Temporal relations between plasma concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol-17beta, progesterone, prolactin, and alpha-melanocyte-stimulating hormone during the follicular, ovulatory, and early luteal phase in the bitch

Compared with other domestic animals, relatively little is known about the changes in, and temporal relations between, reproductive hormones around the time of ovulation in the domestic bitch. Therefore, plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin (PRL), and alpha-melanocyte-stimulating hormone (alpha-MSH) were determined one to six times daily from the start of the follicular phase until 5 days after the estimated day of ovulation in six Beagle bitches. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. A pre-ovulatory PRL or alpha-MSH surge was not observed. The pre-ovulatory FSH and LH surges started concomitantly in four bitches, but in two bitches the FSH surge started 12 h earlier than the LH surge. The FSH surge (110+/-8 h) lasted significantly longer than the LH surge (36+/-5 h). In contrast with the pre-ovulatory FSH surge, the pre-ovulatory LH surge was bifurcated in four of six bitches. The mean plasma LH concentrations before (1.9+/-0.4 microg/L) and after (1.9+/-0.3 microg/L) the LH surge were similar, but the mean plasma FSH concentration before the FSH surge (1.6+/-0.3 U/L) was significantly lower than that after the FSH surge (3.1+/-0.2 U/L). In most bitches the highest plasma estradiol-17beta concentration coincided with or followed the start of the pre-ovulatory LH surge. In five of the six bitches the plasma progesterone concentration started to rise just before or concurrently with the start of the LH surge. In conclusion, the results of this study provide evidence for the differential regulation of the secretion of LH and FSH in the bitch. In addition, the interrelationship of the plasma profiles of estradiol-17beta and LH suggests a positive feedback effect of estradiol-17beta on LH surge release. The start of the pre-ovulatory LH surge is associated with an increase in the plasma progesterone concentration in this species.     

Effects of luteinizing hormone, progesterone, testosterone, estradiol and corticosterone on ovulation and luteinizing hormone release in hens treated with aminoglutethimide

Aminoglutethimide (AG), an inhibitor of steroidogenesis, was administered s.c. to 5 groups of laying hens at a dose of 200 mg AG/kg body weight 9 h before expected midsequence ovulation. This dose has previously been demonstrated to consistently block ovulation. The injection of AG was followed by s.c. injections of: Group 1, 1.0 mg progesterone; Group 2, 0.1 mg estradiol-17 beta; Group 3, 1.5 mg corticosterone, all at 6 h prior to expected ovulation; Group 4, 1.0 mg testosterone at both 8 h and 5 h before expected ovulation; and Group 5, 25 micrograms of ovine luteinizing hormone (LH) at 8 and 50 micrograms ovine LH at 6 h before expected ovulation. For each group, 4 control hens were injected with AG and the appropriate vehicle. Blood samples were taken at 1- or 2-h intervals from the time of AG injection to the expected time of ovulation. The hens were killed 4 h after expected ovulation and examined for the occurrence of ovulation. In all hens injected with vehicle, ovulation and the preovulatory surges of progesterone, testosterone, estradiol-17 beta and LH were inhibited. The plasma concentration of corticosterone was not reduced following an injection of AG. Four of 6 hens ovulated in response to injection of ovine LH, although neither endogenous LH nor progesterone were released. Thus, LH appears to play a direct role in follicular rupture and extrusion of the ovum. The administration of progesterone induced a significant and prolonged rise in LH, restoring AG-blocked ovulation in all hens treated (n = 6). Injections of testosterone restored LH release in all hens and ovulation in 2 of 7 hens treated. Three of 7 hens ovulated in response to the corticosterone injection. A preovulatory rise in LH was not observed, indicating that corticosterone may exert its ovulation-inducing effect directly on the mature follicle. Estradiol-17 beta did not restore LH release or ovulation in any of the hens treated with AG.     

Effects of buserelin injection and deslorelin (GnRH-agonist) implants on plasma progesterone, LH, accessory CL formation, follicle and corpus luteum dynamics in Holstein cows

The influence of Buserelin injection and Deslorelin (a GnRH analogue) implants administered on Day 5 of the estrous cycle on plasma concentrations of LH and progesterone (P4), accessory CL formation, and follicle and CL dynamics was examined in nonlactating Holstein cows. On Day 5 (Day 1 = ovulation) following a synchronized estrus, 24 cows were assigned randomly (n = 4 per group) to receive 2 mL saline, i.m. (control), 8 micrograms, i.m. Buserelin or a subcutaneous Deslorelin (DES) implant in concentrations of 75 micrograms, 150 micrograms, 700 micrograms or 2100 micrograms. Blood samples were collected (for LH assay) at 30-min intervals for 2 h before and 12 h after GnRH-treatment from cows assigned to Buserelin, DES-700 micrograms and DES-2100 micrograms treatments and thereafter at 4-h intervals for 48 h. Beginning 24 h after treatment, ovaries were examined by ultrasound at 2-h intervals until ovulation was confirmed. Thereafter, ultrasonography and blood sampling (for P4 assay) was performed daily until a spontaneous ovulation before Day 45. A greater release of LH occurred in response to Deslorelin implants than to Buserelin injection (P < 0.01). Basal levels of LH between 12 and 48 h were higher in DES-700 micrograms group than in DES-2100 micrograms and Buserelin (P < 0.05). The first wave dominant follicle ovulated in all cows following GnRH treatment. Days to CL regression did not differ between treatments, but return to estrus was delayed (44.2 vs 27.2 d; P < 0.01) in cows of DES-2100 micrograms group. All GnRH treatments elevated plasma P4 concentrations, and the highest P4 responses were observed in the DES-700 micrograms and DES-2100 micrograms groups. The second follicular wave emerged earlier in GnRH-treated than in control cows (9.9 vs 12.8 d; P < 0.01). However, emergence of the third dominant follicle was delayed in cows of DES-2100 micrograms treatment (37.0 d) compared with DES-700 micrograms (22.2 d), Buserelin (17.8 d) or control (19.0 d). In conclusion, Deslorelin implants of 700 micrograms increased plasma P4 and LH concentrations and slightly delayed the emergence of the third dominant follicle. On the contrary, Deslorelin implants of 2100 micrograms drastically altered the P4 profiles and follicle dynamics.     

Bromocriptine-induced premature oestrus is associated with changes in the pulsatile secretion pattern of follicle-stimulating hormone in beagle bitches

The secretory profiles of LH and FSH were investigated before and during the administration of bromocriptine in six beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 6 h every 2 weeks until the next ovulation. Bromocriptine treatment was started 100 days after ovulation. Both before and after bromocriptine treatment, LH and FSH pulses occurred together. The mean duration of the FSH pulse (120 min) was significantly longer than that of the LH pulse (80 min). The interoestrous interval in the bitches treated with bromocriptine was significantly shorter than that of the preceding cycle (160 +/- 3 versus 206 +/- 24 days). The mean basal plasma FSH concentration (7.4 +/- 0.6 versus 6.1 +/- 0.7 iu l-1) and the mean area under the curve for FSH (46.6 +/- 4.7 versus 40.4 +/- 4.4 iu l-1 in 6 h) increased significantly after the start of the bromocriptine treatment. In contrast, the differences in mean basal plasma LH concentration (2.1 +/- 0.2 versus 2.0 +/- 0.2 micrograms l-1) and the mean area under the curve for LH (19.0 +/- 3.1 versus 19.5 +/- 2.5 micrograms l-1 in 6 h) between the day before and 14 days after the start of the bromocriptine treatment were not significant. Bromocriptine administration also lowered the mean amplitude of the FSH pulse and shortened the mean duration of the FSH pulse, without influencing the LH pulse. In addition to demonstrating the concurrent pulsatile secretion of LH and FSH, the results of the present study demonstrate that the bromocriptine-induced shortening of the interoestrous interval in the bitch is associated with an increase in plasma FSH concentration without a concomitant increase in plasma LH concentration. This finding indicates that treatment with the dopamine agonist bromocriptine increase plasma FSH to a concentration that results in the enhancement of follicle development.     

Plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2-alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross-bred bitches

The canine corpus luteum (CL) typically sustains elevated plasma progesterone concentrations for 2 months or more, with a peak approximately 15-25 days after ovulation, followed by a slow decline. The processes involved in the slow, protracted regression of the CL over the remaining 1.5-2-month period in nonpregnant bitches and until shortly prepartum in pregnant bitches are not well characterized. The rapid luteolysis that occurs immediately prepartum appears to be a result of a prepartum rise in peripheral PGF. The potential role of PGF in the slow regression process in the several weeks preceding parturition and in nonpregnant bitches after 15-25 days after ovulation is not known. Therefore, plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2-alpha (PGFM), progesterone (P4) and estradiol (E2) were determined and compared in bitches during nonpregnant diestrus (n = 9) or pregnancy (n = 8). During the gradual decrease in plasma concentrations of progesterone in both groups, the P4 pattern appeared unrelated to changes in either E2 or PGFM concentrations. The PGFM pattern was different between diestrus and pregnant bitches (P > 0.01); there was an apparent progressive but slow increase in PGFM in pregnant bitches from Days 30 to 60, followed by a large increase prior to parturition; concentrations declined immediately postpartum. However, there were no increases in PGFM during the same interval in nonpregnant bitches. Mean estradiol concentrations were sporadically elevated during the last third of pregnancy and less so in nonpregnant diestrus; there was no acute prepartum increase in estradiol associated with the PGFM increase. In summary, although there were no apparent changes in peripheral PGF2alpha concentration involved in regulating the slow protracted phase of luteal regression in nonpregnant bitches, modest increases in PGFM may play a role in ovarian function after mid-gestation in pregnant bitches. Furthermore, the acute prepartum rise in PGFM was not dependent on any concomitant increase in estradiol concentrations.     

Differential regulation of the secretion of luteinizing hormone and follicle-stimulating hormone around the time of ovulation in the bitch

Plasma concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined 3-6 times daily in six Beagle bitches from the start of the follicular phase until 5 d after the estimated day of ovulation. The aim of the study was to gain more detailed information regarding the changes in and the temporal relation between these hormones around the time of ovulation. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. The mean duration of the pre-ovulatory FSH surge (110 +/- 8 h) was significantly longer than that of the pre-ovulatory LH surge (36 +/- 5 h). The FSH surge started concomitantly with the pre-ovulatory LH surge in four bitches, and 12 h before the start of the LH surge in the other two bitches. The pre-ovulatory LH surge had a bifurcated pattern in four bitches. The mean plasma LH concentration before (1.9 +/- 0.4 microg/L) and after (1.9 +/- 0.3 microg/L) the pre-ovulatory LH surge were similar. The mean plasma FSH concentration during the period 72-28 h before the pre-ovulatory LH surge (1.6 +/- 0.3 U/L) was lower (P < 0.001) than that during the period 100-144 h after the pre-ovulatory LH surge (3.1 +/- 0.2U/L). In conclusion, this study demonstrated concurrent pre-ovulatory surges of FSH and LH and provided more evidence for differential regulation of the secretion of FSH and LH.     

The effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations

The effects of anticoagulant, storage time, storage temperature, and assay method, on laboratory measurements of blood progesterone concentrations of dogs is unclear; these factors have had a dramatic effect on blood progesterone concentrations in other species (particularly cows). In six experiments, we determined the effects of assay technique (chemiluminescence versus radioimmunoassay (RIA)), storage time, and temperature, as well as the use of heparinized plasma versus serum (coagulated blood) on measured progesterone concentrations of bitches. The studies showed that: (a) RIA measured significantly higher serum progesterone concentration (SPC) than chemiluminescence; (b) refrigeration of whole blood during the first 2 h after sample collection significantly decreased measured SPC; (c) progesterone concentration in heparinized plasma was not affected by storage temperature of whole blood for at least 5 h; (d) refrigeration of whole, clotted blood did not affect SPC, provided that samples were held at room temperature for the first 2 h after collection. These findings are of particular importance when blood samples are collected for determination of the initial rise in SPC that is associated with the LH surge in estrous bitches.     

Radioreceptor assay for cynomolgus monkey serum luteinizing hormone

Serum luteinizing hormone (LH) of cynomolgus monkeys was measured by radioreceptor assay (RRA). The assay system consisted of 100 microliter of standard or unknown samples, 100 microliter of a receptor preparation and 100 microliter of 125I-LH (LER-960). Dispersed interstitial cells of mature rat testes were suspended in Tris-HCl buffer containing MgSO4 (5 mM), bovine serum albumin (0.1%) and sucrose (0.3 M) and used as the receptor preparation. 125I-LH was dissolved in the same buffer. The incubation was carried out at 37 degrees C for 2 to 3 hr. The nonspecific inhibitory effect of sera in the radioreceptor assay system was compensated for by using LH-free diluent prepared by heat treatment of pooled sera. Validation of the assay demonstrated good reliability in terms of accuracy, precision and sensitivity (equivalent to 3.1 micrograms LER-907/ml of serum). Recovery experiments were performed by adding known amounts of cynomolgus pituitary LH to the normal serum and mean recovery and the standard deviation of the mean was 86 +/- 13%. The intra- and inter-assay coefficients of variation were 7 and 9%, respectively. By using this RRA system, cyclic changes in serum LH during the menstrual cycle were determined in three adult female cynomolgus monkeys. These results indicate that this RRA system is useful in measuring serum LH in female cynomolgus monkeys.     

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.     

Ovulation induction in anestrous bitches by pulsatile administration of gonadotropin-releasing hormone

Preliminary studies in anestrous Beagle bitches demonstrated that a single injection of gonadotropin-releasing hormone (150 micrograms) produced a rapid, physiological rise in serum estradiol lasting 1-3 days while progesterone remained below 1 ng/ml, whereas serial injections of FSH rapidly produced greater elevations in estradiol and a rapid rise in progesterone over 2 ng/ml. Consequently, attempts to induce fertile ovulation by means of pulsatile intravenous administration of GnRH (1 pulse/1.5 hours for 6-12 days; 0.04-0.43 micrograms/kg body weight/pulse) were conducted in eight anestrous bitches. Willingness to mate, serum progesterone levels and results of mating were monitored. In six of the eight bitches, vulval and vaginal signs of proestrus occurred by Day 2-4 after initiation of treatment (Day 0); but, two bitches showed negligible responses. In five of the six bitches in which proestrus was induced, behavioral (n = 4) and vaginal (n = 5) correlates of early estrus occurred by Day 5-7 of treatment and breedings occurred over a period of 4-12 days. Following onset of estrus, four of the five bitches had increases in serum progesterone levels between Days 14 and 18 after initiation of treatment (and 4-11 days after cessation of treatment); three of them became pregnant and whelped normal litters (ranging from 9 to 11 pups). The fifth bitch did not have elevated progesterone during the induced estrus, and upon return to estrus one month later was successfully bred and whelped a normal litter of 10 pups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of luteinizing hormone and oestradiol in plasma and response to injection of gonadotrophin-releasing hormone analogue at selected stages of anoestrus in domestic bitches.

Concentration of plasma luteinizing hormone (LH) and oestradiol concentrations and responses to a standard challenge with a gonadotrophin-releasing hormone (GnRH) analogue were measured at certain stages of anoestrus during consecutive cycles in five beagle bitches. Blood samples were collected every 20 min for 6h followed immediately by injection of GnRH analogue (0.16 micrograms i.v.) and collection of further samples after 10, 20, 40 and 60 min. Five, 10, 17 and three such sampling sequences were obtained during the luteal phase, transition to anoestrus, anoestrus and pro-oestrus respectively (i.e. 154-71, 114-44, 85-11 and 7-1 days before the preovulatory LH peak, respectively). Pulsatile LH secretion occurred spontaneously at all stages of the luteal phase and anoestrus and there was no effect of cycle stage on mean LH concentration or variability. In contrast, oestradiol could not be detected in most samples from early and mid-anoestrus until approximately one month before the preovulatory LH peak, after which average oestradiol concentration and between sample variability appeared to increase. Mean (+/- SEM) oestradiol concentration for all samples collected from 100-75, 74-50, 49-25, 24-10 and 9-1 days before LH peak was 1.4 +/- 0.1, 1.3 +/- 0.1, 2.4 +/- 0.3, 11.0 +/- 1.4 and 36.0 +/- 3.2 pg ml-1, respectively. Plasma LH concentration increased in all bitches after GnRH analogue injection (2.7 +/- 0.7 ng ml-1 at t = 0, 12.5 +/- 1.0 ng ml-1 at t = 10 min, mean +/- SEM, n = 35) regardless of cycle stage.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pituitary-ovarian axis in dogs with remnant ovarian tissue

It can be difficult to confirm the presence of remnant ovarian tissue (ROT) in bitches that are presumed to be ovariohysterectomised. A GnRH stimulation test can be used to distinguish ovariectomised bitches from those in anoestrus, but it is uncertain whether the GnRH-induced changes in plasma LH and oestradiol concentrations that occur in intact bitches also occur in ROT-bitches. We report here eighteen ROT-bitches and compare the results of GnRH stimulation tests with those of six ovariectomised and six bitches in anoestrus.The basal (n = 17) and/or GnRH-stimulated (n = 18) plasma oestradiol concentration was above the detection limit of the assay, i.e., < 7 pmol/l, in all ROT-bitches but below the detection limit in all ovariectomised bitches. Basal plasma LH concentration was significantly higher in ROT-bitches (4.1 ± 0.7 μg/L) than those in anoestrus (0.64 ± 0.04 μg/L), and significantly lower than in ovariectomised bitches (20.2 ± 3.6 μg/L). Basal plasma LH concentration was relatively high in bitches in which there was a long interval between ovariectomy and appearance of oestrus. GnRH administration resulted in a significant increase in plasma LH and oestradiol concentrations in ROT-bitches. The GnRH-induced increase and subsequent decline in plasma LH concentration were significantly less in ROT-bitches than in either ovariectomised bitches or those in anoestrus. The GnRH-induced increase in plasma oestradiol concentration was significantly smaller in ROT-bitches than in those in anoestrus.In conclusion, the results of this study demonstrate that in dogs ROT is associated with noticeable changes in the pituitary-ovarian axis and suggest that a GnRH stimulation test may be used to distinguish between completely ovariectomised bitches and those with ROT.     

Acute-phase protein profile during gestation and diestrous: proposal for an early pregnancy test in bitches

Early pregnancy diagnosis in bitches has special relevance for adequate medical assistance to assure normal gestation, diagnosis of abortion or embryonic resorption, undesirable pregnancy interruption and dog owners wishing to assure medical assistance during parturition and to program participation of females in dog shows. The aim of this study was to verify acute-phase protein profile variation as a consequence of generalized inflammatory reaction due to embryonic endometrial invasion and use alterations as a method for early pregnancy diagnosis. Also the relationship between hormonal status and acute-phase proteins concentrations was assessed. Weekly serum samples were collected from 20 non-pregnant (NP) bitches and 20 pregnant females (P) to determine levels of fibrinogen, haptoglobin, ceruloplasmin, seromucoid, glycoprotein, alpha(2) globulin, progesterone and estradiol-17beta. No correlation was found between the implantation sites formed (number of pups born) and the hepatic stimulus for the acute-phase protein production. The conclusions are that acute-phase proteins can be used as an early pregnancy test for bitches from the 3rd week of gestation (14th-21st day post LH peak) for haptoglobin assay (values above 112.42 mg/dl of HbCN binding capacity), from the 4th to the 6th week (21st-42nd day post LH peak) for ceruloplasmin (values above 12.76+/-5.29 U/l), from the 4th week (21st-28th day post LH peak) for glycoprotein (values above 13.67%) and from the 4th week of gestation (21st-28th day post LH peak) for alpha(2) globulin (values above 0.61 g/dl). Fibrinogen and seromucoid increased in the P group from the 5th to the 6th week, respectively, thus not being suitable as parameters for an early pregnancy diagnosis. Relationship between ceruloplasmin and estradiol-17beta and seromucoid and progesterone were verified. For the acute-phase protein test it is important to verify bitches' healthy condition and to assure the precise mating dates to avoid false-positive and -negative results, respectively.     

Methylation in bovine luteal cells as a regulator of luteinizing hormone action

Experiments were conducted to determine if methylation is a part of the mechanism by which luteinizing hormone (LH) and epinephrine stimulate progesterone production by dispersed bovine luteal cells. Corpora lutea (CL) were collected from 24 Holstein heifers on Day 10 of the estrous cycle and dispersed with collagenase. Net progesterone accumulation, representing total progesterone synthesized by 10(6) cells during a 2-h incubation was determined. Cells from 7 CL were treated with 0 and 5 ng LH, in the presence and absence of methylation inhibitor, S-adenosyl-homocysteine (SAH, 1 mM). LH-stimulated progesterone production was inhibited (P less than 0.05) in the presence of SAH(209 +/- 19 vs. 119 +/- 7 ng/10(6) cells). In the absence of LH, progesterone production was unaffected (87 +/- 22 vs. 68 +/- 28) by SAH. Cells from 4 CL were treated with 10 micrograms epinephrine or 10 micrograms isoproterenol with and without SAH. Both epinephrine and isoproterenol-stimulated progesterone production was inhibited (P less than 0.05) by the presence of SAH (204 +/- 24 vs. 125 +/- 18 and 198 +/- 15 vs. 130 +/- 8). Progesterone production by cells from 4 CL was unaffected by the presence of SAH when treated with Medium 199 (M199) (75 +/- 32), 10 micrograms cholera toxin, which directly stimulates adenylate cyclase on the cytoplasmic side of plasma membranes (168 +/- 19), or 3 mM dibutyryl cAMP (210 +/- 40).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of exogenous progestin on the occurrence of proestrous or estrous signs, plasma concentrations of luteinizing hormone and estradiol in deslorelin (GnRH agonist) treated anestrous bitches

The objectives of this study were to confirm: (i) whether progestin treatment suppressed GnRH agonist-induced estrus in anestrous greyhound bitches; and (ii) the site of progestin action (i.e. pituitary, ovary). All bitches received a deslorelin implant on Day 0 and blood samples were taken from -1 h to +6 h. Five bitches were treated with megestrol acetate (2 mg/kg orally once daily) from -7 d to +6 d (Group 1) and 10 bitches were untreated controls (Group 2). Proestrous or estrous signs were observed in 4 of 5 bitches in Group 1, and 4 of 10 bitches in Group 2 (P = 0.28). The plasma LH responses (area under the curve from 0 to 6h after implantation) were higher (P = 0.008) in Group 2 than in Group 1. Plasma LH responses were similar (P = 0.59) in bitches showing signs of proestrus or estrus (responders) and in non-responders. The plasma estradiol responses (calculated as for LH response) were greater in Group 1 than in Group 2 (P = 0.048), and in responders than in non-responders (P = 0.02). In conclusion: (i) progestin treatment (a) did not suppress the incidence of bitches showing deslorelin-induced proestrus or estrus, and (b) was associated with a reduced pituitary responsiveness and an increased ovarian responsiveness to deslorelin treatment; (ii) the occurrence of proestrous or estrous signs reflected increased ovarian responsiveness to induced gonadotrophin secretion and not increased pituitary responsiveness to deslorelin.     

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus)

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.     

Sodium cloprostenol administered at a continuous low dosage induces polydipsia and suppresses luteal function in early dioestrous bitches.

The aim of this study was to determine whether sodium cloprostenol administered at a continuous low dosage induced luteolysis and polydipsia in early dioestrous bitches. Sodium cloprostenol was administered subcutaneously to greyhounds at doses of 4.04-5.19 microg/kg/day (treated group, n=5) or 0 microg/kg/day (control group, n=5) delivered by mini-osmotic pumps for 7 days. The treated bitches and two of the control bitches were in early dioestrus (Days 5-14, and 6 and 10, respectively) when the mini-osmotic pump was inserted (Day 0). Concentrations of plasmatic progesterone were measured in dioestrous bitches each day from Day -2 to 7, and then weekly until Day 90. Daily intake of water was ascertained in all bitches from Day -2 until Day 10, and their weight was measured on Days -2, 6 and 13. Biochemical analyses on plasma for concentrations of urea and glucose, and urinalyses were performed on all bitches before (Day -1), during (Day 4) and after treatment (Day 10). Concentrations of plasmatic progesterone declined dramatically and rapidly in treated bitches after Day 0 to <2.9 ng/ml but were not similarly affected in the dioestrous control bitches. However, in three of five treated bitches, concentrations of plasmatic progesterone increased to >1 ng/ml in the period from Day 10 to 90 indicating that luteolysis was incomplete. All treated bitches were polydipsic (intake of water >100 ml/kg/day) for 2-6 days during the period of treatment, and for 0-2 days immediately after treatment (Days 7 and 8). One control bitch was polydipsic on Days -2, -1 and 0. The treated bitches were also polyuric since they were hyposthenuric (<1.007, n=4) or isothenuric (1.010, n=1) on Day 4, their weight did not increase and no gastrointestinal or respiratory effects were observed. The control bitches were always hypersthenuric when measured during and after treatment (>1.021). Biochemical analyses of plasma and other data obtained from urinalyses did not reveal any differences between groups. This study indicated that sodium cloprostenol administered at a continuous low dosage induced polydipsia and suppressed luteal function in early dioestrous bitches.     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1 alpha on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF1 alpha were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 microgram PGI2, 10 micrograms PGI2, and 10 micrograms PGI2 plus 5 ng LH averaged 99 +/- 42, 353 +/- 70, 152 +/- 35, 252 +/- 45, and 287 +/- 66 ng/ml (n = 4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.     

Suppression of luteal function in dogs by luteinizing hormone antiserum and by bromocriptine

Beagle bitches were treated with equine anti-LH serum (ALHS) or the dopamine agonist bromocriptine at selected times during the 2-month luteal phase of the ovarian cycle or pregnancy. After a single injection of ALHS (10 ml, i.m.) at Day 42 of pregnancy (N = 2) or the ovarian cycle (N = 3), progesterone was reduced (P less than 0.05) to 7-24% of preinjection values within 1-2 days, and by 4-8 days returned to levels not different from those in control bitches treated with normal horse serum. Injections of bromocriptine (0.1 mg/kg, i.m.) daily for 6 days caused abrupt declines in progesterone which lasted 6-8 days in bitches treated at Day 8 or 22 of pregnancy (N = 5). In bitches treated at Day 42 of pregnancy (N = 3) or in non-pregnant cycles (N = 4) the bromocriptine treatment caused declines (P less than 0.05) in progesterone which were permanent, extensive (less than 2 ng/ml), and therefore abortive. The declines in progesterone in response to immunoneutralization of LH and to prolactin-lowering doses of a dopamine agonist demonstrate that normal luteal function in dogs requires both LH and prolactin.