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.     

Hormonal concentrations in bitches with primary uterine inertia

Normal labor is accompanied by sequential changes in blood concentrations of prostaglandin F2α (measured as 15-ketodihydro-PGF2α = PGFM), progesterone, estradiol, oxytocin, vasopressin, and of elevated cortisol levels. The aim of this study was to investigate hormone concentrations in dogs diagnosed with primary uterine inertia before and during treatment by cesarian section. The hypothesis was the dogs would have abnormally low plasma concentrations in one or several of the hormones involved in parturition. The study comprised seven bitches with total primary uterine inertia (dystocia group) treated with cesarian section and six healthy bitches (control group) subjected to planned cesarean section. Blood samples were taken before anesthesia, before surgery started, on delivery of the first puppy and on delivery of the last puppy. The progesterone:PGFM ratio in plasma was higher in the dystocia group than in the control group, but the serum estradiol concentration did not differ between groups. The plasma concentrations of oxytocin and vasopressin increased in both groups when the first puppies were delivered, but both hormones were more elevated in the control group than in the dystocia group on delivery of the last puppies. The plasma cortisol concentration increased to the same level in both groups. In conclusion, the ratio between progesterone and PGFM was higher and the oxytocin and vasopressin concentrations lower in the dystocia dogs than in the control dogs. The findings indicate that these hormones are involved in the pathophysiology of total primary uterine inertia in bitches.     

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 progesterone, prolactin and relaxin in the luteal phase and pregnancy in normal and short-cycling German Shepherd dogs

Twenty-two nonpregnant and 19 pregnant German Shepherd dogs were assigned to either a control group or a suspected short-cycling group, based on the interestrous interval (> or = 6 month and < 5 month, respectively) and data from previous pregnancies. Blood serum concentrations of progesterone and prolactin were determined from days 5 to 60 (day 0 = ovulation) for characterization of luteal function. In pregnant bitches, placental integrity was additionally assessed by relaxin concentrations. The nonpregnant, suspected short-cycling bitches had significantly lower progesterone concentrations than the controls, indicating decreased luteal activity both in the autonomous and prolactin-dependent period. In the pregnant suspected short-cycling bitches, unavoidable progesterone supplementation prevented assessment of luteal function; it may have suppressed prolactin secretion (significantly lower prolactin concentrations from days 20 to 60, compared with the pregnant control group), but deficient prolactin secretion affecting luteal function cannot be excluded. The significantly lower relaxin concentrations, together with a high incidence of embryonic death found in the pregnant, suspected short-cycling group, may indicate loss of placental integrity and may have caused decreased prolactin concentrations.     

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.     

Effects of dose and duration of continuous GnRH-agonist treatment on induction of estrus in beagle dogs: competing and concurrent up-regulation and down-regulation of LH release

Dose-response estrus-induction trials were conducted during anestrus in 93 treated and 6 control bitches, a continuous administration of the GnRH-agonist lutrelin with a potency 150 x GnRH, and at six different doses from 0.2 to 4.8 microg/kg/d for 7-14 days in 15 groups of six to eight dogs each in defined stages of natural or pharmacologically determined anestrus. Agonist treatment induced clinically and cytologically normal proestrus (in 89% of cases) within 4.8 +/- 0.2 x days, and resulted in behavioral estrus (71%), spontaneous late-proestrus LH (and FSH) surges, ovulation (59%) and pregnancy (44%) in a dose dependent manner. Outcomes of ovulation and pregnancy in most cases required that the dose be sufficiently large enough to routinely stimulate a large initial increase in LH and FSH (i.e., > or = 0.6 microg/kg/d), and of sufficient duration (i.e., > 7 days) to ensure that supra-basal gonadotropin levels persistedntil no longer needed for spontaneous continuation of an induced proestrus. Success additionally required that the GnRH dose be modest enough (i.e., < 1.8 microg/kg/d) to not excessively down-regulate spontaneous pre-ovulatory surge release of gonadotropin or be removed shortly before or at the time when the LH surges typically occurred (10-13 days after initiation of treatment). The 1.8 microg dose was compared to saline to assess the time course of its down-regulation action on serum LH in six ovariohysterectomized bitches compared to four saline-related controls. Results in intact bitches receiving the 1.8-microg doses demonstrated an LH-releasing effect for 10-11 days that overlapped a period of obvious down-regulation seen with the same dose after 3 days in the ovariohysterectomized bitches. In the latter, however, complete down-regulation to anestrus-like values did not occur until after 18-21 days of treatment. A dose of 0.6 microg/kg/d for 12 days yielded the best estrus-induction results, including pregnancy rates of 100% in six bitches treated in natural-anestrus bitches, six bitches in which anestrus had been advanced by a luteolytic prostaglandin treatment and in six bitches in which anestrus had been extended by progesterone implants administered for 3 months. Although lutrelin is not commercially available, these results provide guidelines for the development of estrus-inducing protocols with other GnRH-agonists of known biopotencies.     

Reproductive hormone concentrations during estrus,pregnancy, and pseudopregnancy in the Labrador bitch

Serum luteinizing hormone (LH), estrone, estradiol-17β, and progesterone were measured during the estrous cycle, pregnancy and parturition in seven adult pregnant Labrador bitches and during the estrous cycle and one gestation length equivalent in six adult pseudopregnant bitches. Although the duration of proestrus was similar in both groups, the duration of estrus was longer in the bitches that subsequently became pregnant. Mean serum LH concentrations were similar in both groups during most of the study. However, during Weeks 6 to 9 after the preovulatory LH peak, serum LH concentration was higher in both pseudopregnant and pregnant groups of animals and declined to basal levels thereafter. Mean serum estrone concentrations in the pregnant animals were higher than those of pseudopregnant animals and remained elevated throughout gestation, followed by a decline at whelping. Serum estradiol-17β levels were higher during the 4 wkimmediately following ovulation in the pseudopregnant group compared with those observed in pregnant animals. Serum progesterone concentrations generally remained higher during pseudopregnancy compared with those of the pregnant animals during gestation.In conclusion, a major difference between pregnant and pseudopregnant bitches is a pregnancy-specific elevation in estrone levels. The placenta may be a likely source of estrone during pregnancy.     

Estrus induction in Beagle bitches with the GnRH-agonist implant containing 4.7 mg Deslorelin

Estrogens, gonadotrophins, dopamine agonists, gonadotrophin releasing hormone (GnRH) and its agonists have been used for estrus induction in bitches. A long acting GnRH agonist implant (4.7 mg Deslorelin; Suprelorin®, Virbac) with a continuous hormone release has been developed for suppression of sexual function in male dogs. In this study we administered the Deslorelin implant placed subcutaneously on the medial side of the leg to induce estrus in 11 anestrous Beagle bitches (group A). 6 Beagle bitches (group B) with a spontaneous estrous cycle were used as controls. The progress of pre-estrus and estrus was documented by behaviour, vaginoscopy, vaginal cytology and progesterone concentration. In group A a bloody vaginal discharge was detected on average 4.8 (range 3-10) d after application of the implant. At this moment implants were removed under local anaesthesia. Pre-estrus lasted for an average of 4.5 d (range 1-12). All bitches showed estrous signs and ovulated. The ovulation took place on day 8.2 (range 4-15) after start of pre-estrus. In group B pre-estrus lasted for 7.5 d (range 6-9), and the mean day of ovulation was day 11 (range 9-13). As a consequence of ovulation, progesterone serum concentrations exceeded 10 ng/ml during or after the time of ovulation in all bitches. All bitches were bred to fertile Beagle stud dogs or inseminated with fresh semen intravaginally. Between days nine and 19 after ovulation all bitches underwent ovariohysterectomy. The uterine horns were flushed and flushes were examined for ova or embryos. The pregnancy rate in group A was 63.6% and in group B 66.7%. Despite the significantly shorter period of pre-estrus a fertile estrus could be induced in 7 out of 11 treated bitches. Induction of a fertile estrus can be achieved with a GnRH-implant—already registered for the use in male dogs—placed subcutaneously on the medial side of the leg.     

Effects of aglépristone, a progesterone receptor antagonist, administered during the early luteal phase in non-pregnant bitches

Aglépristone, a progesterone receptor antagonist, was administered to six non-pregnant bitches in the early luteal phase in order to determine its effects on the duration of the luteal phase, the interestrous interval, and plasma concentrations of progesterone and prolactin. Aglépristone was administered subcutaneously once daily on two consecutive days in a dose of 10 mg/kg body weight, beginning 12 +/- 1 days after ovulation. Blood samples were collected before, during, and after administration of aglépristone for determination of plasma progesterone and prolactin concentrations. The differences in mean plasma concentration of progesterone and of prolactin before, during, and after treatment were not significant. Also, the duration of the luteal phase in the six treated bitches (72 +/- 6 days) did not differ significantly from that in untreated control dogs (74 +/- 4 days ). However, the intervals during which plasma progesterone concentration exceeded 64 and 32 nmol/l were significantly shorter in the six treated bitches than in untreated control dogs. The interestrous interval was significantly shorter in beagle bitches treated with aglépristone (158 +/- 16 days) than in the same group prior to treatment (200 +/- 5 days ). It is concluded that administration of aglépristone during the early luteal phase in the non-pregnant bitch affects progesterone secretion, but not sufficiently to shorten the luteal phase. The shortening of the interestrous interval suggests that aglépristone administered in the early luteal phase influences the hypothalamic-pituitary-ovarian axis.     

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.     

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate)

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.     

Plasma relaxin levels in pregnant and lactating dogs

The pattern of plasma relaxin has been studied during pregnancy and following parturition in two breeds of dogs, Labrador retrievers and beagle hounds. Blood samples were collected at weekly intervals following mating and during pregnancy, parturition, and lactation. Relaxin, progesterone, and estradiol-17 beta were determined by specific double antibody radioimmunoassays. Immunoreactive relaxin (IR) was not detectable in plasma of male dogs, bitches in anestrous, or pseudopregnant bitches that had undergone an infertile mating. IR was first detectable in plasma in the third or fourth week of gestation in retrievers and beagles. IR levels rose to a peak of 4-5 ng/ml in both breeds. The peak plasma levels were reached 2-3 wk before whelping and declined significantly prior to that event. IR then persisted during lactation at a level of 0.5-2 ng/ml for 4-9 wk, but was significantly higher (p less than 0.01) at all time periods and persisted longer in labradors than in beagles. The secretion of relaxin did not parallel that of progesterone, which was highly elevated in the first samples drawn (during the first week of pregnancy), remained high through 5 or 6 wk of gestation, then slowly declined until the time of parturition, becoming undetectable during lactation. Plasma estradiol-17 beta was low after the second week of pregnancy in both breeds of dogs and became undetectable during lactation. The source of relaxin in the dog is not known currently, and its sites of secretion and role in pregnancy are currently under investigation in our laboratories. The dog is the first species in which plasma IR is detectable during lactation using antibody R6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs

Five pregnant beagle bitches were treated with 2.5 mg mifepristone/kg body weight, twice a day, for 4.5 days starting at Day 32 of gestation. Results of fetal ultrasonography and assay of serum progesterone concentrations every 2-4 days were compared to those in 5 control bitches. Mifepristone resulted in a premature (P less than 0.01) termination of pregnancy (36 +/- 1 vs 65 +/- 1 days), without side effects. The antiprogestagen also caused progesterone to decline to less than 1 ng/ml by Day 40-45 after the preovulatory LH peak (vs 64-67 days in controls) and reduced (P less than 0.05) mean concentrations on Days 34-50 (2.2 +/- 0.5 vs 6.3 +/- 0.3 ng/ml). The results suggest that antiprogestagen therapy is a safe means to terminate unwanted pregnancy in dogs, and that luteal function in pregnant bitches is dependent on luteotrophic support that is blocked by antiprogestagen treatment, directly or indirectly, due to termination of pregnancy.     

Hormonal changes in spontaneous and aglépristone-induced parturition in dogs

To increase our understanding of the endocrine changes associated with parturition in dogs, plasma concentrations of progesterone (P4), 15-ketodihydroprostaglandin F(2alpha) (PGFM), estradiol-17-beta (E2beta), cortisol, ACTH, prolactin (PRL), LH, and FSH were measured in six spontaneously whelping bitches and in six bitches in which parturition was induced with the progesterone-receptor blocker aglépristone on day 58 of pregnancy. Expulsion of pups in the induced group took place in the presence of P4 concentrations that were still elevated. PGFM concentrations increased before parturition in both groups, but levels were lower in the induced bitches. PGFM levels reached a maximum in both groups during parturition and quickly decreased in the spontaneously whelping group after parturition, but remained elevated in the induced group. In both groups, cortisol concentrations reached similar maximum levels during the last 30 h before the onset of expulsion. During the 3 days postpartum, cortisol concentrations were higher in the induced group. The highly variable ACTH concentrations did not differ significantly throughout the study within or between groups. In both groups, E2beta concentrations decreased and PRL concentrations increased between the late gestational period and the 30-h period before parturition. Concentrations of both LH (spontaneously whelping group) and FSH (both groups) decreased between late gestation and the postpartum period. The results of this study illustrate the hormonal changes around parturition in the bitch, and reveal that aglépristone-induced parturition is associated with still incomplete luteolysis, an altered PGFM profile, and elevated postpartum cortisol concentrations as compared with spontaneously whelping dogs.     

Effects of the 3β-hydroxysteroid dehydrogenase inhibitor trilostane on luteal progesterone production in the dog

Interference with the pregnancy-maintaining influence of progesterone is the basis of most methods for termination of unwanted pregnancy in dogs. The currently available methods are based on induction of luteolysis or blocking of the progesterone receptor. Inhibition of progesterone synthesis using a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD) could be another strategy to terminate unwanted pregnancies.In this study we investigated the effects of the 3β-HSD inhibitor trilostane on corpus luteum function in non-pregnant bitches. Trilostane was administered orally for seven consecutive days in either the pituitary-independent part of the luteal phase (PIP, start of treatment on D11 after ovulation, n = 6) or the pituitary-dependent part (PDP, start of treatment on D31 after ovulation, n = 6), in an oral dose of about 4.5 mg/kg bw, twice daily. Results were compared with those obtained in control bitches (n = 6). ACTH stimulation tests were performed to assess adrenocortical reserve capacity.Trilostane caused no apparent side effects and ACTH stimulation tests revealed good suppression of cortisol secretion. Trilostane also caused a significant decrease in plasma progesterone concentration. When it was stopped during PIP, progesterone secretion was completely restored and there was no difference in the length of the luteal phase between those dogs and control dogs (99 days, range 70-138 d and 99 d, range 60-112 d, respectively). When trilostane was stopped during PDP there was no post-treatment recovery of progesterone secretion and although the luteal phase tended to be shorter (66 d, range 41-101 d) the difference was not significant (P = 0.09). Plasma prolactin concentration did not increase after the trilostane-induced decrease in plasma progesterone. The interoestrous interval in dogs treated during PIP (234 d, range 175-269 d) or PDP (198 d, range 120-287 d) was not significantly shorter than the control interval (247 d, range 176-313 d).In conclusion, trilostane treatment was effective in decreasing plasma progesterone concentration in bitches during the luteal phase, but the dose regimen used in this study produced less clear-cut inhibition of ovarian steroidogenesis than have other strategies to decrease plasma progesterone concentration. Further studies are warranted to determine whether trilostane can be used to terminate unwanted pregnancy in the bitch without inducing adrenocortical insufficiency.     

Peripheral plasma levels of oestradiol-17 beta and progesterone in the bitch during the oestrous cycle, in normal pregnancy and after dexamethasone treatment.

Plasma oestradiol-17 beta concentrations in Labradors increased during pro-oestrus to an average maximal concentration of of 79-7 +/- 10-9 (S.D.) pg/ml, and then fell rapidly. In 6/7 bitches the peak occurred within 1 day of oestrus. No consistent changes in plasma oestradiol levels were observed during pregnancy and at parturition and the values were similar to those in late anoestrus. Plasma progesterone levels did not increase markedly during pro-oestrus. At oestrus, progesterone values rose and maximal concentrations, which varied from about 20 to about 55 ng/ml, were reached within a few days of the oestradiol peak. Plasma progesterone decreased in late pregnancy and in one of the three bitches studied in detail low or undetectable levels were reached 10 days before parturition. In the other two bitches an abrupt decrease in progesterone occurred just before parturition. Dexamethasone treatment (2 X 5 mg daily for 10 days) from Day 30 of pregnancy resulted in intrauterine death and resorption of the fetuses in the two bitches studied. Treatment from about Day 45 resulted in the birth of dead fetuses at Days 55 and 59 of pregnancy. The changes in plasma oestradiol levels were very small. No changes in plasma progesterone levels were seen when dexamethasone was given in late pregnancy, but an accelerated decline occurred after treatment in mid-pregnancy.     

Induction of canine pyometra by inoculation of Escherichia coli into the uterus and its relationship to reproductive features

To characterize oestrus-related factors affecting the induction of and recovery from pyometra in bitches, 60 clinically healthy beagle bitches were used for induction of pyometra by inoculation of Escherichia coli into the uterus during oestrous and metoestrous stages. The animals were classified into the following six groups according to inoculation time: Days 1-10, 11-20, 21-30, 31-40, 41-50 and 51-60 after LH surge. The incidence of pyometra during the periods Days 11-20 and 21-30 after LH surge was 90.9% and 78.9% respectively, while that during Days 1-10 and 51-60 after LH surge was less than 20%, and the patterns of the incidence of pyometra and the serum progesterone levels were similar. There was no difference in the incidence of pyometra induced in bitches less than 5 years old compared to bitches over 6 years old. Oestrus in all of the bitches with pyometra induced by E. coli returned with or without PGF 2alpha treatment, unlike in bitches with spontaneous pyometra. The duration of the oestrous cycle in the non-treated and PGF 2alpha-treated groups was 231.4+/-55.2 days and 162.1+/-40.6 days (P < 0.001), respectively, and there was no difference in the rate of return of oestrus between the two groups. The conception rate in all of the bitches in which oestrus had returned was 81.8%. The above findings indicate that the period during which severe pyometra could be induced was limited to the early stage in metoestrus.     

Aglepristone (RU534) administration to non-pregnant bitches in the mid-luteal phase induces early luteal regression

The effect of the antiprogestagen aglepristone (10 mg/kg bw), administered at days 29 and 30 following the estimated day of LH surge (day 0), on corpora lutea (CL) function was examined during the diestrus phase of non-pregnant bitches. Aglepristone shortened (P < 0.01) the luteal phase and complete luteolysis (progesterone <2 ng/mL) was observed at days 40.8 ± 3.5 and 71.5 ± 4.6 (means ± SD; n = 9/group) in treated and control bitches, respectively. Peripheral estradiol-17β concentrations declined from 91.5 ± 14.3 pg/mL at day 9 to 50 pg/mL at day 18, remaining at approximately the same levels thereafter in both treated and control bitches. Intraluteal in vitro synthesis of progesterone and estradiol-17β released by CL explanted at day 38 from control bitches (511.9 ± 285.6 and 40.7 ± 17.2 pg/mg protein, respectively) did not differ from that of treated. From day 38, intraovarian hemodynamic variables (arterial blood flow, systolic peak, and end-diastolic velocities), monitored by color-coded and pulsed Doppler, decreased more steeply (P < 0.01) in aglepristone-treated (n = 4) than in control (n = 4) bitches, whereas the resistance index increased (P < 0.01) in treated animals. All the blood flow parameters were undetectable at 60 ± 3.6 and 68 ± 2.0 days (medians ± SD) after LH peak in treated and control bitches, respectively. In conclusion, aglepristone administration to dogs during the mid-luteal phase markedly accelerates the luteolytic process which is accompanied by a parallel decline in ovarian blood flow supply with a shift from approximately 8 to 10 days.     

Immunohistochemical determination of estrogen receptor-α in canine vaginal biopsies throughout proestrus,estrus, and early diestrus

The aim of this study was to describe the presence of estrogen receptor-α (ERα) in several vaginal histological compartments in healthy adult bitches throughout three estrous cycle stages (proestrus, estrus, and early diestrus) and to relate ERα presence with serum progesterone and estradiol-17β concentrations. For this purpose, serial blood samples and vaginal biopsies were taken from five bitches every 48 hours, starting at the clinical onset of proestrus, marked by the beginning of serosanguineous vaginal secretion. Serum progesterone and estradiol-17β concentrations were determined by RIA, whereas detection of steroid receptors was carried out through immunohistochemistry. Subjective image analysis was conducted by two independent observers in the following histological compartments: superficial, intermediate, and deep epithelia and superficial (loose) and deep (dense) stroma (connective tissue). Nuclear ERα immunoreactivity was detected in every histological compartment and estrous cycle stage studied. ERα expression varied among histological compartments and during stages of the cycle. Receptor expression was associated with estradiol-17β and progesterone serum profiles. Most relevant cyclic changes were detected in the superficial and deep epithelia and in the dense connective tissue. The highest ERα expression was detected during diestrus, although each compartment had a different pattern throughout the other cycle stages. Thus, vaginal ERα expression in the bitch varied throughout proestrus, estrus, and early diestrus according to the histological compartment involved.     

Influence of season and parity on the recovery of in vivo canine oocytes by flushing fallopian tubes

Assisted reproductive technology (ART) in dogs largely depends on the in vivo matured oocytes due to lack of a suitable in vitro maturation system. The present study evaluated the technique of flushing fallopian tubes to collect in vivo matured canine oocytes by laparotomy, and determined the effects of seasons, and parity of donor bitches on the success of oocyte retrieval. Oocytes were retrieved from anesthetized bitches by laparotomy. About 7 ml of TCM-199 supplemented with HEPES was used to flush each individual fallopian tube. Oocytes were categorized as good, fair, poor, immature or aged based on the nuclear stage, cumulus cell layers, color and homogeneity of ooplasm. Oocytes categorized as being good or fair were considered usable, while poor, aged or immature oocytes were considered unusable for ART. A significantly higher number of oocytes per bitch were retrieved during the spring (11.2) compared to the winter (7.9). The oocyte recovery rates were 89.4, 92.2, 89.7 and 89.3% for spring, summer, autumn, and winter, respectively. The highest percentage of usable oocytes (74.7%) was retrieved during autumn (P>0.05). The number of oocytes was influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches (10.3) compared to nulliparous bitches (7.7). The percentage of usable oocytes was more in multipara (71.5%) compared to nullipara (64.7%) (P>0.05). Collection of in vivo produced oocytes by laparotomy represents a potential source of matured oocytes for ART in dogs.