DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 8(4):573-585,1991
EFFECT OF CONSTANT INFUSION OF OXYTOClN ON LUTEAL LIFESPAN AND OXYTOClN-INDUCED RELEASE OF PROSTAGLANDIN F2~ IN HEIFERS 1 S.L. Lutz% M.F. Smith% D.H. Keisler and H.A. Garverick Department of Animal Sciences, University of Missouri, Columbia, MO 65211 Received May 21, 1991
ABSTRACT Two experiments were conducted to determine whether constant infusion of oxytocin would prolong the luteal phase and inhibit uterine prostaglandin F2tx (PGF2ct) secretion in heifers. In Experiment 1, twelve heifers, treated with saline (SAL) or oxytocin (OXY) via jugular cannulae infusions (INF) or osmotic minipumps (OMP), were allotted at estrus into four treatment groups (n = 3). Treatments were: SAL-INF, SAL-OMP, OXY-INF and OXY-OMP. Physiological saline or oxytocin was given from Days 10 to 23 (Day 0 = estrus) of the estrous cycle. Method of treatment (jugular cannula infusion or osmotic minipump) had no effect (P > 0.05) on estrous cycle length or pattern of secretion of progesterone; therefore, data were pooled. Estrous cycle lengths were extended (P < 0.01) for heifers which received oxytocin (25.3 + 0.4 d) compared to saline (20.5 + 0.4 d). Luteolysis did not occur in oxytocin-treated heifers until after treatment ceased. Experiment 2 was designed and conducted identically to Experiment 1 with the addition of a "challenge" injection of oxytocin (100 IU oxytocin, i.v.) given on Day 16 of the estrous cycle. Treatment of heifers with oxytocin extended (P < 0.05) estrous cycle length by an average of 3 d compared to heifers treated with saline. The "challenge" injection induced (P < 0.05) secretion of PGF2cz (as measured by the stable PGF2ct metabolite, 15-keto-13,14-dihydro-PGF2~t) in saline-treated but not oxytocin-treated heifers. In both Experiment 1 and 2, serum concentrations of FSH were elevated (P < 0.05) in oxytocin-treated heifers. No increase was obseryed for LH or prolactin. The rise in estradiol-1713 at luteolysis was not affected (P > 0.10) by treatment. In summary, constant infusion of oxytocin extended luteal lifespan, prolonged secretion of progesterone, and inhibited oxytocin-induced secretion of PGF2tx. Constant infusion of oxytocin did not affect serum concentrations of estradiol-1713, LH or prolactin; however, serum concentrations of FSH were elevated during the oxytocin treatment period. INTRODUCTION Recent evidence indicates that the corpus luteum of the cow (1,2) and sheep (2,3) synthesizes and secretes oxytocin. Although the physiological role of oxytocin in luteal function of ruminants remains unclear, evidence suggests that it may be directly and(or) indirectly involved in luteolysis (4). Injections of oxytocin during the early luteal phase reduced luteal lifespan in cattle (5,6,7) and goats (8); however, this effect was abolished by hysterectomy (5,7). Both active and passive immunization against oxytocin delayed luteolysis in cyclic sheep (9,10,11) and goats (12). The luteolytic action of oxytocin may be mediated through prostaglandin F2cz (PGF2ct), since injections of oxytocin increased plasma concentrations of PGF2o~ in uterine intact heifers (13). Furthermore, oxytocin treatment increased secretion of PGF2o~ by the uterus (14) and PGF2~ injections increased plasma concentrations of oxytocin (I 5,16). The physiological role of this positive feedback loop may be to ensure luteal regression. During the late luteal phase, endometrial oxytocin receptor concentrations increase to peak at estrus in both sheep (17) and cattle (18,19). At the time of luteolysis, oxytocin and PGF2tz are secreted in a simultaneous pulsatile manner in both sheep (20,21,22) and cattle (23). McCracken et al. (24) have proposed that pulsatile secretion of oxytocin and subsequent down regulation of uterine oxytocin receptors are required for the pulsatile secretion of PGF2ct observed at luteolysis. Flint and Sheldrick (25) reported that constant (non-pulsaCopyright © 1991 Butterworth-Heinemann
573
0739-7240/91/$3.00
574
LUTZ ET AL.
tile) infusion of oxytocin during the luteal phase of the estrous cycle, delayed return to estrus in cyclic sheep. However, the effect of constant infusion of oxytocin on luteal lifespan and uterine secretion of PGF2c~ in cattle is not clear (26,27,28). Therefore, the first objective of this study was to determine the effect of constant infusion of oxytocin on luteal lifespan and secretion of progesterone, estradiol-1713, FSH, LH and prolactin in cattle (Experiment 1). A second objective was to determine whether constant infusion of oxytocin would inhibit the oxytocin-induced secretion of PGF:~ [as measured by the stable PGF2c~ metabolite, 15-keto-13,14-dihydro-PGF2~ (PGFM)] on Day 16 of the bovine estrous cycle (Experiment 2).
MATERIALS AND METHODS Animals and Management. Estrus was synchronized in twenty-four Holstein (n = 16) and Guernsey (n = 8) heifers [12 heifers per experiment - Holstein (n = 8) and Guernsey (n = 4), 12 to 14 months of age weighing ~350 kg] with two 25 mg injections (i.m.) of PGF2c~ (Lutalyse, The UpJohn Company, Kalamazoo, MI) given fifteen d apart. Heifers were visually observed for estrous behavior for 4 d following each injection. In Brahman heifers, the corpus luteum formed following PGF2~-induced luteolysis contained fewer small and large iuteal cells and had a lower response to LH in vitro compared to corpora lutea formed following spontaneous regression (29). If PGF2c~-induced luteolysis did alter luteal cell populations of the subsequently formed corpora lutea in the present study, the effect should have been similar for both the saline and oxytocin-treated heifers. Furthermore, two injections of PGF2ot do not reportedly reduce fertility (30). During both experiments, heifers were housed in individual tie stalls, fed a ration consisting primarily of corn, cottonseed hulls and soybeans twice daily and had fresh water available ad libitum. Experimental Design. Both experiments were designed as 2 × 2 factorials involving treatment (saline or oxytocin) and method of treatment (jugular infusion or osmotic minipump). Experiment 1: Twelve heifers were allotted, by breed and weight, into four treatment groups (n = 3). Treatments were: Group 1 (saline-infusion); Group 2 (saline-osmotic minipump); Group 3 (oxytocin-infusion); Group 4 (oxytocin-osmotic minipump). Treatment with saline or oxytocin began on Day 10 (Day 0 = estrus) of the estrous cycle. A computer-controlled infusion system (31) programmed to deliver either sterile physiological saline (0.9% NaC1) or oxytocin (Sigma Chemical Co., St. Louis, MO; 218 ng oxytocin/h/ kg body weight dissolved in saline) was connected to heifers in Groups 1 and 3, respectively. Osmotic minipumps (Alzet Model 2m12, Alza Corporation, Palo Alto, CA) filled with either saline or 26 mg oxytocin (13 mg/ml) dissolved in saline were placed subcutaneously (under local anesthesia) in the axillary region of heifers in Groups 2 and 4, respectively. On Day 23 of the estrous cycle (treatment day 13), the computer-controlled infusion system was disconnected and the osmotic minipumps were removed. Luteolysis was determined to have occurred when serum concentrations of progesterone had fallen below 1 rig/ ml. Experiment 2: The second experiment was designed and conducted as previously described for Experiment 1 with the addition of an intravenous injection of 100 IU of oxytocin (20 IU/ml) given to all heifers (n = 12) on Day 16 of the estrous cycle. Oxytocin has been used to test the ability of the uterus to secrete PGF2c~ both in vivo (32,33) and in vitro (34,35). Response to the challenge injection of oxytocin was assessed by measuring peripheral concentrations of the stable PGF2~ metabolite, 15-keto-13,14-dihydro-PGF2o~ (PGFM), which has been shown to accurately reflect changes in uterine PGF2ot secretion (36,37). Blood Sampling. Blood samples (10 ml) for both experiments were collected via jugular cannulae daily from Day 3 to 9 and twice daily from Day 10 to 27 of the estrous cycle.
OXYTOCIN AND LUTEAL FUNCTION IN HEIFERS
575
Samples were allowed to clot at room temperature for ~2 hr then centrifuged at 4 C (3000 x g) for 30 min. Serum was divided into 2 aliquots and stored at -20 C for subsequent analysis. Serum concentrations of oxytocin, progesterone, estradiol-17~, FSH, LH and prolactin were determined by radioimmunoassay. Blood samples (5 ml) following the Day 16 challenge injection of oxytocin in Experiment 2 were collected -60, -45, -30, -15, 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 and 180 minutes from the time of injection and serum concentrations of oxytocin, PGFM, FSH and prolactin were determined by radioimmunoassay. Radioimmunoassays. Serum concentrations of oxytocin were determined by a radioimmunoassay procedure developed and validated in this laboratory. A standard solution of oxytocin (400 pg/ml) was prepared using oxytocin acetate salt (Sigma Chemical Co., St. Louis, MO) dissolved in 1% gel-phosphate buffered saline. Oxytocin standard was pipetted in triplicate such that the standard curve contained the following amounts: 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0 and 200.0 pg/tube. A standard serum pool was prepared by adding oxytocin standard to a bovine serum pool. Serial dilutions of the "spiked" standard serum pool (2.5 lal to 500 l.tl) were parallel to the standard curve and recovery of oxytocin from "spiked" sera was 90%. Serum concentrations of oxytocin were determined in duplicate 250 ~tl samples. Oxytocin antiserum (rabbit anti-oxytocin, 100 ~tl of 1:10,000 dilution, Arnel Products Co. Inc., New York, NY) was added to each assay tube except total and non-specific binding tubes. Assay tubes were incubated at 4 C for 48 hr after which 100 lxl of ['25I]iodotyrosyF oxytocin (3 pg/tube; Amersham Corporation, Arlington Heights, IL) was added to each tube. Assay tubes were incubated at 4 C for an additional 12 hr, then 100 ~1 of sheep anti-rabbit (IgG fraction) pre-precipitated antisera was added to each tube except total tubes. Assay tubes were mixed, incubated at room temperature for 10 min and centrifuged at 4 C (3200 x g) for 30 min. Supematant was discarded and radioactivity in the precipitate was counted for 1 min/tube in a gamma counter. Minimum detectable concentration of oxytocin was 1.87 pg/tube. Inter- and intra-assay coefficients of variation were 18.2% and 14.4%, respectively. Serum concentrations of the following hormones were determined by radioimmunoassay procedures previously validated in our laboratory: progesterone (38); estradiol-17~ (31); FSH (39); LH (40); prolactin (31) and PGFM (33). Minimum detectable concentrations of the hormones in serum were 2.05 pg, 1.93 pg, 2.66 ng, 10.25 ng, 1.19 ng and 3.86 pg/tube, respectively. Inter-assay coefficients of variation were 13.9%, 18.3%, 10.2%, 16.8%, 17.2% and 4.1%, respectively. Intra-assay coefficients of variation were 8.8%, 11.3%, 12.8%, 10.3%, 9.2% and 6.7%, respectively. Statistical Analyses. Effects of treatments on estrous cycle length were analyzed by a 2 x 2 factorial analysis of variance (41). Effects of treatments on serum concentrations of oxytocin, progesterone, estradiol-17~, FSH, LH and prolactin were analyzed by analysis of variance for a split plot in time design with repeated measurements (42). Effects of treatments on serum concentrations of estradiol-1713 at luteolysis and effects of the challenge injection of oxytocin on serum concentrations of PGFM, FSH and prolactin were also analyzed by analysis of variance for a split plot in time design. RESULTS Experiment 1. Treatment of heifers with oxytocin extended (P < 0.01) estrous cycle length by an average of 4.7 d compared to heifers treated with saline (Table 1). Serum concentrations of progesterone were not affected by initiation of oxytocin treatment on Day 10 of the estrous cycle and were maintained at elevated levels in oxytocin-treated heifers until treatment ceased on Day 23 of the estrous cycle (Figure 1). Return to estrus after delayed luteolysis occurred within 3 d after cessation of oxytocin treatment. Method of treatment (jugular infusion v s . osmotic minipump) had no effect (P > 0.05) on
576
L U T Z ET AL.
TABLE 1. EFFECT OF SALINE AND OXYTOCIN TREATMENTS (DAYs l 0 TO 23 OF THE ESTROUS CYCLE) ON ESTROUS CYCLE LENGTH
Estrous Cycle Length (days) ~ Treatment
Experiment 1
Experiment 2
Saline
20.5 _+ 0 . 4
18.6 + 0.8
Oxytocin
2 5 . 2 + 0.4*
21.6 _+ 0.8*"
" v a l u e s are m e a n _+ SE. *P < 0.01 c o m p a r e d w i t h s a l i n e v a l u e . **P < 0 . 0 5 c o m p a r e d w i t h s a l i n e v a l u e .
luteal lifespan; therefore, endocrine data were pooled. Serum concentrations of oxytocin were similar (P > 0.10) for saline and oxytocin treated heifers prior to treatment, 36.6 + 5.7 pg/ml and 20.3 + 2.5 pg/ml, respectively. After initiation of treatment, serum concentrations of oxytocin increased (P < 0.01) to 182.5 + 10.1 pg/ml in oxytocin-treated heifers compared to 24.8 + 2.1 pg/ml in saline-treated heifers (Figure 1). Serum concentrations of oxytocin remained elevated throughout the treatment period (Days 10 to 23 of the estrous cycle) in heifers receiving oxytocin via computer-controlled jugular infusion (Figure 1C); whereas, concentrations of oxytocin steadily declined over the treatment period in heifers receiving oxytocin via osmotic minipump (Figure IB). Serum concentrations of estradiol-17[3, LH and prolactin were similar (P > 0.10) for saline and oxytocin treated heifers prior to and during the treatment period (Table 2). Constant infusion of oxytocin had no effect (P > 0.10) on the rise in estradiol-17~, following the decline in progesterone, observed at luteolysis (Figure 2).
0
600
6
400
4
200
2
E v
~" z o
"I.-' u3 L,-I (.9 0
,~
0
O
B
_A E c~
400
6
4
200
© l--
0
>X 0
2 0 10
z
600
8
400
6 4
200
2 0
5
I
I
I
I
I
I
6
9
12
15
18
21
24
27
DAY OF ESTROUS CYCLE Figure I. Mean serum concentrations of progesterone (, ) and oxytocin ( ) in heifers treated with saline (A, n = 6) or oxytocin (B, osmotic minipump, n = 3: C, jugular infusion, n = 3) in Experiment I. Treatment period (Days 10 to 23 of the estrous cycle) is indicated by the solid bar.
OXYTOCIN AND LUTEAL FUNCTION IN HEIFERS
577
TABLE 2. EFFECT OF SALINEAND OXYTOCIN TREATMENTS(DAYs 10 TO 23 OF THE ESTROUS CYCLE) ON SERUM CONCENTRATIONSAOF ESTRADIOL-1715,L H AND PROLACTIN
Estradiol-I 7~ (pglml)
Treatment
LH (ng/ml)
Prolactin (ng/ml)
Pre-TreatmenP Treatment~ Pre-Treatment Treatment Pre-Treatment
Treatment
Ex~fimentl Saline Oxytocin
1.11±0.2 1.60±0.2
1.60±0.2 2.01±0.2
1.26±0.1 1.18±0.1
1.13±0.1 1.57±0.1
24.36±2.1 19.93±1.6
23.28±1.1 21.53±1.4
Experiment2 Saline Oxytocin
0.69±0.1 0.48±0.1
0.66±0.1 0.45±0.1
0.92±0.1 0.94±0.1
0.80±0.1 1.09±0.1
39.14±4.1 40.54±3.5
29.32±2.5 36.02±4.2
~Values are mean _+SE. bPre-treatment value is mean + SE for Days 6 to 10 of the estrous cycle. ~Treatment value is mean + SE for Days 11 to 15 of the estrous cycle.
Serum concentrations of F S H were similar (P > 0.10) for saline and oxytocin treated heifers prior to treatment, 35.4 + 3.4 ng/ml and 32.9 + 2.5 ng/ml, respectively. However, after initiation of treatment, serum concentrations of FSH increased (P < 0.05) to 64.5 + 4.5 ng/ml in oxytocin-treated heifers compared to 24.6 + 1.7 ng/ml in saline-treated heifers (Figure 3). The increase observed in oxytocin-treated heifers occurred within 2.5 d after initiation of oxytocin treatment and persisted until treatment ceased on Day 23 of the estrous cycle. Serum concentrations of F S H returned to baseline within 2.5 d following cessation of oxytocin treatment. The increase in serum concentrations of FSH which occurred coincident with oxytocin treatment could not be attributed to FSH contamination or oxytocin
10
10 8
6
6
4
4
2
E
2 0
o
,.,
13
8
z
6
6
r~
4
4
~
0
a~ l--
v~
LJJ 0
E
m
5-
0
2
2
~
0
0
a:
Vol. 8(4):573-585,1991
EFFECT OF CONSTANT INFUSION OF OXYTOClN ON LUTEAL LIFESPAN AND OXYTOClN-INDUCED RELEASE OF PROSTAGLANDIN F2~ IN HEIFERS 1 S.L. Lutz% M.F. Smith% D.H. Keisler and H.A. Garverick Department of Animal Sciences, University of Missouri, Columbia, MO 65211 Received May 21, 1991
ABSTRACT Two experiments were conducted to determine whether constant infusion of oxytocin would prolong the luteal phase and inhibit uterine prostaglandin F2tx (PGF2ct) secretion in heifers. In Experiment 1, twelve heifers, treated with saline (SAL) or oxytocin (OXY) via jugular cannulae infusions (INF) or osmotic minipumps (OMP), were allotted at estrus into four treatment groups (n = 3). Treatments were: SAL-INF, SAL-OMP, OXY-INF and OXY-OMP. Physiological saline or oxytocin was given from Days 10 to 23 (Day 0 = estrus) of the estrous cycle. Method of treatment (jugular cannula infusion or osmotic minipump) had no effect (P > 0.05) on estrous cycle length or pattern of secretion of progesterone; therefore, data were pooled. Estrous cycle lengths were extended (P < 0.01) for heifers which received oxytocin (25.3 + 0.4 d) compared to saline (20.5 + 0.4 d). Luteolysis did not occur in oxytocin-treated heifers until after treatment ceased. Experiment 2 was designed and conducted identically to Experiment 1 with the addition of a "challenge" injection of oxytocin (100 IU oxytocin, i.v.) given on Day 16 of the estrous cycle. Treatment of heifers with oxytocin extended (P < 0.05) estrous cycle length by an average of 3 d compared to heifers treated with saline. The "challenge" injection induced (P < 0.05) secretion of PGF2cz (as measured by the stable PGF2ct metabolite, 15-keto-13,14-dihydro-PGF2~t) in saline-treated but not oxytocin-treated heifers. In both Experiment 1 and 2, serum concentrations of FSH were elevated (P < 0.05) in oxytocin-treated heifers. No increase was obseryed for LH or prolactin. The rise in estradiol-1713 at luteolysis was not affected (P > 0.10) by treatment. In summary, constant infusion of oxytocin extended luteal lifespan, prolonged secretion of progesterone, and inhibited oxytocin-induced secretion of PGF2tx. Constant infusion of oxytocin did not affect serum concentrations of estradiol-1713, LH or prolactin; however, serum concentrations of FSH were elevated during the oxytocin treatment period. INTRODUCTION Recent evidence indicates that the corpus luteum of the cow (1,2) and sheep (2,3) synthesizes and secretes oxytocin. Although the physiological role of oxytocin in luteal function of ruminants remains unclear, evidence suggests that it may be directly and(or) indirectly involved in luteolysis (4). Injections of oxytocin during the early luteal phase reduced luteal lifespan in cattle (5,6,7) and goats (8); however, this effect was abolished by hysterectomy (5,7). Both active and passive immunization against oxytocin delayed luteolysis in cyclic sheep (9,10,11) and goats (12). The luteolytic action of oxytocin may be mediated through prostaglandin F2cz (PGF2ct), since injections of oxytocin increased plasma concentrations of PGF2o~ in uterine intact heifers (13). Furthermore, oxytocin treatment increased secretion of PGF2o~ by the uterus (14) and PGF2~ injections increased plasma concentrations of oxytocin (I 5,16). The physiological role of this positive feedback loop may be to ensure luteal regression. During the late luteal phase, endometrial oxytocin receptor concentrations increase to peak at estrus in both sheep (17) and cattle (18,19). At the time of luteolysis, oxytocin and PGF2tz are secreted in a simultaneous pulsatile manner in both sheep (20,21,22) and cattle (23). McCracken et al. (24) have proposed that pulsatile secretion of oxytocin and subsequent down regulation of uterine oxytocin receptors are required for the pulsatile secretion of PGF2ct observed at luteolysis. Flint and Sheldrick (25) reported that constant (non-pulsaCopyright © 1991 Butterworth-Heinemann
573
0739-7240/91/$3.00
574
LUTZ ET AL.
tile) infusion of oxytocin during the luteal phase of the estrous cycle, delayed return to estrus in cyclic sheep. However, the effect of constant infusion of oxytocin on luteal lifespan and uterine secretion of PGF2c~ in cattle is not clear (26,27,28). Therefore, the first objective of this study was to determine the effect of constant infusion of oxytocin on luteal lifespan and secretion of progesterone, estradiol-1713, FSH, LH and prolactin in cattle (Experiment 1). A second objective was to determine whether constant infusion of oxytocin would inhibit the oxytocin-induced secretion of PGF:~ [as measured by the stable PGF2c~ metabolite, 15-keto-13,14-dihydro-PGF2~ (PGFM)] on Day 16 of the bovine estrous cycle (Experiment 2).
MATERIALS AND METHODS Animals and Management. Estrus was synchronized in twenty-four Holstein (n = 16) and Guernsey (n = 8) heifers [12 heifers per experiment - Holstein (n = 8) and Guernsey (n = 4), 12 to 14 months of age weighing ~350 kg] with two 25 mg injections (i.m.) of PGF2c~ (Lutalyse, The UpJohn Company, Kalamazoo, MI) given fifteen d apart. Heifers were visually observed for estrous behavior for 4 d following each injection. In Brahman heifers, the corpus luteum formed following PGF2~-induced luteolysis contained fewer small and large iuteal cells and had a lower response to LH in vitro compared to corpora lutea formed following spontaneous regression (29). If PGF2c~-induced luteolysis did alter luteal cell populations of the subsequently formed corpora lutea in the present study, the effect should have been similar for both the saline and oxytocin-treated heifers. Furthermore, two injections of PGF2ot do not reportedly reduce fertility (30). During both experiments, heifers were housed in individual tie stalls, fed a ration consisting primarily of corn, cottonseed hulls and soybeans twice daily and had fresh water available ad libitum. Experimental Design. Both experiments were designed as 2 × 2 factorials involving treatment (saline or oxytocin) and method of treatment (jugular infusion or osmotic minipump). Experiment 1: Twelve heifers were allotted, by breed and weight, into four treatment groups (n = 3). Treatments were: Group 1 (saline-infusion); Group 2 (saline-osmotic minipump); Group 3 (oxytocin-infusion); Group 4 (oxytocin-osmotic minipump). Treatment with saline or oxytocin began on Day 10 (Day 0 = estrus) of the estrous cycle. A computer-controlled infusion system (31) programmed to deliver either sterile physiological saline (0.9% NaC1) or oxytocin (Sigma Chemical Co., St. Louis, MO; 218 ng oxytocin/h/ kg body weight dissolved in saline) was connected to heifers in Groups 1 and 3, respectively. Osmotic minipumps (Alzet Model 2m12, Alza Corporation, Palo Alto, CA) filled with either saline or 26 mg oxytocin (13 mg/ml) dissolved in saline were placed subcutaneously (under local anesthesia) in the axillary region of heifers in Groups 2 and 4, respectively. On Day 23 of the estrous cycle (treatment day 13), the computer-controlled infusion system was disconnected and the osmotic minipumps were removed. Luteolysis was determined to have occurred when serum concentrations of progesterone had fallen below 1 rig/ ml. Experiment 2: The second experiment was designed and conducted as previously described for Experiment 1 with the addition of an intravenous injection of 100 IU of oxytocin (20 IU/ml) given to all heifers (n = 12) on Day 16 of the estrous cycle. Oxytocin has been used to test the ability of the uterus to secrete PGF2c~ both in vivo (32,33) and in vitro (34,35). Response to the challenge injection of oxytocin was assessed by measuring peripheral concentrations of the stable PGF2~ metabolite, 15-keto-13,14-dihydro-PGF2o~ (PGFM), which has been shown to accurately reflect changes in uterine PGF2ot secretion (36,37). Blood Sampling. Blood samples (10 ml) for both experiments were collected via jugular cannulae daily from Day 3 to 9 and twice daily from Day 10 to 27 of the estrous cycle.
OXYTOCIN AND LUTEAL FUNCTION IN HEIFERS
575
Samples were allowed to clot at room temperature for ~2 hr then centrifuged at 4 C (3000 x g) for 30 min. Serum was divided into 2 aliquots and stored at -20 C for subsequent analysis. Serum concentrations of oxytocin, progesterone, estradiol-17~, FSH, LH and prolactin were determined by radioimmunoassay. Blood samples (5 ml) following the Day 16 challenge injection of oxytocin in Experiment 2 were collected -60, -45, -30, -15, 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 and 180 minutes from the time of injection and serum concentrations of oxytocin, PGFM, FSH and prolactin were determined by radioimmunoassay. Radioimmunoassays. Serum concentrations of oxytocin were determined by a radioimmunoassay procedure developed and validated in this laboratory. A standard solution of oxytocin (400 pg/ml) was prepared using oxytocin acetate salt (Sigma Chemical Co., St. Louis, MO) dissolved in 1% gel-phosphate buffered saline. Oxytocin standard was pipetted in triplicate such that the standard curve contained the following amounts: 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0 and 200.0 pg/tube. A standard serum pool was prepared by adding oxytocin standard to a bovine serum pool. Serial dilutions of the "spiked" standard serum pool (2.5 lal to 500 l.tl) were parallel to the standard curve and recovery of oxytocin from "spiked" sera was 90%. Serum concentrations of oxytocin were determined in duplicate 250 ~tl samples. Oxytocin antiserum (rabbit anti-oxytocin, 100 ~tl of 1:10,000 dilution, Arnel Products Co. Inc., New York, NY) was added to each assay tube except total and non-specific binding tubes. Assay tubes were incubated at 4 C for 48 hr after which 100 lxl of ['25I]iodotyrosyF oxytocin (3 pg/tube; Amersham Corporation, Arlington Heights, IL) was added to each tube. Assay tubes were incubated at 4 C for an additional 12 hr, then 100 ~1 of sheep anti-rabbit (IgG fraction) pre-precipitated antisera was added to each tube except total tubes. Assay tubes were mixed, incubated at room temperature for 10 min and centrifuged at 4 C (3200 x g) for 30 min. Supematant was discarded and radioactivity in the precipitate was counted for 1 min/tube in a gamma counter. Minimum detectable concentration of oxytocin was 1.87 pg/tube. Inter- and intra-assay coefficients of variation were 18.2% and 14.4%, respectively. Serum concentrations of the following hormones were determined by radioimmunoassay procedures previously validated in our laboratory: progesterone (38); estradiol-17~ (31); FSH (39); LH (40); prolactin (31) and PGFM (33). Minimum detectable concentrations of the hormones in serum were 2.05 pg, 1.93 pg, 2.66 ng, 10.25 ng, 1.19 ng and 3.86 pg/tube, respectively. Inter-assay coefficients of variation were 13.9%, 18.3%, 10.2%, 16.8%, 17.2% and 4.1%, respectively. Intra-assay coefficients of variation were 8.8%, 11.3%, 12.8%, 10.3%, 9.2% and 6.7%, respectively. Statistical Analyses. Effects of treatments on estrous cycle length were analyzed by a 2 x 2 factorial analysis of variance (41). Effects of treatments on serum concentrations of oxytocin, progesterone, estradiol-17~, FSH, LH and prolactin were analyzed by analysis of variance for a split plot in time design with repeated measurements (42). Effects of treatments on serum concentrations of estradiol-1713 at luteolysis and effects of the challenge injection of oxytocin on serum concentrations of PGFM, FSH and prolactin were also analyzed by analysis of variance for a split plot in time design. RESULTS Experiment 1. Treatment of heifers with oxytocin extended (P < 0.01) estrous cycle length by an average of 4.7 d compared to heifers treated with saline (Table 1). Serum concentrations of progesterone were not affected by initiation of oxytocin treatment on Day 10 of the estrous cycle and were maintained at elevated levels in oxytocin-treated heifers until treatment ceased on Day 23 of the estrous cycle (Figure 1). Return to estrus after delayed luteolysis occurred within 3 d after cessation of oxytocin treatment. Method of treatment (jugular infusion v s . osmotic minipump) had no effect (P > 0.05) on
576
L U T Z ET AL.
TABLE 1. EFFECT OF SALINE AND OXYTOCIN TREATMENTS (DAYs l 0 TO 23 OF THE ESTROUS CYCLE) ON ESTROUS CYCLE LENGTH
Estrous Cycle Length (days) ~ Treatment
Experiment 1
Experiment 2
Saline
20.5 _+ 0 . 4
18.6 + 0.8
Oxytocin
2 5 . 2 + 0.4*
21.6 _+ 0.8*"
" v a l u e s are m e a n _+ SE. *P < 0.01 c o m p a r e d w i t h s a l i n e v a l u e . **P < 0 . 0 5 c o m p a r e d w i t h s a l i n e v a l u e .
luteal lifespan; therefore, endocrine data were pooled. Serum concentrations of oxytocin were similar (P > 0.10) for saline and oxytocin treated heifers prior to treatment, 36.6 + 5.7 pg/ml and 20.3 + 2.5 pg/ml, respectively. After initiation of treatment, serum concentrations of oxytocin increased (P < 0.01) to 182.5 + 10.1 pg/ml in oxytocin-treated heifers compared to 24.8 + 2.1 pg/ml in saline-treated heifers (Figure 1). Serum concentrations of oxytocin remained elevated throughout the treatment period (Days 10 to 23 of the estrous cycle) in heifers receiving oxytocin via computer-controlled jugular infusion (Figure 1C); whereas, concentrations of oxytocin steadily declined over the treatment period in heifers receiving oxytocin via osmotic minipump (Figure IB). Serum concentrations of estradiol-17[3, LH and prolactin were similar (P > 0.10) for saline and oxytocin treated heifers prior to and during the treatment period (Table 2). Constant infusion of oxytocin had no effect (P > 0.10) on the rise in estradiol-17~, following the decline in progesterone, observed at luteolysis (Figure 2).
0
600
6
400
4
200
2
E v
~" z o
"I.-' u3 L,-I (.9 0
,~
0
O
B
_A E c~
400
6
4
200
© l--
0
>X 0
2 0 10
z
600
8
400
6 4
200
2 0
5
I
I
I
I
I
I
6
9
12
15
18
21
24
27
DAY OF ESTROUS CYCLE Figure I. Mean serum concentrations of progesterone (, ) and oxytocin ( ) in heifers treated with saline (A, n = 6) or oxytocin (B, osmotic minipump, n = 3: C, jugular infusion, n = 3) in Experiment I. Treatment period (Days 10 to 23 of the estrous cycle) is indicated by the solid bar.
OXYTOCIN AND LUTEAL FUNCTION IN HEIFERS
577
TABLE 2. EFFECT OF SALINEAND OXYTOCIN TREATMENTS(DAYs 10 TO 23 OF THE ESTROUS CYCLE) ON SERUM CONCENTRATIONSAOF ESTRADIOL-1715,L H AND PROLACTIN
Estradiol-I 7~ (pglml)
Treatment
LH (ng/ml)
Prolactin (ng/ml)
Pre-TreatmenP Treatment~ Pre-Treatment Treatment Pre-Treatment
Treatment
Ex~fimentl Saline Oxytocin
1.11±0.2 1.60±0.2
1.60±0.2 2.01±0.2
1.26±0.1 1.18±0.1
1.13±0.1 1.57±0.1
24.36±2.1 19.93±1.6
23.28±1.1 21.53±1.4
Experiment2 Saline Oxytocin
0.69±0.1 0.48±0.1
0.66±0.1 0.45±0.1
0.92±0.1 0.94±0.1
0.80±0.1 1.09±0.1
39.14±4.1 40.54±3.5
29.32±2.5 36.02±4.2
~Values are mean _+SE. bPre-treatment value is mean + SE for Days 6 to 10 of the estrous cycle. ~Treatment value is mean + SE for Days 11 to 15 of the estrous cycle.
Serum concentrations of F S H were similar (P > 0.10) for saline and oxytocin treated heifers prior to treatment, 35.4 + 3.4 ng/ml and 32.9 + 2.5 ng/ml, respectively. However, after initiation of treatment, serum concentrations of FSH increased (P < 0.05) to 64.5 + 4.5 ng/ml in oxytocin-treated heifers compared to 24.6 + 1.7 ng/ml in saline-treated heifers (Figure 3). The increase observed in oxytocin-treated heifers occurred within 2.5 d after initiation of oxytocin treatment and persisted until treatment ceased on Day 23 of the estrous cycle. Serum concentrations of F S H returned to baseline within 2.5 d following cessation of oxytocin treatment. The increase in serum concentrations of FSH which occurred coincident with oxytocin treatment could not be attributed to FSH contamination or oxytocin
10
10 8
6
6
4
4
2
E
2 0
o
,.,
13
8
z
6
6
r~
4
4
~
0
a~ l--
v~
LJJ 0
E
m
5-
0
2
2
~
0
0
a:
