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The effect of days postpartum, indomethacin and oxytocin on prostaglandin metabolite concentrations in postpartum suckled beef cows
THERIOGENOLOGY
THE EFFECT OF DAYS POSTPARTUM, INDOMETHACIN AND OXYTOCIN ON PROSTAGLANDIN METABOLITE CONCENTRAgf$NS IN POSTPARTUM SUCKLED BEEF COWS=' T. R. Troxel,d M. J. Opsomere D. J. Kesler
and
Department
of Animal Science University of Illinois Urhana, IL 61801
Received
for publication: Accepted:
January 30, 1984 May LL, 1984
ABSTRACT In Experiment 1, blood samples were collected on days 1, 4, 7, 10, 13, lb, 19, 22, and 25 postpartum from the jugular veins of 10 suckled beef cows to determine 13, 14-dihydro-15-keto prostaglandin F20 (PGFM) concentrations during the early postpartum period. PGFM concentrations on days 1 and 4 were 207.8 + 33.9 and 283.6 + 45.6 pg/ml and then declined linearly (r = -0.71; P < 0.05) to 44.1 + 5.7 and-44.0 + 5.3 pg/ml on days 22 and 25 postpartum. Two groups of post;artum (25.3 + 8.5 and 37.7 + 1.1 days) suckled beef cows (10 cows/group) were used in th< second experi;ent. Five cows of each group received intrauterine infusions of indomethacin for 5.5 days while the other five cows of each group served as controls. All cows had calves removed at the time of the last indomethacin infusion and were subcutaneously administered oxytocin six hours later. During the infusion period, PGFM concentrations decreased (P < 0.01) across time for both groups of Concentrations of PGFM increased (P < 0.05) after lndomethacin-treated cows. oxytocin treatment for both groups of control and indomethacin-treated cows, hut concentrations were higher for the control cows than for the indomethacin-treated cows. Key Words: Postpartum Suckled Beef cows, Indomethacin, Prostaglandin Metabolite
Oxytocin,
a
The prostaglandin metabolite assay and the validation of this assay were conducted under the supervision of Drs. J. E. Hixon and P. G. Weston in their blahoratory. The authors appreciate their generosity. The crystalline 13, 14-dihydro-15-keto prostaglandin F2u and the 13, 14-dihydro-15-keto prostaglandin F2a antisera were generously supplied hy Dr. K. T. Kirton (The Upjohn Co.). 'This research was conducted as part of regional research project NC-113 d"Methods for Improvement of Fertility of Cows Postpartum." Texas Agricultural Extension Service, P. 0. Drawer 1849, Uvalde, TX 78801 eAnimal Science Research Center, University of Missouri, Columbia, MO 65211
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THERIOGENOLOCY INTRODUCTION -_ Several treatments such as gonadotropin releasing hormone steriods (g-11) have been used beef cows. The duration of the nursing (3) and GnRH treatment
early weaning (l), limited nursing (2,3), (GnRH) treatment (4-9) and a combination of sex to initiate ovarian cycles in anestrous suckled luteal phases after early weaning (1). limited (5,6,8), however, has been shown to he shorter and progesterone secretion is less than during the luteal phase of the normal estrous cycle. The incidence of the short luteal phase in postpartum beef cows has been reported to be 70 to 80% after early weaning and GnRH treatment (1,6,8,12) regardless of the interval postpartum when treated. These short luteal phases are generally 7 to 10 days as compared with a normal estrous cycle length of 20 to 21 days. Short luteal phases also occur spontaneously in postpartum beef cows and are most frequently observed between the first and second estrus detected after parturition (1). Since ova resulting from the induced ovulations appear to be capable of being fertilized, failure to sustain pregnancy appears to be due to early corpus luteurn regression (12,13). The exact cause of the short luteal phase in the postpartum cow is not clearly understood. One explanation may be the presence of a luteolytic factor such as prostaglandin F n(PCF2ry). Thatcher et al. (14) reported elevated levels of 13, 14-dihy f ro-15-keto PCF2a (PGFM) during start of the early postpartum period of milked dairy cows, and they reported that PGFMwas produced by the uterus (15). Only when PGFM levels decreased, about 10 to 20 days postpartum, was there a detected rise in progesterone levels in milked dairy cows. PGFM concentrations, however, have not been determined in early postpartum suckled beef COWS. Furthermore, it has not been demonstrated if indomethacin will suppress the oxytocin-inducible release of PGFM. Therefore, objectives of profile of PGFM concentrations postpartum period of suckled indomethacin, a prostaglandin concentrations in postpartum
these experiments were 1) to determine the in jugular vein serum during the early beef cows and 2) to determine the effect of synthesis inhibitor, and oxytocin on PGFM suckled beef cows. MATERIALS AND METHODS -a
Experiment 1. Ten mature Hereford cows from the University of Illinois beef herd were used in this experiment. Blood samples were collected via jugular venipuncture on days 1, 4, 7, 10, 13, 16, 19, 22 and 25 postpartum in order to determine PGFM concentrations during the early postpartum period. Calves were allowed to suckle throughout the blood samplinR period. All blood samples were assayed for PGFM.
Ten mature Hereford cows from the University of Illinois Experiment 2. beef herd (Group 1) and 10 mature crossbred beef cows from the University of Illinois Dixon SprinEs Agricultural Center (Group 2) were used in this study. Both groups of cows received the same treatment. Five cows from each group received intrauterine infusions of indomethacin for 5.5 days beginning on days 23 to 27 (25.3 + 0.5 days) postpartum and on days 32 to 41 (37.7 f. 1.1 days) postpartum for the first and second group of cows, respectively. Indomethacin (40 mR) was administered by a pipet via the cervix into each uterine horn two times a day at 12-hour intervals. Therefore, each indomethacin-treated cow received 160 mg of indomethacin per day. The indomethacin was dissolved in 0.4
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ml acetone and diluted to a volume of 5 ml with 0.2 M phosphate buffer pH 7.2 (8.0 mg/ml) (16). The other five cows from each group received no treatment and served as controls. At the time of the last infusion (morning of the sixth day), calves were removed from all COWS. Oxytocin (150 IU) was administered subcutaneously to all cows six hours after calf removal. Blood samples were collected via jugular venipuncture into evacuated tubes immediately before the morning infusions and immediately before the oxytocin injections. Additional samples were collected 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0 hours after oxytocin treatment. All of the blood samples were assayed for PGFM. General Procedures. Blood samples from all experiments were placed in crushed ice immediately after collection and then placed in a cold room (4 C) for 24 hours. The serum was then harvested and frozen until it was assayed for PGFM concentrations. Cows in Exp. 1 and Exp. 2 (Group 1) were fed corn stalks to appetite plus 22.5 kg of corn silage per day for about 30 days before calving. During the experimental periods (after calving), 11.3 kg of grass hay was provided per head per day. During the winter, cows in Exp. 2 (Group 2) had continuous Starting access to fescue hay and were fed corn silage to appetite. approximately 30 days before calving and continuing throughout the experimental period, these cows were fed 0.45 kg of soybean meal. Assay of Hormones. Serum PGFM concentrations were quantified by radioimmunoassay. PGFM was extracted from duplicate 500-~1 aliquots of serum. After the addition of 50 nl of 0.5 N acetic acid and 4 ml of ethyl acetate, The serum layer samples were shaken on a mechanical shaker for 15 minutes. was frozen in a mixture of solid CO and methanol for two minutes before decanting the solvent fraction into'disposable culture tubes. Extracts were dried under a stream of air at 37 C. Standard curves were established for each assay in quadruplet at concentrations of 0, 5, 10, 25, 50, 100, 200, 300, 400 and 500 pg/ml of crystalline PGFM dissolved in tris buffer (0.1% gelatin, 0.01% thimerosal, pH 7.4). Cross-reaction of PGFM antisera has been reported to be 0.1% for PGF~'Y, 15.3% for 15-keto-PGF2cr and 100% for 13, 14-dihydro-15-keto-PGF20 (17). The antisera was used at a dilution of 1:5,000.00. When 5, 10, 25, 50 and 100 pg/ml (dissolved in 100 ~1 of tris buffer) of exogenous 13, 14-dihydro-15keto-PGF2n were added to 400 ~1 of plasma from a postpartum cow, the percentage recovery was 97.0, 99.5, 105.8, 101.4 and 89.1, respectively (X = 38.6; n = 17). The least amount of PGFM which inhibited (P < 0.05) binding of H-PGFM to antibody was 5 pg/ml (n=6). Parallelism was determined by quantifying PGFM in different volumes of pooled early postpartum and pooled diestrous blood serum. When volumes of 250, 350, 500 and 650 1~1 of pooled diestrous serum was assayed, PGFM values were 5.9 + 0.6, 5.9 + 0.9, 6.2 + 1.4 When volumes oT' 125, 250,-500 and 755 ~1 and 6.2 2 0.5 pg/ml, respectively. of pooled early postpartum serum were assayed, PGFM values were 99.1 + 18.1, 90.5 + 9.2, 105.5 + 7.9 and 94.2 2 2.2 pg/ml, respectively. Extracts-of both pools-of serum were parallel (P < 0.01; r = 0.97) to the standard cgrve. Recovery aliquots were included by the addition of Q,30,000 cpm of H PGFM to The recovery rates were 74.2 + 1.1%. pooled cow serum.
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Each Samples were assayed for PGFM in three replicates of the PGFM assay. assay included the complete set of serum samples from each cow. Pooled serum was used for calculations of the inttaassay coefficient of variation (11.0%). The mean of the pool sample was 48.3 2 2.0 pg/ml. Duplicated estimates of hormone concentrations were Data analysis. The inttaassay and interassay coefficients of averaged before analysis. variation were determined by the procedure of Rodbatd (18). The PGFM concentrations from day 4 until day 22 postpartum (Exp. 1) were analyzed by linear regression (19). The PGFM concentrations during the infusion period (Exp. 2) and after oxytocin treatment for both groups of cows (Exp. 2) were analyzed by split-plot analysis of variance as described by Gill and Hafs (20) due to repeated sampling of cows. Standard errors were determined from the error mean square as described by Steel and Torrie (19). RESULTS Experiment 1. Concentrations of PGFM from parturition to day 25 postpartum ate shown in Figure 1. PGFM concentrations on daJs 1 and 4 wete 207.8 + 33.9 and 283.6 + 45.6 pgiml. Concentrations of PGFM then declined linearly (t = -0.71; P < 0.01) to 44.1 2 5.7 and 44.0 2 5.3 pg/ml on days 22 and 25 postpartum. Experiment 2. The concentrations of PGFM for the first and second groups of postpartum cows are shown in Figures 2 and 3. During the period of indomethacin treatment, PGFM concentrations in the treated cows decreased across time for both groups of cows (Group 1, P < 0.05; Group 2, P < 0.01). In addition, a treatment by time interaction (P < 0.01) was also detected in Concentrations of PGFM increased (P < both groups of cows during this period. 0.05) after oxytocin treatment for both control and indomethacin-treated cows. However, after oxytocin treatment for the first group of postpartum cows, concentrations of PGFM were higher (P < 0.05) for the control cows than for the indomethacin-treated cows. Although not significant (P > O.lO), the same trend was observed for the second group of postpartum cows-
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Days
Figure 1.
AUGUST
PoslportUm
Mean concentrations e S E) of PGFM in serum of suckled beef cows (Exp. 1) from parturition to day 15 postpartum.
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DD~S of lndometbxin
Figure
192
2.
Tnatment
Hours After Oxytcdn Treatment
Mean concentrations of PGFM in serum of cows in Group 1 (Exp. 2) during indomethacin treatment and after oxytocin treatment. Standard errors of the Values are means of five observations. difference between two treatment means for a given time during indomethacin treatment and after oxytocin treatment are 11.2 and Standard errors of the difference between two time means 7.5. for one treatment during indomethacin treatment and after oxytocin treatment are 7.2 and 5.7.
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,I1
10
-----
gure 3.
T
--Control lndomefhacin
Mean concentrations of PGFM in serum of cows in Group 2 (Exp. 2) during indomethacin treatment and after oxytocin treatment. Values are means of five observations. Standard errors of the difference between two treatment means for a given time during indomethacin treatment and after oxytocin treatment are 9.9 and 8.3. Standard errors of the difference between two time means for one treatment during indomethacin treatment and after oxytocin treatment are 5.3 and 9.4.
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THERIOGENOLOCY DISCUSSION At approximately seven days prepartum, PGFM concentrations gradually increased inthe dairy cow (14), with a major increase between day 1 and 4 postpartum (14). After a peak of PGFM on day 4 postpartum, concentrations then declined linearly to day 15 postpartum in the milked dairy cow (14) and to day 22 postpartum in the suckled beef cow in the present experiment. Guilbault et al. (15) demonstrated that PGFM was produced by the postpartum uterus. In the study by Thatcher et al. (14) and in Exp. 1, blood samples were collected only once a day. Kindahl et al. (21) demonstrated that PGFM concentrations have transitory changes. Therefore, there apparently are factors that will stimulate transitory increases in PGFM concentrations. Oxytocin is one compound that has been shown to be capable of inducing transient increases in prostaglandin concentrations in cyclic cows (22,23). When oxytocin was administered to postpartum cows, a similar increase in PGFM concentrations was detected. Therefore, it is possible that a transient increase in PGFM concentrations may occur after the oxytocin release associated with the suckling stimulus. Indomethacin suppressed basal concentrations and the oxytocin-induced release of PGFM. It was not surprising to also detect a rise in PGFM concentrations in the indomethacin-treated cows. The dosage may have been too low to suppress this release or the interval between indomethacin infusions may have been too long. The same treatment procedure, however, prolonged luteal lifespan in postpartum beef cows (25). In any event, PGFM concentrations were suppressed in indomethacin-treated cows. Several investigators (5,6,8) have demonstrated a high incidence (70% to 80%) of short luteal phases in anestrous postpartum suckled beef cows induced to ovulate with GnRH treatment. Resler et al. (24) demonstrated that corpora lutea induced from GnRH-induced ovulations did not become responsive to luteinizing hormone in vitro and the corpora lutea did not continue to develop beyond day 5. When indomethacin was administered to postpartum suckled beef cows with corpora lutea induced by GnRH treatment, PGFM concentrations were suppressed and corpora lutea lifespan was prolonged (25). In summary, concentrations of PGFM were elevated in the postpartum suckled beef cow and oxytocin induced a release of PGFM. Indomethacin suppressed both basal concentrations of PGFM and the oxytocin induced release of PGFM. We demonstrated in another experiment that suppression of PGFM concentrations enhanced the function and lifespan of corpora lutea induced postpartum suckled beef cows (25). Therefore, one cause of short luteal phases in postpartum beef cows is luteolytic prostaglandins.
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REFERENCES 1.
Odde, L. C., Ward, H. S., Kiracofe, G. H., McKee, R. M., and Kittok, R. J. Short estrous cycles and associated serum progesterone levels in beef COWS. Theriogenology -14:105-112 (1980).
2.
Randel, R. D. and Welker, G. A. Once daily suckling efEects on cow-calf performance. J. Anim. Sci. -43:301 (Abstr.) (1976). Flood, P. F., Manns, J. G., Humphrey, W. D., and Mapletoft, R. J. The first corpus luteum of the postwrtum beef COW. Prog. for Sot. Study of Fertil., p. 30 (Abstr.) (1979). Britt, J. H., Kiser, T. E., Seguin, B. E., Hafs, H. D., Oxender, W. D., suckling COWS. J. and Kitchie, H. D. Fertility after GnRH and PGF2ain Anim. Sci. -41:345 (Abstr.) (1975). Webb, R., Lamming, G. E., Haynes, N. B., Hafs, H. D., and Man%, J. G. Response of cyclic and postpartum suckled cows to injections of synthetic LH-RH. J. Reprod. Fert. %:203-210 (1977). Lishmann, A. W., Allison, P. M. J., Fogwell, R. L., Butcher, R. L., and Inskeep, E. K. Follicular development and function of induced corpora lutea in underfed postpartum anestrous beef COWS. J. Anim. Sci. -48:867-876 (1979).
7.
Fonseca, F. A., Britt, J. H., Kosugiyama, M. K., Ritchie, H. D., and Dillard E. V. Ovulation, ovarian function, and reproductive performance after treatment with GnRH in postpartum suckled cows. Theriogenology -13:171-181 (1980).
8.
Kesler, D. J., Troxel, T. R., and Hixon, D. L. Effect of days postpartum and exogenous GoRH on reproductive hormones and ovarian changes in postpartum suckled beef cows. Theriogenology -13:287-296 (1980).
9.
Troxel, T. R., Ke‘sler, D. J., Noble, R. C., and Carlin, S. E. Ovulation and reproductive hormones following steroid pretreatment, calf removal J. Anim. Sci. -51:652-659 and GnRH in postpartum suckled beef cows. (1980).
10.
Walters, D. L., Songster, B., Valencia, M., Burrell, W. C. and Wiltbank, J. N. Steroids in con.juction with 48 hr calf removal on early weaning in thin anpstrous COWS. J. Anim. Sci. -45 (Suppl. I):215 (Abstr.) (1977).
11.
Smith, M. F., Rurrell, W. C., Shipp, L. D., Short, L. R., Songstet, W. M ., and Wiltbank, J. N. Hormone treatments and use of calf removal in postpartum heef COWS. _I. Anim. Sci. -48:1285-1294 (1979).
12.
Troxel, T. R., Cmarik, C. F., Ott, R. S., Lock, T. F., and Kesler, D. J. The effect of method of GnRH administration and short-term calf removal on ovarian function and reproductive performance in postpartum suckled Theriogenology beef cows administered PCF2u for estrous synchronization. -20:417-433 (1983).
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THERIOCENOLOCY 13.
Ramirez-Godinez, J. A., Kiracofe, G. H., Carnahan, D. L., Spire, M. F., Beeman, K. B., Stevenson, J. S., and Schallis, R. R. Evidence for ovulation and fertilization in heef cows with short estrous cycles. Theriogenology -17:409-414 (1982).
14.
Thatcher, W. W., Wilcox, C. J., Collier, K. J., Eley, 0. S., and Head, H. H. Bovine conceptus--maternal interactions during the pre- and postpartum periods. J. Dairy Sci. -63:1530-1540 (1980).
15.
Guilbault, L. A., Thatcher, W. W., Drost, M., Hopkins, S., and Foster, D. 8. A uterine prolapse and hysterectomy model to characterize PGFM dynamics in the early postpartum period. J. Anim. Sci. -53 (Suppl. I):323 (Abstr.) (1981).
lb.
Lewis, P. E. and Warren, J. E. Effect of indomethacin on luteal function J. Anim. Sci. -45:763-767 (1977). in ewes and heifers.
17.
Cornette, J. C., Harrison, K. L., and Kirton, K. T. Measurements of prostaglandin F a metabolites by radioimmunoassay. Prostaglandins 1:155-164 (1974$.
18.
Rodbard, D. Statistical aspects of Radioimmunoassay. In: Ode11 and Daughaday (Ed.). Principles of Competitive Protein Binding Assays, V. J. B. Lippincott Co., Philadelphia, 1971, p. 220.
19.
Steel, R. G. D. and Torrie, J. H. Principles and Procedures Statistics. McGraw-tfill Book Co., New York (1960).
2v.
Gill, J. L. and Hafs, H. D. Analysis of repeated measurements animals. J. Anim. Sci. -33:331-336 (1971).
21.
Kindahl, H., Edqvist, L.-E., Granstrom, E., and Bane, A. The release of prostaglandin F2a as reflected by 15-keto-13, 14-dihydroprostaglandin F2a in the peripheral circulation during normal luteolysis in heifers. Prostaglandins -11:871-X78 (1976).
22.
Newcomh, R., Booth, W. D., and Brown, L. E. A. The effect of oxytocin treatment on the levels of prostaglandin F in the blood of heifers. J. Reprod. Fert. -49:17-24 (1977).
23.
Milvae, R. A. and Hansel, W. Concurrent uterine venous and ovarian arterial prostaglandin F concentrations in heifers treated with oxytocin. J. Reprod. Fert. -60:7-15 (1980).
24.
Kesler, D. J., Weston, P. G., Pimentel, C. A., Troxel, T. L.. and Hixon, J. E. Diminution of the in vitro response hormone by copora lutea induced by gonadotropin releasing treatment of postpartum suckled beef cows, J. Anim. Sci. (1981).
25.
196
of
of
R., Vincent, D. to luteinizing hormone
-53:749-754
Troxel, T. R. and Kesler, D. J. The effect of indomethacin and oxytocin on prbstaglandin metabolfte concentrations in postpartum suckled beef cows. J. Anim. Sci. (In press).
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1984 VOL. 22 NO. 2
THE EFFECT OF DAYS POSTPARTUM, INDOMETHACIN AND OXYTOCIN ON PROSTAGLANDIN METABOLITE CONCENTRAgf$NS IN POSTPARTUM SUCKLED BEEF COWS=' T. R. Troxel,d M. J. Opsomere D. J. Kesler
and
Department
of Animal Science University of Illinois Urhana, IL 61801
Received
for publication: Accepted:
January 30, 1984 May LL, 1984
ABSTRACT In Experiment 1, blood samples were collected on days 1, 4, 7, 10, 13, lb, 19, 22, and 25 postpartum from the jugular veins of 10 suckled beef cows to determine 13, 14-dihydro-15-keto prostaglandin F20 (PGFM) concentrations during the early postpartum period. PGFM concentrations on days 1 and 4 were 207.8 + 33.9 and 283.6 + 45.6 pg/ml and then declined linearly (r = -0.71; P < 0.05) to 44.1 + 5.7 and-44.0 + 5.3 pg/ml on days 22 and 25 postpartum. Two groups of post;artum (25.3 + 8.5 and 37.7 + 1.1 days) suckled beef cows (10 cows/group) were used in th< second experi;ent. Five cows of each group received intrauterine infusions of indomethacin for 5.5 days while the other five cows of each group served as controls. All cows had calves removed at the time of the last indomethacin infusion and were subcutaneously administered oxytocin six hours later. During the infusion period, PGFM concentrations decreased (P < 0.01) across time for both groups of Concentrations of PGFM increased (P < 0.05) after lndomethacin-treated cows. oxytocin treatment for both groups of control and indomethacin-treated cows, hut concentrations were higher for the control cows than for the indomethacin-treated cows. Key Words: Postpartum Suckled Beef cows, Indomethacin, Prostaglandin Metabolite
Oxytocin,
a
The prostaglandin metabolite assay and the validation of this assay were conducted under the supervision of Drs. J. E. Hixon and P. G. Weston in their blahoratory. The authors appreciate their generosity. The crystalline 13, 14-dihydro-15-keto prostaglandin F2u and the 13, 14-dihydro-15-keto prostaglandin F2a antisera were generously supplied hy Dr. K. T. Kirton (The Upjohn Co.). 'This research was conducted as part of regional research project NC-113 d"Methods for Improvement of Fertility of Cows Postpartum." Texas Agricultural Extension Service, P. 0. Drawer 1849, Uvalde, TX 78801 eAnimal Science Research Center, University of Missouri, Columbia, MO 65211
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THERIOGENOLOCY INTRODUCTION -_ Several treatments such as gonadotropin releasing hormone steriods (g-11) have been used beef cows. The duration of the nursing (3) and GnRH treatment
early weaning (l), limited nursing (2,3), (GnRH) treatment (4-9) and a combination of sex to initiate ovarian cycles in anestrous suckled luteal phases after early weaning (1). limited (5,6,8), however, has been shown to he shorter and progesterone secretion is less than during the luteal phase of the normal estrous cycle. The incidence of the short luteal phase in postpartum beef cows has been reported to be 70 to 80% after early weaning and GnRH treatment (1,6,8,12) regardless of the interval postpartum when treated. These short luteal phases are generally 7 to 10 days as compared with a normal estrous cycle length of 20 to 21 days. Short luteal phases also occur spontaneously in postpartum beef cows and are most frequently observed between the first and second estrus detected after parturition (1). Since ova resulting from the induced ovulations appear to be capable of being fertilized, failure to sustain pregnancy appears to be due to early corpus luteurn regression (12,13). The exact cause of the short luteal phase in the postpartum cow is not clearly understood. One explanation may be the presence of a luteolytic factor such as prostaglandin F n(PCF2ry). Thatcher et al. (14) reported elevated levels of 13, 14-dihy f ro-15-keto PCF2a (PGFM) during start of the early postpartum period of milked dairy cows, and they reported that PGFMwas produced by the uterus (15). Only when PGFM levels decreased, about 10 to 20 days postpartum, was there a detected rise in progesterone levels in milked dairy cows. PGFM concentrations, however, have not been determined in early postpartum suckled beef COWS. Furthermore, it has not been demonstrated if indomethacin will suppress the oxytocin-inducible release of PGFM. Therefore, objectives of profile of PGFM concentrations postpartum period of suckled indomethacin, a prostaglandin concentrations in postpartum
these experiments were 1) to determine the in jugular vein serum during the early beef cows and 2) to determine the effect of synthesis inhibitor, and oxytocin on PGFM suckled beef cows. MATERIALS AND METHODS -a
Experiment 1. Ten mature Hereford cows from the University of Illinois beef herd were used in this experiment. Blood samples were collected via jugular venipuncture on days 1, 4, 7, 10, 13, 16, 19, 22 and 25 postpartum in order to determine PGFM concentrations during the early postpartum period. Calves were allowed to suckle throughout the blood samplinR period. All blood samples were assayed for PGFM.
Ten mature Hereford cows from the University of Illinois Experiment 2. beef herd (Group 1) and 10 mature crossbred beef cows from the University of Illinois Dixon SprinEs Agricultural Center (Group 2) were used in this study. Both groups of cows received the same treatment. Five cows from each group received intrauterine infusions of indomethacin for 5.5 days beginning on days 23 to 27 (25.3 + 0.5 days) postpartum and on days 32 to 41 (37.7 f. 1.1 days) postpartum for the first and second group of cows, respectively. Indomethacin (40 mR) was administered by a pipet via the cervix into each uterine horn two times a day at 12-hour intervals. Therefore, each indomethacin-treated cow received 160 mg of indomethacin per day. The indomethacin was dissolved in 0.4
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ml acetone and diluted to a volume of 5 ml with 0.2 M phosphate buffer pH 7.2 (8.0 mg/ml) (16). The other five cows from each group received no treatment and served as controls. At the time of the last infusion (morning of the sixth day), calves were removed from all COWS. Oxytocin (150 IU) was administered subcutaneously to all cows six hours after calf removal. Blood samples were collected via jugular venipuncture into evacuated tubes immediately before the morning infusions and immediately before the oxytocin injections. Additional samples were collected 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0 hours after oxytocin treatment. All of the blood samples were assayed for PGFM. General Procedures. Blood samples from all experiments were placed in crushed ice immediately after collection and then placed in a cold room (4 C) for 24 hours. The serum was then harvested and frozen until it was assayed for PGFM concentrations. Cows in Exp. 1 and Exp. 2 (Group 1) were fed corn stalks to appetite plus 22.5 kg of corn silage per day for about 30 days before calving. During the experimental periods (after calving), 11.3 kg of grass hay was provided per head per day. During the winter, cows in Exp. 2 (Group 2) had continuous Starting access to fescue hay and were fed corn silage to appetite. approximately 30 days before calving and continuing throughout the experimental period, these cows were fed 0.45 kg of soybean meal. Assay of Hormones. Serum PGFM concentrations were quantified by radioimmunoassay. PGFM was extracted from duplicate 500-~1 aliquots of serum. After the addition of 50 nl of 0.5 N acetic acid and 4 ml of ethyl acetate, The serum layer samples were shaken on a mechanical shaker for 15 minutes. was frozen in a mixture of solid CO and methanol for two minutes before decanting the solvent fraction into'disposable culture tubes. Extracts were dried under a stream of air at 37 C. Standard curves were established for each assay in quadruplet at concentrations of 0, 5, 10, 25, 50, 100, 200, 300, 400 and 500 pg/ml of crystalline PGFM dissolved in tris buffer (0.1% gelatin, 0.01% thimerosal, pH 7.4). Cross-reaction of PGFM antisera has been reported to be 0.1% for PGF~'Y, 15.3% for 15-keto-PGF2cr and 100% for 13, 14-dihydro-15-keto-PGF20 (17). The antisera was used at a dilution of 1:5,000.00. When 5, 10, 25, 50 and 100 pg/ml (dissolved in 100 ~1 of tris buffer) of exogenous 13, 14-dihydro-15keto-PGF2n were added to 400 ~1 of plasma from a postpartum cow, the percentage recovery was 97.0, 99.5, 105.8, 101.4 and 89.1, respectively (X = 38.6; n = 17). The least amount of PGFM which inhibited (P < 0.05) binding of H-PGFM to antibody was 5 pg/ml (n=6). Parallelism was determined by quantifying PGFM in different volumes of pooled early postpartum and pooled diestrous blood serum. When volumes of 250, 350, 500 and 650 1~1 of pooled diestrous serum was assayed, PGFM values were 5.9 + 0.6, 5.9 + 0.9, 6.2 + 1.4 When volumes oT' 125, 250,-500 and 755 ~1 and 6.2 2 0.5 pg/ml, respectively. of pooled early postpartum serum were assayed, PGFM values were 99.1 + 18.1, 90.5 + 9.2, 105.5 + 7.9 and 94.2 2 2.2 pg/ml, respectively. Extracts-of both pools-of serum were parallel (P < 0.01; r = 0.97) to the standard cgrve. Recovery aliquots were included by the addition of Q,30,000 cpm of H PGFM to The recovery rates were 74.2 + 1.1%. pooled cow serum.
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Each Samples were assayed for PGFM in three replicates of the PGFM assay. assay included the complete set of serum samples from each cow. Pooled serum was used for calculations of the inttaassay coefficient of variation (11.0%). The mean of the pool sample was 48.3 2 2.0 pg/ml. Duplicated estimates of hormone concentrations were Data analysis. The inttaassay and interassay coefficients of averaged before analysis. variation were determined by the procedure of Rodbatd (18). The PGFM concentrations from day 4 until day 22 postpartum (Exp. 1) were analyzed by linear regression (19). The PGFM concentrations during the infusion period (Exp. 2) and after oxytocin treatment for both groups of cows (Exp. 2) were analyzed by split-plot analysis of variance as described by Gill and Hafs (20) due to repeated sampling of cows. Standard errors were determined from the error mean square as described by Steel and Torrie (19). RESULTS Experiment 1. Concentrations of PGFM from parturition to day 25 postpartum ate shown in Figure 1. PGFM concentrations on daJs 1 and 4 wete 207.8 + 33.9 and 283.6 + 45.6 pgiml. Concentrations of PGFM then declined linearly (t = -0.71; P < 0.01) to 44.1 2 5.7 and 44.0 2 5.3 pg/ml on days 22 and 25 postpartum. Experiment 2. The concentrations of PGFM for the first and second groups of postpartum cows are shown in Figures 2 and 3. During the period of indomethacin treatment, PGFM concentrations in the treated cows decreased across time for both groups of cows (Group 1, P < 0.05; Group 2, P < 0.01). In addition, a treatment by time interaction (P < 0.01) was also detected in Concentrations of PGFM increased (P < both groups of cows during this period. 0.05) after oxytocin treatment for both control and indomethacin-treated cows. However, after oxytocin treatment for the first group of postpartum cows, concentrations of PGFM were higher (P < 0.05) for the control cows than for the indomethacin-treated cows. Although not significant (P > O.lO), the same trend was observed for the second group of postpartum cows-
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Days
Figure 1.
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Mean concentrations e S E) of PGFM in serum of suckled beef cows (Exp. 1) from parturition to day 15 postpartum.
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DD~S of lndometbxin
Figure
192
2.
Tnatment
Hours After Oxytcdn Treatment
Mean concentrations of PGFM in serum of cows in Group 1 (Exp. 2) during indomethacin treatment and after oxytocin treatment. Standard errors of the Values are means of five observations. difference between two treatment means for a given time during indomethacin treatment and after oxytocin treatment are 11.2 and Standard errors of the difference between two time means 7.5. for one treatment during indomethacin treatment and after oxytocin treatment are 7.2 and 5.7.
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,I1
10
-----
gure 3.
T
--Control lndomefhacin
Mean concentrations of PGFM in serum of cows in Group 2 (Exp. 2) during indomethacin treatment and after oxytocin treatment. Values are means of five observations. Standard errors of the difference between two treatment means for a given time during indomethacin treatment and after oxytocin treatment are 9.9 and 8.3. Standard errors of the difference between two time means for one treatment during indomethacin treatment and after oxytocin treatment are 5.3 and 9.4.
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THERIOGENOLOCY DISCUSSION At approximately seven days prepartum, PGFM concentrations gradually increased inthe dairy cow (14), with a major increase between day 1 and 4 postpartum (14). After a peak of PGFM on day 4 postpartum, concentrations then declined linearly to day 15 postpartum in the milked dairy cow (14) and to day 22 postpartum in the suckled beef cow in the present experiment. Guilbault et al. (15) demonstrated that PGFM was produced by the postpartum uterus. In the study by Thatcher et al. (14) and in Exp. 1, blood samples were collected only once a day. Kindahl et al. (21) demonstrated that PGFM concentrations have transitory changes. Therefore, there apparently are factors that will stimulate transitory increases in PGFM concentrations. Oxytocin is one compound that has been shown to be capable of inducing transient increases in prostaglandin concentrations in cyclic cows (22,23). When oxytocin was administered to postpartum cows, a similar increase in PGFM concentrations was detected. Therefore, it is possible that a transient increase in PGFM concentrations may occur after the oxytocin release associated with the suckling stimulus. Indomethacin suppressed basal concentrations and the oxytocin-induced release of PGFM. It was not surprising to also detect a rise in PGFM concentrations in the indomethacin-treated cows. The dosage may have been too low to suppress this release or the interval between indomethacin infusions may have been too long. The same treatment procedure, however, prolonged luteal lifespan in postpartum beef cows (25). In any event, PGFM concentrations were suppressed in indomethacin-treated cows. Several investigators (5,6,8) have demonstrated a high incidence (70% to 80%) of short luteal phases in anestrous postpartum suckled beef cows induced to ovulate with GnRH treatment. Resler et al. (24) demonstrated that corpora lutea induced from GnRH-induced ovulations did not become responsive to luteinizing hormone in vitro and the corpora lutea did not continue to develop beyond day 5. When indomethacin was administered to postpartum suckled beef cows with corpora lutea induced by GnRH treatment, PGFM concentrations were suppressed and corpora lutea lifespan was prolonged (25). In summary, concentrations of PGFM were elevated in the postpartum suckled beef cow and oxytocin induced a release of PGFM. Indomethacin suppressed both basal concentrations of PGFM and the oxytocin induced release of PGFM. We demonstrated in another experiment that suppression of PGFM concentrations enhanced the function and lifespan of corpora lutea induced postpartum suckled beef cows (25). Therefore, one cause of short luteal phases in postpartum beef cows is luteolytic prostaglandins.
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REFERENCES 1.
Odde, L. C., Ward, H. S., Kiracofe, G. H., McKee, R. M., and Kittok, R. J. Short estrous cycles and associated serum progesterone levels in beef COWS. Theriogenology -14:105-112 (1980).
2.
Randel, R. D. and Welker, G. A. Once daily suckling efEects on cow-calf performance. J. Anim. Sci. -43:301 (Abstr.) (1976). Flood, P. F., Manns, J. G., Humphrey, W. D., and Mapletoft, R. J. The first corpus luteum of the postwrtum beef COW. Prog. for Sot. Study of Fertil., p. 30 (Abstr.) (1979). Britt, J. H., Kiser, T. E., Seguin, B. E., Hafs, H. D., Oxender, W. D., suckling COWS. J. and Kitchie, H. D. Fertility after GnRH and PGF2ain Anim. Sci. -41:345 (Abstr.) (1975). Webb, R., Lamming, G. E., Haynes, N. B., Hafs, H. D., and Man%, J. G. Response of cyclic and postpartum suckled cows to injections of synthetic LH-RH. J. Reprod. Fert. %:203-210 (1977). Lishmann, A. W., Allison, P. M. J., Fogwell, R. L., Butcher, R. L., and Inskeep, E. K. Follicular development and function of induced corpora lutea in underfed postpartum anestrous beef COWS. J. Anim. Sci. -48:867-876 (1979).
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Fonseca, F. A., Britt, J. H., Kosugiyama, M. K., Ritchie, H. D., and Dillard E. V. Ovulation, ovarian function, and reproductive performance after treatment with GnRH in postpartum suckled cows. Theriogenology -13:171-181 (1980).
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Kesler, D. J., Troxel, T. R., and Hixon, D. L. Effect of days postpartum and exogenous GoRH on reproductive hormones and ovarian changes in postpartum suckled beef cows. Theriogenology -13:287-296 (1980).
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Troxel, T. R., Ke‘sler, D. J., Noble, R. C., and Carlin, S. E. Ovulation and reproductive hormones following steroid pretreatment, calf removal J. Anim. Sci. -51:652-659 and GnRH in postpartum suckled beef cows. (1980).
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Walters, D. L., Songster, B., Valencia, M., Burrell, W. C. and Wiltbank, J. N. Steroids in con.juction with 48 hr calf removal on early weaning in thin anpstrous COWS. J. Anim. Sci. -45 (Suppl. I):215 (Abstr.) (1977).
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Smith, M. F., Rurrell, W. C., Shipp, L. D., Short, L. R., Songstet, W. M ., and Wiltbank, J. N. Hormone treatments and use of calf removal in postpartum heef COWS. _I. Anim. Sci. -48:1285-1294 (1979).
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Troxel, T. R., Cmarik, C. F., Ott, R. S., Lock, T. F., and Kesler, D. J. The effect of method of GnRH administration and short-term calf removal on ovarian function and reproductive performance in postpartum suckled Theriogenology beef cows administered PCF2u for estrous synchronization. -20:417-433 (1983).
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Ramirez-Godinez, J. A., Kiracofe, G. H., Carnahan, D. L., Spire, M. F., Beeman, K. B., Stevenson, J. S., and Schallis, R. R. Evidence for ovulation and fertilization in heef cows with short estrous cycles. Theriogenology -17:409-414 (1982).
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Thatcher, W. W., Wilcox, C. J., Collier, K. J., Eley, 0. S., and Head, H. H. Bovine conceptus--maternal interactions during the pre- and postpartum periods. J. Dairy Sci. -63:1530-1540 (1980).
15.
Guilbault, L. A., Thatcher, W. W., Drost, M., Hopkins, S., and Foster, D. 8. A uterine prolapse and hysterectomy model to characterize PGFM dynamics in the early postpartum period. J. Anim. Sci. -53 (Suppl. I):323 (Abstr.) (1981).
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Lewis, P. E. and Warren, J. E. Effect of indomethacin on luteal function J. Anim. Sci. -45:763-767 (1977). in ewes and heifers.
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Cornette, J. C., Harrison, K. L., and Kirton, K. T. Measurements of prostaglandin F a metabolites by radioimmunoassay. Prostaglandins 1:155-164 (1974$.
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Rodbard, D. Statistical aspects of Radioimmunoassay. In: Ode11 and Daughaday (Ed.). Principles of Competitive Protein Binding Assays, V. J. B. Lippincott Co., Philadelphia, 1971, p. 220.
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Steel, R. G. D. and Torrie, J. H. Principles and Procedures Statistics. McGraw-tfill Book Co., New York (1960).
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Gill, J. L. and Hafs, H. D. Analysis of repeated measurements animals. J. Anim. Sci. -33:331-336 (1971).
21.
Kindahl, H., Edqvist, L.-E., Granstrom, E., and Bane, A. The release of prostaglandin F2a as reflected by 15-keto-13, 14-dihydroprostaglandin F2a in the peripheral circulation during normal luteolysis in heifers. Prostaglandins -11:871-X78 (1976).
22.
Newcomh, R., Booth, W. D., and Brown, L. E. A. The effect of oxytocin treatment on the levels of prostaglandin F in the blood of heifers. J. Reprod. Fert. -49:17-24 (1977).
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Milvae, R. A. and Hansel, W. Concurrent uterine venous and ovarian arterial prostaglandin F concentrations in heifers treated with oxytocin. J. Reprod. Fert. -60:7-15 (1980).
24.
Kesler, D. J., Weston, P. G., Pimentel, C. A., Troxel, T. L.. and Hixon, J. E. Diminution of the in vitro response hormone by copora lutea induced by gonadotropin releasing treatment of postpartum suckled beef cows, J. Anim. Sci. (1981).
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Troxel, T. R. and Kesler, D. J. The effect of indomethacin and oxytocin on prbstaglandin metabolfte concentrations in postpartum suckled beef cows. J. Anim. Sci. (In press).
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