Continuous infusion of relaxin on periparturient progesterone, oxytocin and relaxin plasma concentrations and time of parturition in beef heifers

SCIENCE Animal Reproduction Science 46 ( 1997) 15-25

Continuous infusion of relaxin on periparturient progesterone, oxytocin and relaxin plasma concentrations and time of parturition in beef heifers K.H. Smith a,1,A.I. Musah a*2,S.-J. Cho a, C. Schwabe b, L.L. Anderson a3* aDepartment

of Animal Science, Iowa State University, Ames IA 5001 l-3150.

b Department of Biochemistry and Moleculur Biology, Medical 29425-221

USA

University of South Carolina,

Charleston SC

I, USA

Accepted 9 September 1996

Abstract These studies were designed to determine whether continuous i.v. infusion of increasing dosages of porcine relaxin during late pregnancy in beef heifers would influence circulating blood concentrations of relaxin, progesterone and oxytocin, and time of onset of parturition. Beef heifers were bred by artificial insemination and, on Day 277, fitted with indwelling jugular cannulas for hormone infusion and blood sampling from Day 277 to Day 286. Intravenous infusion of purified porcine relaxin (pRLX, 3OOOUmg-‘) was started in heifers (n = 8) at increasing dosages (2OOU h-’ on Days 277 and 278, 300U h- ’ on Days 279 and 280, 5OOUh-’ on Day 281, 600U h- ’ on Day 282, and 700 Uh- ’ on Days 283-286). Phosphate-buffered saline (PBS, lOm1 h- ‘) was infused during these same times to control animals (n = 6). Relaxin treatment steadily increased the circulating plasma concentration of immunoreactive relaxin to more than 120ngml-’ compared with less than O.Sngml-’ in PBS-treated controls. Relaxin infusion in increasing dosages over the treatment time was associated with a significant decrease (P < 0.01) in plasma progesterone concentration compared with the PBS controls. The rate of change in progesterone levels between pRLX and PBS groups differed (P < 0.05) at 300U h-‘, 600U h- ’

* Corresponding author. ’Present address: MSD AGVET Division, Merck and Co., Inc., 4620 West Roundup Road, Bismark, ND 58501, USA. ’ Present address: Department of Biological Sciences, College of Science and Technology, St. Cloud State University, St. Cloud, MN 56301, USA. 0378-4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01616-S

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K.H. Smith et al./Animal Reproduction Science 46 (1997) 15-25

and 700U h-t dosage intervals, respectively. Plasma levels of oxytocin at 4 h intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS. Mean concentrations of oxytocin in PBS control heifers peaked at 0.95 pgml- ’during the corresponding infusion of 700 U h- ’ pRLX, which peaked at 0.77 pg ml- ’. Although continuous i.v. infusion of relaxin resulted in a decrease in circulating blood levels of progesterone, it did not significantly reduce the interval between the beginning of pRLX treatment and parturition compared with the PBS-infused control heifers. These results indicate that continuous i.v. infusion

of high levels of porcine relaxin resulted in a decrease in progesterone secretion in late pregnant beef heifers.0 1997 Elsevier Science B.V. Keywords:

Relaxin; Progesterone;Oxytocin; Pregnancy; Heifer; Parturition

1. Introduction Relaxin @LX), a peptide hormone with partial structural homology to insulin, is produced in abundant quantities by corpora lutea on the ovaries during late pregnancy in the pig (Anderson et al., 1973; Haley et al., 1987). Relaxin induces marked cervical dilatation, pelvic relaxation, separation of the pubic symphysis, and interpubic ligament formation in several mammalian species (Anderson, 1987; Sherwood, 1994). Although the pig is a rich source of this hormone, only a single copy of the RLX-like gene with a short open reading frame corresponding to part of the relaxin A-chain is present in sheep (Roche et al., 1993). There is little evidence supporting the biosynthesis of a RLX-like hormone in cattle (Hartung et al., 1995). Ruminants do respond to i.v. or i.m. injection of porcine relaxin, however, by hormone-specific quiescence of myometrial activity (Porter et al., 1981) in sheep and cattle, and by cervical softening and dilatation in cattle (Musah et al., 1986, 1987a,b) and sheep (Gazal et al., 1993). The biology of relaxin differs in many respects between ruminants and nonruminants. Immunoreactive concentration of circulating RLX is much less in ruminants than in nonruminants. Relaxin administration to late pregnant dairy and beef heifers has been shown to result in decreased circulating concentration of progesterone and earlier onset of parturition compared with vehicle treated controls (Anderson et al., 1995). These results were based on the hormone being injected intramuscularly or introduced into the cervical OSonce or twice at a dosage of 3000-5000U. The objective of the present study was to determine the effects of continuous infusion of increasing dosages of porcine relaxin for several days on hormone secretion profiles associated with late pregnancy and on parturition in beef heifers. These periparturient hormones include progesterone, oxytocin and immunoreactive relaxin.

2. Materials and methods 2.1. Animals Fourteen primiparous crossbred beef heifers approaching their first calving were bred by artificial insemination at estrus (Day 0) from 171-189 Julian days. Gestation

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17

averaged 283 days in this herd. During the early stages of pregnancy (spring, summer and autumn) heifers were maintained on pasture at the Rhodes Research Farm. The heifers were fed a ration of mixed silage supplemented with hay during the winter months. On Day 275, the heifers were transferred to the Animal Reproduction Laboratory at Ames, acclimated in confinement pens for hormone treatments and calving, and fed corn (2.3 kg per animalday-‘), hay and fresh water ad libitum. 2.2. Experimental groups The experimental design is presented in Fig. 1. On ,Day 275, heifers were assigned randomly to one of two treatment groups: porcine relaxin (pRLX), n = 8; and phosphate-buffered saline controls (PBS), n = 6. They were bilaterally fitted with indwelling catheters (i.d. 0.125 cm; o.d. 0.225 cm; Tygon Microbore Tubing, No. 14-170 SE, Fisher Scientific Co., Pittsburgh, PA) into the external jugular veins. The control group received a continuous infusion of 0.01 M PBS, pH 7.0, at a rate of 10 ml h- ’. Relaxin used in this study was extracted from ovaries of pregnant pigs and purified according to procedures described by Bbllesbach and Schwabe (1985). Highly purified porcine relaxin (pRLX, 3OOOUmgg’> was dissolved in 0.01 M PBS, pH 7.0, and infused in a graded dosage scheme. Infusion pumps (Infusion/Withdrawal Pump, Harvard Apparatus Inc., Mills, MA) were calibrated to deliver 10 ml h- ’. The duration of pRLX or PBS infusion was lOdays, beginning 6days before expected parturition at 06:OOh and terminating at either parturition or Day 286, if parturition had not occurred. The relaxin was infused in graded dosages throughout the time course of the experiment (Fig. 1). The dosage schedule for the pRLX group was as follows: 200 U h- ’ of pRLX on Days 277 and 278; 300 U h- ’ on Days 279 and 280; 500 U h- ’ on Day 281; 600 U h-l on Day 282; 700U h- I on Day 283 (estimated parturition), until the end of treatment. The control group was infused with PBS from Day 277 to Day 286. The heifers were monitored continuously to precisely determine the time of parturition,

EXPERIMENTAL

DESIGN 700

Cannulation PRLX n=8

+

PBS n=6

+

/

1

I

-48 II

Fig. 1. Experimental or phosphate-buffered

2oo

I 3oo ’ purified porcine relaxin, U h-l

I

I

I

I

/

I,

I

I

1

phosphate I,

I

-24

I,

0 277

design for late pregnant saline (PBS).

I

I

I

I

buffer saline, 10 ml h-l

I /

I

,

,

I

I

I

I

I,

I,

1,

I,

I,,

24 48 72 96 120 144 168 192 Hours from treatment

11 I I I I I t II

215

I

I I II

279 281 Day of gestation

II

beef heifers intravenously

II/II

II

I

infused with porcine relaxin (pRLX)

18

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dystocia, calf birth weight, and placental delivery. Dystocia was evaluated on the basis of number and percentage of heifers requiring assistance. Dystocia score was defined as: 1, none; 2, hand pull; 3, chain-hand pull; 4, chain-jack pull; 5, Caesarean section; 6, feteotomy. 2.3. Radioimmunoassay of progesterone, relaxin, and oxytocin in peripheral plasma Hourly blood samples (10ml) were collected via a jugular catheter beginning at 06:OOh before pRLX or PBS infusion began, as described previously (Musah et al., 1989). Blood was collected in 16mm X 100mm heparinized Vacutainors (Becton Dickson Vacutainor Systems, Rutherford, NJ), maintained on ice up to 1 h, and centrifuged (2OOOg). Plasma from these samples was frozen and stored at - 20°C for radioimmunoassay @IA) of progesterone, relaxin, and oxytocin. Progesterone was determined in duplicate 200 p,l aliquots of plasma with RIA procedures described by Musah et al. (1987a) using a fully characterized antibody (GDN-337; Gibori et al., 1977). Recovery after extraction with benzene-hexane (1:2 v/v) was 89 + 2.6% (mean f SE), and the minimal detectable concentration of progesterone was 0.25 ng ml- ’. The inter- and intra-assay coefficients of variation were 8.9% (n = 9) and 10.5% (n = 361, respectively; nonspecific binding was 3.1%. Immunoreactive relaxin was determined in duplicate aliquots of 25-100 p.1 plasma in a double antibody RIA by using 1251-labeledmonotyrosylated pRLX (Schwabe, 1983) and antiporcine relaxin serum (R6; Dr. B.G. Steinetz) using the procedure previously described (Musah et al., 1987a). Assay sensitivity was 35 pg ml- ’ plasma. To determine the accuracy and precision of this assay, quantities of 0.20, 0.50, 1.00, 1.50, 2.00, 3.00, 4.00, and 5.OOng per tube were added in quadruplicate aliquots of 100 p1 peripheral plasma from an ovariectomized cow. Means (+ SE) for within-assay comparisons of relaxin in peripheral plasma for RLX-treated and PBS control heifers were 1.I3 f O.O16ngml-‘, 1.03 f O.O09ngml-‘, 1.19 f O.O15ngml-‘, 1.05 f O.O12ngml-‘, and 1.08 f O.O07ngml-‘, with intra-assay variances of 2.90%, 1.80%, 3.22%, 1.18%, and 1.15%, respectively. Inter-assay mean comparisons of relaxin in RLX-treated and PBS control heifers was 1.10 f 0.03 ngml- ’ with a covariance of 5.90% (n = 5). Oxytocin extracted from plasma was quantified by a sensitive RIA method (Musah et al., 1989) using synthetic oxytocin (Syntocinon, lOUSPml- ‘, No. 895-J5087, Sandoz Pharmaceuticals, East Hanover, NJ) for the standard curve (0.2, 0.4, 0.8, 1.O, 2.0, 4.0, 6.0, 8.0, 10.0, 16.0, 20.0, 30.0, 40.0, and 50.0 pg per 2OOl~l), oxytocin antibody (from Professor D. Schams, Technische Universidt, Mlinchen, Germany, K#8, E.15, 1.7 g) and labeled oxytocin (100 t~l; 3OOOcountsmin- ’ per 100 ~1; [ 1251]OT NBX-187, 2200 Ci mM- ’, NEN Research Products, DuPont, Wilmington, DE). The maximum binding was 42-44%, nonspecific binding was 1.O-1.6%, and assay sensitivity was 0.01 pg per tube. Intra-assay coefficients of variation of two internal standards were 13.7% and 18.6% (n = 4). Interassay coefficient of variation was 7.0% (n = 4). 2.4. Statistical analysis Experimental units were the individual cows, and they were randomly assigned to treatments. Data were analyzed with a split plot analysis of variance, and the general

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linear model (GLM) and Student’s f-test for continuous variables were used for comp~sons between trea~ent groups (Spector et al., 1985; Snedecor and Cochran, 1989). Hormone reagent was the main plot, with time subplots for evaluation of time and time X ~eatment interaction.

3. Results 3.1. E#ecr

of relaxin

on par~r~tion in beef heifers

Continuous infusion of increasing dosages of purified pRLX (range from 4800 to 16 800 U per 24 h) into primiparous beef heifers during late pregnancy did not affect day of calving as compared with the controls receiving an infusion of PBS (P > 0.051 (Table 1). The duration of delivery was similar in pRLX- and PBS-treated heifers (Table 1). Only two of the 14 pRLX- and PBS-treated heifers required manual assistance for calf delivery. Dystocia score did not differ between the two groups. There was no incidence of retained placentae beyond 24 h postpartum in experimental or control heifers. Catves from dams in both treatment groups were considered as normal. 3.2. EfSect of continuous infusion ofpRLX on plasma concentrations of immunoreactive relaxin

Peripheral plasma concentrations of immunoreactive relaxin in these late pregnant beef heifers are presented in Fig. 2. Pretreatment concentrations of immunoreactive relaxin remained similar (P > 0.05) between pRLX and PBS groups (0.4 + 0.07 ng ml- ’ and 0.3 + 0.02 ngml- ‘1. Immunoreactive relaxin in the PBS control heifers did not change 1 h after treatment (0.3 f 0.01 ng ml- ’) until the end of the PBS infusion 2 16 h later (0.3 k 0.01 ng ml- ’). However, continuous infusion of pRLX resulted in increased immunoreactive relaxin concentration in peripheral plasma (P < 0.01) as the dosage of pRLX (U h- ’> was increased. The rate of change of immunoreactive levels of relaxin in the pRLX group vs. the PBS controls differed (P < 0.05) at hormone dosage intervals of 200, 500 and 700 U h- ’. Mean plasma concentrations of immunoreactive relaxin during each successive pRLX dosage infused were significantly different (P < 0.001; 4.4 f

Table 1 Effect of continuous Treatment

pRLX PBS

infusion of porcine relaxin on duration of pregnancy

No. of heifers

8 6

Pregnancy

in beef heifers

Duration of delivery (min)

Dystocia

(days)

First treatment to calving a (h)

No.

score

282& 1.4 281 f 1.3

131rt20 102f26

87+ 15 761t 18

2 0

2.6 f 0.42 2.5 f 0.34

a Porcine relaxin (3OOOUmg-‘1 dissolved in 0.01 MPBS was infused continuously parturition at increasing dosages from 200 to 7OOUh-‘. Controls received a continuous lOmlh_‘. Values are mean *SE.

from Day 277 to infusion of PBS at

20

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0.1 -I 8

I 0

24

40

56

72

66

104

120

136

162

166

104

200

216

Time in hours Fig. 2. Immunoreactive relaxin concentration (ngml- ‘) in peripheral plasma of beef heifers during continuous i.v. infusion of pRLX (3OOOUmg- ‘, n = 8) or PBS (n = 6) from Day 277 to Day 286 of pregnancy. Values are mean &SE.

0.35ngml-’ at 3OOUh-‘; 9.3 +0.75ngml-’ at 3OOUh-‘; 21 +2.0ngml-’ at 5OOUh-‘; 33 It 4.3ngml-’ at 6OOUh-‘) with a peak of 77 f 4ngml-’ during the infusion of 700 U h- ’ of pRLX. Mean concentrations of immunoreactive relaxin in PBS control heifers were significantly lower during infusion of pRLX at dosages of 2007OOUh-‘. 3.3. Effect of continuous infusion of pRL.X on peripheral plasma progesterone concentra tions Changes in progesterone concentrations in peripheral blood plasma are presented in 3. During the pretreatment period, mean ( f SE) progesterone concentrations were 4.2 f 0.38 ng ml-’ with a range of 2.2-7.2ng ml-’ in the 14 beef heifers. Mean deviations of progesterone from pretreatment means remained similar (P > 0.05) among both groups before the infusion of pRLX or PBS. Infusion of pRLX resulted in an increase (P < 0.05) in mean plasma progesterone concentrations during infusion of the 200-300 U h- ’ dosages. Progesterone decreased ( P < 0.05) during continuous infusion of 500-7OOUh- ’ pRLX compared with PBS controls. The rate of change in progesterone levels between 300, 600 and 7OOUh-I pRLX and PBS groups differed (P < 0.05). The profile of progesterone (ng ml-’ ) during 1OOhbefore parturition is shown in Fig. 4 for both groups.

Fig.

K.H.

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(1997)

15-25

156

172

21

188

204

220

Time in hours

Fig. 3. Progesterone concentration in peripheral plasma (ngml- ‘) as deviation from pretreatment means during continuous i.v. infusion of increasing dosages of pRLX (3000 Umg- ’, n = 8) or PBS (n = 6) from Day 277 (Oh) to Day 286 (220h) of pregnancy in beef heifers. Values arc mean ) SE.

-c pRLX

0-i -100

f -90

-80

-70

-60

-50

40

30

-20

-10

0

Hours from parturition

Fig. 4. Progesterone concentration in peripheral plasma (ngml- ’) during IOOh before parturition in pRLX (3OOOUmg-‘, n = 8) and PBS (n = 6) i.v. infusion. Values are mean f SE.

K.H. Smith et al./Animal Reproduction Science46 (1997) 15-25

22

2.5 +pRLX --PBS

I’ E $5 P e r” 81 c

IO.,

0

-m

-160

-144

428

112

-96

-80

-64

-48

-32

-16

0

Hours from parturition

Fig. 5. Oxytocin concentration (pgml-‘) in peripheral plasma during continuous i.v. infusion of increasing dosages of pRLX (3O@O U mg - ’, n = 8) and PBS (n = 6) in late pregnant beef heifers from time of parturition (Oh). Values are mean *SE.

3.4. Effect of continuous infusion of pRLX on peripheral plasma oxytocin concentrations

Oxytocin levels at 4h intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS (Fig. 5). Oxytocin in control heifers peaked at 0.95 f 0.10 pg ml - ’ during the 72 h before parturition, corresponding to infusion of 500~6OOU h- ’ in the pRLX-treated group, which peaked at 0.77 f 0.07 pg ml-‘. Plasma oxytocin increased simultaneously during the corresponding infusion period of 200-500 U h- ’ in the PBS control heifers (P < O.OS>,and during the same period in the pRLX-treated heifers (P < 0.01). In one control heifer a spike release of oxytocin (3.8 pgml- ’) was detected, but there were no peak releases of oxytocin determined in any other heifers in that group.

4. Discussion The main finding was that continuous i.v. infusion of purified pRLX at increasing dosages ranging from 200 to 700 U h- ’over several days significantly decreased plasma progesterone concentration, but did not result in earlier parturition compared with PBS-infused control heifers. The results presented here indicate that continuous infusion of pRLX into late pregnant beef heifers maintained greatly elevated circulating relaxin concentrations to parturition. This imposed RLX profile in cattle mimicked the endogenous prepartum profiles of circulating concentrations of relaxin found in other species,

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such as the pig (Felder et al., 1986). Increasing circulating concentrations of endogenous relaxin during late pregnancy have been shown to be associated with unimpaired normal parturition in this species, as evidenced by an increased number of stillboms when pigs are treated with antiporcine relaxin antibody (Dlamini et al., 1993; Cho et al., 1996). In cattle, relaxin given intramuscularly at a rate of 6000U on Day 275 resulted in a transient decrease in circulating progesterone concentration and earlier calving (Musah et al., 1986, 1987a,b). The continuous infusion of high dosages of pRLX in the present study may have decreased uterine myometrial activity. In contrast with previous findings, gestation was not shortened in the present study, nor in that by Caldwell et al. (1988) who injected intramuscularly 3000U relaxin 10days before expected calving. Thus a single injection of pRLX too many days before expected parturition or continuous infusion of high dosages of pRLX for several days did not result in earlier calving. The incidence of dystocia was 60% in the PBS-treated control cows in the Caldwell et al. (1989) study, however, which is in marked contrast to 14% dystocia in the 14 beef heifers of the present study. Furthermore, in the present study, there was no incidence of retained placentae in either group and calving was similar in both RLXand PBS-treated heifers. A temporal relationship exists between the graded rise in plasma relaxin concentration and the abrupt decrease in progesterone in response to infused porcine relaxin. This is consistent with earlier findings by Musah et al. (1986, 1987a), who found a significant decrease in circulating progesterone as early as 24h after deposition of relaxin in a gel into the cervical OSon Day 278, and again, at 90min after i.m. injection on Day 273. The dosage, time and route of relaxin treatment are crucial in determining whether complete luteal demise or temporary suppression of luteal progesterone secretion without induction of parturition will occur in cattle. It has been postulated that oxytocin of luteal cell origin may play a role in the regression of the bovine corpus luteum (Hansel and Dowd, 1986). In the present study, plasma oxytocin concentrations increased near parturition, but were similar when compared with PBS-treated controls. No spike releases of oxytocin were detected as compared with prior studies where a bolus i.v. or i.m. injection of relaxin (i.e. up to 9000U) induced single and multiple peaks of oxytocin that ranged from 10 to 42 pg ml- ’ (Musah et al., 1989); peak relaxin concentrations occurred within 30min after hormone treatment. However, detection of oxytocin peaks in the present study may not have been revealed because of the infrequency of plasma sampling intervals. Furthermore, the infusion of increasing dosages of relaxin may have subdued oxytocin peak releases during the 216h of continuous pRLX treatment. It has been shown that oxytocin is released intermittently, and in cattle plasma oxytocin levels appear to be low or undetectable during late pregnancy, but are high during the expulsive phase of labor (Schams et al., 1979). Cultured luteal cells obtained from different stages of gestation in cattle secrete oxytocin, and relaxin induced a dose-dependent suppression of oxytocin release into the culture medium (Musah et al., 1990a). Relaxin also caused a dose-dependent decrease in progesterone release into the cell culture medium, which was attenuated in bovine luteal cells from late stages of pregnancy (Musah et al., 1990b). Intravenous infusion of relaxin into rats was found by Summerlee et al. (1984) to suppress the release of oxytocin and delay delivery. Those workers postulated that

24

K.H. Smith et a/./Animal Reproduction Science 46 (1997) IS-25

relaxin may affect the central nervous system to inhibit oxytocin release in late pregnant

rats. Other observations revealed that treatment with relaxin either by i.m. injection or combined with i.v. infusion will cause spike releases of plasma oxytocin in cattle (Musah et al., 1989). The discrepancies between these results may relate to the origin of endogenous oxytocin, whether the source is hypothalamic or luteal. In summary, the results presented here indicate that the continuous infusion of porcine relaxin during late pregnancy results in a decrease in progesterone secretion in cattle.

Acknowledgements We thank M.E. Shell, T.J. Randall and K.E. Langner for excellent technical assistance. Porcine relaxin antiserum was generously provided by Dr. B.G. Steinetz, Laboratory for Experimental Medicine and Surgery in Primates, New York University Medical Center, Tuxedo, NY. Oxytocin antibody was a gift from Professor D. Schams, Technisthe Universitit, Mbnchen, Germany. This is Journal Paper J-16863 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA (Projects 3223, 3316 and 2273, the last a contributing project to North Central Regional Project NC-l 13 Methods for Improvement of Fertility in Cows Postpartum), supported by Hatch Act and State of Iowa funds. These studies were supported in part by the US Department of Agriculture, NRICGP Competitive Grant 93-37203-8965. All experiments in this study were performed by following the standards established by the Animal Welfare Act and NIH Guide for the Care and Use of Laboratory Animals, Publications 85-23, and were approved by the Iowa State University Animal Care Advisory Committee.

References Anderson, L.L., 1987. Regulation of relaxin secretion and its role in pregnancy. Adv. Exp. Med. Biol., 219: 421-463. Anderson, L.L., Ford, J.J., Melampy, R.M. and Cox, D.F., 1973. Relaxin in porcine corpora lutea during pre.gnancy and after hysterectomy. Am. J. Physiol., 225: 1215-1219. Anderson, L.L., C&al, O.S., Dlamini, B. and Li, Y., 1995. The role of relaxin in ruminants. In: A.H. MacJ_e.mran, G.W. Tregear and G.D. Bryant-Greenwood (Editors), Progress in Relaxin Research. World Scientific Publishing Co., Singapore, pp. 428-438. Btillesbach, E.E. and Schwabe, C., 1985. Naturally occurring porcine relaxins and large scale preparation of the B29 hormone. Biochemistry, 24: 7717-7722. Caldwell, R.W., Anthony, R.V., Whittier, J.C., Smith, M.F. and Morrow, R.E., 1988. Will porcine relaxin induce parturition in beef cows when administered lOdays prepartum? J. Anim. Sci.. 66(Suppl. 1): 150 (Abstr. 141). Caldwell, R.W., Anthony, R.V., Bellows, R.A. and Hall, J.A., 1989. Administration of porcine relaxin to primigravid beef heifers at either four or seven days prepartum. Biol. Reprod.. 4&Suppl. 1): 70 (Abstr. 68). Cho, S-J., Jacobson, C.D. and Anderson, L.L., 1996. Acute effects of antiporcine relaxin on progesterone and relaxin secretion, and parturition in gilts. J. Anim. Sci.. 74(Suppl. 1): 73 (Abstr. 198). Dlamini, B., Schwabe, C., Klindt, J., Jacobson, CD. and Anderson, L.L., 1993. Antiporcine relaxin given during late pregnancy delays parturition in the pig. Endocrinology, 130: 108 (Abstr. 231). Felder, K.J., Molina, J.R., Benoit, A.M. and Anderson, L.L., 1986. Precise timing for peak relaxin and decreased progesterone secretion after hysterectomy in the pig. Endocrinology, 119: 1502-1509.

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