxin assay. Numerous sets of samples were found to contain elevated concentrations of relaxin, even in the -10 and -5 min pretreatment samples. In Experiment 1, Period 3, one cow (336) had apparent pretreatment relaxin concentrations of 11 to 15 @ml. In Period 4, this same animal treated intracervically had apparent pretreatment concentrations of relaxin of 27 to 29 ng/ml and post treatment values that ranged from 19 to 32 ng/ml over the 48-h period. Concentrations of relaxin of this magnitude normally were seen only within the fist 30 min following intravenous administration. In Period 5, plasma samples from Cow 336, (this time serving as a control receiving only the beeswax/oil carrier) had an apparent pretreatment concentration of relaxin of 1 r&ml, which decreased over the next 24 h. However, at 36 h, the apparent concentration of relaxin rose to 31 nglml. This animal showed a pattern similar to Cow 839, which also had high pretreatment concentrations of 13 @ml in Period 4 and 37 to 40 r&ml in Period 5. Once an animal began to show elevated pretreatment concentrations of relaxin (11.0 rig/ml), its pretreatment concentrations remained elevated in subsequent periods. While only one animal (Cow 336) in Period 3 had high pretreatment concentrations, there were two animals (Cows 336 and 839) in Period 4, and four animals (Cows 245,336,449 and 839) in Period 5 that had elevated pretreatment concentrations of relaxin (Figure 1). This pattern of values with increased frequency of exposure to relaxin treatment suggested the production of antibodies in the cow to the injected porcine relaxin. The presence of [‘2SI]relaxin in the ammonium sulfate precipitates of plasma samples taken prior to treatment indicated the presence of antibodies to porcine relaxin. Plasma samples that, at 1:lO dilution, were able to precipitate 15% or more of the added [rzI]relaxin were considered to contain antibodies to relaxin at a concentration high enough to interfere with the relaxin assay (Figure 5). Data from periods in which cows showed antibody production were not used in the statistical analyses of Experiments 1 and 2. Once a cow developed an antibody response, she continued to show the response in subsequent periods. In addition, with repeated exposures, additional cows demonstrated the antibody response. DISCUSSION This study investigated the effect of different routes of administration of relaxin, and the use of a beeswax/oil carrier to prolong high concentrations of exogenous relaxin in the cow. The beeswax/oil carrier resulted in variation in plasma concentrations of relaxin among cows, which may reflect a lack of uniform distribution of relaxin throughout the carrier and/or differences in absorption of relaxin from the injection site. The use of saline as an alternative carrier resulted in greater similarity of plasma concentrations of relaxin among cows. The rate of release of relaxin from beeswax/oil was diminished, and the maximum concentrations attained were low in relation to those seen after administration of relaxin in saline. In addition, there was no difference between
JUNE 1991 VOL. 36 NO. 6
1141
-.
1 \ \
l/10
\
cow
\
COW
\ \
zg -_ 2
1 1/1000
\ \ \
--._
--_
‘..
\
... .
l/l
.
i
.
,
\
1
\
\
.
\
.
\
‘..
\
l/10
\
cow
.
\
\
\
\ \
.
..
f
..
--
.. *.
* ?
l/1006
-.
sulfate from plasma
l/100
\
270
-.
.* *4
-
--
-0
-.4
..
-------N
\
.. ...
636
Relaxin
cow
---_
*
Anti-porcine
Plasma Concentration
l/100
\
4224
336
Titer of Bovine Plasma
Figure 5. Percentage of El*%]relaxin precipitated by ammonium collected prior to relaxin administration.
Otl/l
20.
40.
60.
c zii looK
F 3!
loo 1
THERIOGENOLOGY the plasma concentrations of relaxin administered intramuscularly in saline or beeswax/oil at 24 h post treatment. Although potentiation is assessed by biological response, and is not solely dependent upon concentrations in the blood, it is not likely that the beeswax/oil carrier would result in potentiation or prolongation of relaxin in the cow in view of these results. We cannot explain the differences seen in the magnitude of circulating porcine relaxin (21 ng/ml) in the studies of Musah et al. (73) compared with those of our study (0.4 ng/ml after intracervical and 12.6 q/ml after intramuscular in saline administration). Caldwell et al. (25) reported a concentration of 7.5 ng/ml 2-h after intramuscular administration of 1 mg porcine relaxin in saline to beef heifers. Although samples were obtained only at 2-h intervals (23, the appearance of the curve depicting the concentration of relaxin in plasma over time was similar to that obtained in our present study. The carrier used for the intracervical deposition and the method and site of deposition are the same in this study as in those of Musah et al. (7,24). It has been demonstrated in humans that the hormone vasopressin injected into the cervical tissue will produce systemic cardiovascular effects (26). However, the authors are unaware of information available on the systemic uptake of protein hormones, other than relaxin, deposited in the cervical lumen. An additional trial conducted to compare concentrations of Circulating relaxin after mid-cervical and uterine body administration revealed similar low systemic uptake ( C.M. Barros, D.L. Paccamonti, and M.J. Fields, unpublished data). One difference between the studies of Musah et al. (724) and this study is the use of nonpregnant versus pregnant cows. The copious mucus produced during estrus which flows toward the vulva may result in the flushing of material placed in the cervix. During pregnancy, the cervical plug could serve to trap substances placed in the cervix. Also, differences in the endocrine state of the pregnant vs nonpregnant cow may have affected the uptake of relaxin from the cervix into the systemic circulation. Musah et al. (7,24) used late-term pregnant beef heifers, animals which would be under a longer period of increasing estrogen influence. The animals used in the present study were estrus induced with PGF, and were under a much shorter period of estrogenic influence. An attempt was made to control the variability of the stage of the estrous cycle by estrus synchronization, but no attempt was made to compare pregnant and nonpregnant cows. However, when four late pregnant Hereford cows received relaxin intracervically, systemic concentrations were similarly low (M. Ombionyo and M.J. Fields, unpublished data). When cows received relaxin repeatedly at 2-wk intervals, antibody production was observed. However, the production of antibody to porcine relaxin did not occur until after the animal was injected intramuscularly with relaxin in 5% beeswax/oil. In Experiment 1, cows whose plasma sample precipitated the highest percentage of [‘~I]relaxin had also been treated with a subcutaneous injection of relaxin in 5% beeswax/oil. In several instances cows developed antibodies to porcine relaxin after only two injections of relaxin, one of which was in oil intramuscularly. Plasma samples from each of those cows, when treated with relaxin in beeswax/oil intramuscularly when examined, demonstrated the presence of antibodies against porcine relaxin. Apparently, the beeswax in the sesame oil carrier acted as an adjuvant, in effect immunizing the cows against
JUNE 1991 VOL. 36 NO. 6
1143
THERIOGENOLOGY relaxin, rather than potentiating or prolonging the effects of relaxin, as seen in mice (13) and guinea pigs (14). This possibility of immunizing cows against relaxin may provide a means for studying the role of relaxin in pregnancy and parturition in this species. The clinical significance of these findings should not be overemphasized since cows would seldom receive repeated injections of relaxin, particularly at such short intervals. In light of the findings in this study, it is also highly unlikely that a beeswax/oil carrier would be used. Production of antibodies to porcine relaxin would not be expected under field conditions, particularly in view of the lack of antibody production when the carriers K-Y gel or saline were utilized. Nonetheless, a preparation of bovine relaxin could be an ideal alternative for clinical uses,such as the induction of parturition in the cow REFERENCES 1.
Downing, S.J. and Sherwood, O.D. The physiological role of relaxin in the pregnant rat. II. The influence of relaxin on uterine contractile activity. Endocrinology l&:1206-1214 (1985).
2.
Nara, B.S., Welk, EA., Rutherford, J.E., Sherwood, O.D., and Fist, N.L. Effect of relaxin on parturition and frequency of live births in pigs. J. Reprod. Fertil. &%359-365 (1982).
3.
Steinetz, B.G., Beach, V.L. and Kroc, R.L. The influence of progesterone, relaxin and estrogen on some structural and functional changes in the pre-parturient mouse. Endocrinology a:271-280 (1957).
4.
Kroc, R.L., Steinetz, B.G. and Beach, V.L. The effects of estrogens, progestagens, and relaxin in pregnant and nonpregnant laboratory rodents. Ann. N.Y. Acad. Sci. =942-980 (1959).
5.
Downing, S.J. and Sherwood, O.D. The physiological role of relaxin in the pregnant rat. I. The influence of relaxin on parturition. Endocrinology 116: 1200- 1205 (1985).
6.
Perezgrovas, R. and Anderson, L.L. Effect of porcine relaxin on cervical dilatation, pelvic area and parturition in beef heifers. Biol. Reprod. %765-776 (1982).
7.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Pelvic development as affected by relaxin in three genetically selected frame sizes of beef heifers. Biol. Reprod. %:363-369 (1986).
8.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Relaxin on induction of parturition in beef heifers. Endocrinology m:1476-1482 (1986).
9.
Musah, A.I., Schwabe, C., Willham, R.L. and Anderson, L.L. Induction of parturition, progesterone secretion, and delivery of placenta in beef heifers given
1144
JUNE 1991 VOL. 36 NO. 6
THERIOGENOLOGY relaxin with cloprostenol or dexamethasone. Biol. Reprod. 37:797-803 (1987). 10.
Hall, V.A., Moore, C.L. and Dracy, A.E. Preliminary report on dilating the cervix of a non-pregnant cow. Proc. SD. Acad. Sci. 37:53-63 (1958).
11.
Eggee, C.J. and Dracy, A.E. Histological study of effects of relaxin on the bovine cervix. J. Dairy Sci. 49:1053-1057 (1966).
12.
Graham, E.F. Some Mechanical and Hormonal Methods of Dilating the Bovine Cervix. MS. Thesis, South Dakota State College, Brookings 1952.
13.
Kliman, B. and Greep, R.O. The enhancement of relax&induced pubic ligament in mice. Endocrinology Q~586-595 (1958).
14.
Steinetz, B.G., Beach, V.L. and Rroc, R.L. The physiology of relaxin in laboratory animals. In: Lloyd, C.W. (ed) Recent Progress in the Endocrinology of Reproduction. Academic Press, New York, 1959, pp. 389-427.
15.
Fields, M.J., Fields, P.A., Castro-Hemandez, A. and Larkin, L.H. Evidence for relaxin in corpora lutea of late pregnant cows. Endocrinology m869-876 (1980).
16.
Fields, M.J., Roberts, R. and Fields, P.A. Gctadecylsilica and carboxymethyl cellulose isolation of bovine and porcine relaxin. Ann. N.Y. Acad. Sci. m:36-46 (1982).
17.
Larkin, L.H., Fields, P.A. and Pardo, R. Mouse uterus bioassay for relaxin. In: Bryant-Greenwood, G.D., Niall, H.D. and Greenwood, F.C. (eds), Relaxin. Elsevier North Holland, New York, 1981, pp. 321-330.
18.
Fields, P.A. and Fields, M.J. Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig. Biol. Reprod. 21169-1179 (1985).
19.
McMurtry, J.P., Kwok, S.C.M. and Bryant-Greenwood, G.D. Target tissues for relaxin identified in vitro with ‘ZSI-labelled porcine relaxin. J. Reprod. Fertil. $$209-216 (1978).
20.
Bolton, A.E. and Hunter, W.M. The labelling of proteins to high specific radioactivities by conjugation to a ‘2SI-containing acylating agent, B&hem. J. 133:529-539 (1973).
21.
Statistical Analysis System, SAS User’s Guide. Statistical Analysis System Institute, Inc., Car-y, NC, 1985.
22.
Baggot, J.D. Principles of pharmacokinetics. In: Principles of Drug Disposition in Domestic Animals: The Basis of Veterinary Clinical Pharmacology. W.B. Saunders Co., Philadelphia, 1977, pp. 158-162.
JUNE 1991 VOL. 36 NO. 6
growth of the
1146
THERIOGENOLOGY 23.
Bryant-Greenwood, G.D. Radioimmunoassay of relaxin. In: Abraham, G.E. (ed) A Handbook of Radioimmunoassay. Marcel Dekker. New York, 1977, pp. 243-273.
24.
Musah, A.I., Schwabe, C. and Anderson, L.L. Acute decrease in progesterone and increase in estrogen secretion caused by relaxin during late pregnancy in beef heifers. Endocrinology -120:317-324 (1987).
25.
Caldwell, R.W., Bellows, R.A., Hall, J.A. and Anthony, R.V. Administration of pig relaxin to beef heifers 4 or 7 days pre partum. J. Reprod. Fertil. 90:165-174 (1990).
26.
Rundqvist, E., Allen, D. and Larsson, G. Comparison between lysine vasopressin and a long-acting analogue (Na-triglycyl-lysine vasopressin) used as local hemostatic agents for conization. Acta Obstet. Gynecol. Stand. 67:301-305 (1988).
1146
JUNE 1991 VOL. 36 NO. 6