Progestin treatment for infertility in bitches with short interestrus interval

The purpose of this study was to investigate if the suppression of estrus by the administration of a synthetic progestin, megestrol acetate or clormadinone acetate, could be an effective treatment to infertility in bitches with shortened interestrus periods and previous infertility. Ten bitches of different breeds and ages, with history of infertility and presenting repeated interestrus intervals of less than 4 months, were treated daily either with megestrol acetate (2 mg/kg, n = 8) or clormadinone acetate (0.5 mg/kg, n = 2) orally for 8 days. The treatments were begun within a maximum of 3 days after the onset of clinical signs of proestrus. Estrus was prevented in all animals and appearance of the following proestrus cycle was observed within 2.7 +/- 0.6 months (mean +/- S.D.) after the beginning of the treatment. When mated during the first post-treatment estrous cycle, bitches became pregnant and whelped normal healthy offspring. No negative side effects were clinically detected over the study period. Our results show that, in bitches with shortened interestrus intervals and previous infertility, suppression of one estrus with synthetic progestins administered at recommended doses, allows fertile breedings on the subsequent cycle, producing litter sizes within the normal range.     

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.     

The use of deslorelin implants for the synchronization of estrous in diestrous bitches

A novel approach to estrous induction in diestrous bitches is described. Twelve spontaneously cycling anestrous bitches served as controls. Thirteen anestrous and 15 diestrous bitches were induced to come into synchronous estrous using prostaglandin (diestrous bitches only) and deslorelin implants (Ovuplant). Implants contained either 2.1 or 1.05 mg deslorelin and were administered beneath the vestibular submucosa. All treated bitches came into estrous, regardless of implant size. Whereas all anestrous bitches ovulated, one of six diestrous bitches treated with the larger implant and three of nine treated with the smaller implant failed to ovulate. Induced bitches generally produced fewer corpora lutea than controls. Sixty-seven percent of control bitches became pregnant, with 0.63 fetuses per corpus luteum, whereas the pregnancy rate and fetuses per corpus luteum were 67 and 70% and 0.42 and 0.55 in the anestrous bitches induced with 1.05 and 2.1 mg deslorelin implants, respectively (not different from controls). Only 2 of 15 induced diestrous bitches conceived a detectable pregnancy, one of which was resorbed. In conclusion, although ovulatory estrous can be induced in bitches that had their most recent ovulation 40-100 days ago, these bitches are very unlikely to become pregnant during the induced estrous. The reason for the poor fertility in these diestrous bitches requires further study.     

Characterization and distribution of gonadotrophs in the pars distalis and pars tuberalis of the equine pituitary gland during the estrous cycle and seasonal anestrus

Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-microm sections using monoclonal antibodies to the LHbeta or FSHbeta subunits and a polyclonal antibody to ovine LHbeta. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 +/- 5. 8% and 19.1 +/- 2.1% of the total cell density in sexually active and anestrous animals, respectively (P: > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 +/- 0.1%) than in anestrous (1.2 +/- 0.05%) mares (P: < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 +/- 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 +/- 1.2%; P: < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.     

Influence of reproductive stage at PRID insertion on synchronization of estrus and ovulation in mares

In the present study, we investigated the effects of reproductive status, size of follicles and plasma progesterone concentrations of mares at PRID insertion on the efficacy of the treatment, estrous cycle patterns, plasma concentrations of progesterone and LH. The progesterone-releasing device (PRID) was administered intravaginally to 28 Haflinger mares for 11 days at different reproductive stages: anestrus (n=6), estrus (n=11) and diestrus (n=11). Plasma concentrations of progesterone at insertion (Day 1) of PRID differed among treatment groups (anestrus: 0.2-0.6 ng mL(-1), estrus: 0.2-0.5 and diestrus: 1.6-10.8 ng mL(-1); P<0.001). Total secretion of progesterone (area under curve (AUC)) during treatment period revealed highest values in diestrus (38.2+/-3.1 ng mL(-1)h(-1)) followed by estrus (25.1+/-2.7) and anestrus (21.0+/-0.4 ng mL(-1)h(-1); P<0.05). Progesterone area under curve (AUC) was positively correlated with initial progesterone concentrations (R=0.5; P<0.05), but it did not correlate with the interval from PRID removal to ovulation. Plasma concentrations of LH during treatment period, were significantly lower in anestrous mares (184.6+/-28.6 ng mL(-1)h(-1)) when compared to estrous and diestrous mares (349.7+/-53.3 and 370.5+/-40.3 ng mL(-1)h(-1); P<0.05). Follicular size at PRID insertion had no effects on the intervals from PRID removal to subsequent estrus and ovulation. Follicle diameters at removal of PRID were significantly correlated with the interval from coil removal to estrus (R=-0.55, P<0.05) and ovulation (R=-0.72, P<0.0004) in cyclic mares. In anestrus 0 of 6 (0%) mares, in estrus 5 of 11 (45.5%) and in diestrus 6 of 11 (54.5%) mares ovulated within a defined interval of 1 day before to 1 day after mean interval from PRID removal to ovulation. In cyclic mares, response to treatment was significantly higher when compared to anestrous mares: almost all mares responded with estrus and ovulation independent from the stage of the estrous cycle at the start of treatment. However, accuracy of synchronization was still unsatisfactory. In cyclic mares, the plasma progesterone concentrations at insertion of PRID seem to be more important for the efficacy of the treatment than the assignment to estrous cycle stages.     

Luteolytic effects of prostaglandin F2alpha on day 8 to 19 corpora lutea in the bitch

Luteolysis was induced in 5 experimental Beagle (8 cycles) and 7 client-owned bitches treated with 150 to 200 microg/kg, sc of prostaglandin F2alpha administered twice daily for 4 d, starting on Days 8 to 19 after the onset of cytological diestrus. Five experimental Beagle bitches had been mated during the estrus preceding treatment, and copulation had been confirmed in 2/7 client-owned bitches presented for termination of unwanted pregnancy. Serum progesterone concentration (mean +/- SD) declined from 26.1 +/- 66 ng/ml before treatment to 0.3 +/- 0.4 ng/ml on the fourth day of treatment One of the 7 client-owned bitches maintained her pregnancy even though serum progesterone concentrations were less than 0.5 ng/ml on the third and fourth day of treatment. Mean (+/- SEM) inter-estrous intervals before and following prostaglandin-induced luteolysis were 207.3 +/- 12.4 (n = 11 cycles in 6 bitches) and 95.5 +/- 20.0 d (n = 6 cycles in the same 6 bitches; P < 0.0001), respectively These results suggest that effective prostaglandin-induced luteolysis can be achieved with administration of 180 microg/kg during the third week of diestrus in pregnant and nonpregnant bitches.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

Effect of a GnRH antagonist on GnRH agonist-implanted anestrous bitches

Various combinations of gonadotropin-releasing hormone (GnRH) antagonists and long-acting GnRH agonists have been assessed in several species to prevent the “flare-up” effect that agonists cause on the pituitary-gonadal axis. To determine the effect of a single administration of the GnRH antagonist acyline in anestrous GnRH agonist-implanted domestic bitches, 19 dogs (canis familiaris) were randomly assigned to receive either 10 mg sc deslorelin acetate (DA; n = 6) or DA combined with 330 μg/kg sc acyline within the first 48 h (DA&ACY; n = 13). These bitches were examined daily for detection of posttreatment flare-up, manifested as an estrous response during the month after treatment. In the DA and DA&ACY groups, an estrous response was detected in 6 of 6 and 9 of 12 (P < 0.5) of the bitches, starting 5.3 ± 1.3 and 10.1 ± 1.8 d (mean ± SEM, P = 0.5), respectively, after treatment. Based on serum progesterone concentrations, ovulation occurred in 6 of 6 and 5 of 9 of these bitches (P = 0.1). None of the dogs had any local or systemic side effects related to the treatments. In five DA and six DA&ACY bitches that could be followed up after the trial, interestrus intervals were 385 ± 22.5 and 330 ± 69.1 d, respectively (P > 0.1). It was concluded that the current antagonist protocol prevented initial ovarian stimulation in one quarter of the treated dogs, whereas the stimulation period was postponed and ovulation was inhibited in approximately half of the remainder.     

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)     

Induction of cyclicity in postpartum anestrous beef cows using progesterone, GnRH and estradiol cypionate (ECP)

The objective of the present study was to determine whether treatment of postpartum multiparous and primiparous anestrous beef cows with an intravaginal progesterone-releasing insert (CIDR) and PGF(2alpha), with and without the addition of GnRH or estradiol cypionate (ECP) at the time of CIDR insertion, is effective in stimulating onset of estrous cycles. Postpartum lactating Angus primiparous (n=47, 2 years of age, 495+/-6 kg) and multiparous (n=76, >or=3 years of age, 553+/-9 kg) cows were assigned by calving date to four blocks spaced 21-day apart. Cows were assigned sequentially by calving date to four treatment groups: (1) PGF(2alpha) (n=30), (2) CIDR-PGF(2alpha) (n=30), (3) GnRH-CIDR-PGF(2alpha) (n=33), and (4) ECP-CIDR-PGF(2alpha) (n=27). Intravaginal CIDR inserts were in place from days -7 to 0. A single 100 microg injection of GnRH or 2 mg ECP were administered on day -7, and 25mg PGF(2alpha) was administered on day 0. Day 0 averaged 38+/-1 day postpartum. Blood samples were collected on days -19, -9, 0, 5, 9, 12, 16, 19, 23, 26, and 30 for determination of plasma progesterone concentrations. Pre-treatment luteal activity (progesterone>or=1 ng/ml) was detected in 19% of primiparous and 8% of multiparous cows. Progesterone concentrations on day 0 were greater (P<0.001) in primiparous (3.2+/-0.3 ng/ml) than multiparous (2.0+/-0.2 ng/ml) cows. Following CIDR withdrawal, progesterone concentrations from days 5 to 30 were used to categorize response profiles as either: (1) treatment-induced onset of estrous cycles, (2) continued anestrus, or (3) spontaneous ovulation and subsequent formation of a CL. Incidence of treatment-induced onset of estrous cycles, which was defined as progesterone concentrations >or=1 ng/ml in three or more consecutive samples from days 9 to 19, was influenced by treatment and parity. Percentages of cows initiating estrous cycles were greater (P<0.001) in the three CIDR-treated groups than in the PGF(2alpha) group (55 and 8%, respectively). Percentages of cows initiating estrous cycles in the CIDR-PGF(2alpha), GnRH-CIDR-PGF(2alpha), and ECP-CIDR-PGF(2alpha) groups were 55, 58, and 52%, respectively. Incidence of treatment-induced estrous cycles in the three CIDR-treated groups of cows was greater (P=0.008) in primiparous (76%) than multiparous (43%) cows. Treatment of postpartum anestrous primiparous and multiparous beef cows with CIDR-PGF(2alpha) approximately 40-day postpartum provides an approach to increase the percentage of cows that have reinitiated estrous cycles by the start of the breeding season.     

The roles of progestagen and uterine irritant in the maintenance of cystic endometrial hyperplasia in the canine uterus

Cystic endometrial hyperplasia (CEH) was induced in the left uterine horns of 14 mature ovariectomised greyhound bitches with an intra-luminal silk suture (uterine irritant) and treatment with estradiol benzoate and megestrol acetate (to simulate stages of a normal canine estrous cycle). Right uterine horns served as suture-free controls. From Day 30 of simulated diestrus, bitches received treatments of suture removal (n = 4), progestagen withdrawal (n = 5) or both (n = 5). Necropsies were performed 3 or 9 weeks later. At 3 weeks, severe cystic endometrial hyperplasia was present in all (6/6) left horns and in no (0/6) right horns. At 9 weeks, the left horns in 5/6 of bitches subjected to progestagen-withdrawal had recovered (in varying degrees) from cystic endometrial hyperplasia, whereas no recovery was evident in the left horns of bitches (n = 2) that continued to receive progestagen. This study demonstrated that: (i) cystic endometrial hyperplasia was reversible upon withdrawal of progestagen; (ii) progestagen maintained cystic endometrial hyperplasia in the presence or absence of irritant; and (iii) persistent endometrial irritation in the absence of progestagen may not maintain cystic endometrial hyperplasia.     

Selective control of the estrous cycle of the dog through suppression of estrus and reduction of the length of anestrus

The objective of this study was to determine the effectiveness of testosterone in suppressing estrus in the bitch, and of cabergoline in shortening the length of the subsequent anestrous period. In Experiment 1, 12 diestrual Beagle bitches were randomly divided into two groups when plasma progesterone (P(4)) concentration was <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with testosterone cypionate every 14 days for a total of 239 days, and bitches in Group 2 served as untreated controls. On Day 274, bitches in both groups were treated with cabergoline for 40 days and blood samples were obtained on Days 274, 276 and 279 for determination of plasma prolactin (PRL) concentrations using RIA. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 2, 12 Greyhound bitches previously treated with testosterone within the last 6 months were randomly divided into two groups. At the initiation of this experiment, P(4) concentration was determined to verify that all bitches had a concentration of <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with cabergoline for 36 days, and bitches in Group 2 (n=6) served as untreated controls. Blood samples were obtained on Days 0, 2 and 5 to determine PRL concentrations. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 1, one bitch (Group 1) exhibited estrus after treatment with testosterone (1mg/kg body weight) for 43 days, and one bitch (Group 1) exhibited estrus after treatment with testosterone (2mg/kg body weight) for 113 days. None of the other four bitches in Group 1 exhibited estrus during the period of testosterone treatment (239 days). All bitches in Group 2 (control) exhibited estrus during the 239 days of the study. In addition, five of the six testosterone-treated bitches showed signs of proestrual bleeding within an average of 12.6 days (range of 5-25 days) after treatment with cabergoline; and, four of the six nontestosterone bitches showed signs of proestrual bleeding within an average of 28 days (range of 6-46 days). Prolactin concentrations in bitches in both Groups 1 and 2 significantly decreased after treatment with cabergoline. In Experiment 2, one of the six bitches showed signs of proestrual bleeding within 15 days after treatment with cabergoline. From the results of this study, it was concluded that exogenous testosterone was moderately effective (66%) in suppressing estrus in Beagle bitches, and cabergoline was effective in shortening the length of the anestrous period of Beagle bitches whose estrous cycle was previously suppressed with exogenous testosterone, but less effective in shortening the length of the anestrous period in Greyhound bitches previously treated with testosterone to suppress estrus.     

Seasonal relationships between dopamine D1 and D2 receptor and equine FSH receptor mRNA in equine ovarian epithelium

Dopamine (DA) blockade during anestrus or early spring transition can facilitate ovarian recrudescence and advance the timing of the first ovulation of the season. Some laboratories have reported variable results using DA antagonists to stimulate follicular growth during the mid-portion of the anestrual period. Differences in DA antagonist efficacy may be due to the FSH secretory status of the anestrous mare and the presence or absence of functional ovarian FSH receptors. We hypothesize that direct ovarian dopaminergic input can affect follicular growth through regulation of FSH receptor (FSHr) populations. To investigate this, the amount of DA D1 and D2 receptor (D1r, D2r) and FSHr mRNA was quantified in ovarian tissues in anestrous and mares expressing estrus at typical intervals that are detected during the breeding season. Ovaries (n=26) were collected from 10 anestrous mares and 13 mares that had initiated estrous cycles (n=8 luteal; n=5 follicular phase). The quantity of D1r and D2r mRNA and FSHr mRNA was determined in cortex of both groups and granulosa/theca (those having initiated estrous cycles) tissues by semi-quantitative polymerase chain reaction using the comparative cycle time method. The reference gene was glyceraldehyde-3-phosphate dehydrogenase. The fold-change for each sample was calculated based on a calibrator sample. Fold-change values for D1r and D2r were the dependent variable and tissue was the independent variable in a one-way ANOVA. Results of fold-change in FSHr were compared by ANCOVA due to unequal sample sizes from each mare. Correlations between receptors within each tissue type were determined. For each receptor type and tissue, correlations between follicular and luteal phases were determined. The fold-change of D1r mRNA was less than D2r mRNA in all tissue types and between seasons. The quantity of D2r message in ovarian cortex was greater (p<0.05) during anestrus than after estrous cycles had been initiated. Fold-change in D2r in granulosa/theca was not different dependant on estrous cycle phase or follicle size. Quantity of FSHr mRNA was less in anestrous ovarian cortex and greater after estrous cycles had been initiated. FSHr mRNA fold-change in the ovarian cortex after estrous cycle initiation was not different between estrous cycle phases, but was greater in smaller (<30mm) follicles compared with larger (>/=30mm) follicles. We have demonstrated an inverse temporal relation between ovarian D2r and FSHr in mares dependant upon season. The functional significance of this relationship deserves further study.     

Effect of stage of anestrus on the induction of estrus by the dopamine agonist cabergoline in dogs

Beagle bitches were administered the dopamine D2 receptor agonist cabergoline in 3 groups of 5 animals each, starting on known days of the estrous cycle. Cabergoline treatment was started in either early anestrus (Days 93 to 108), mid-anestrus (Days 123 to 156), or late anestrus (Days 161 to 192) at doses of 5 ug/kg/d, per os, and was continued until the confirmation of induced proestrus or for 40 d. Reproductive parameters were compared with those in 5 control anestrous bitches (Days 90 to 150). In control bitches, the mean (+/- SEM) interval to the next proestrus (73+/-11 d) resulted in an interestrus interval (192+/-9 d) similar to that of the previous cycles (196+/-11 d). In 14 of the 15 cabergoline-treated bitches, the next proestrus occurred within 4 to 30 d, was premature in early and mid-anestrous bitches and developed with low variability within groups. The resulting intervals to proestrus in bitches treated with cabergoline in early anestrus (20+/-2 d), mid-anestrus (14+/-3 d) and late anestrus (6+/-1 d) resulted in interestrus intervals in those groups of 131+/-5, 166+/-7 and 196+/-2 d, respectively. In response to treatment, interestrus intervals were reduced (P<0.05) and more synchronous (P<0.05) in early and mid-anestrus bitches, and were more synchronous (P<0.05) in late-anestrous bitches compared with those of control bitches or those of the previous cycle. Periovulatory estradiol and progesterone profiles of induced cycles in treated bitches were similar to those of spontaneous cycles in control bitches. Four of 5 control bitches and 12 of the 14 responding cabergoline-treated bitches became pregnant and produced normal litters. Plasma prolactin concentrations at Days 2 and 5 of treatment (0.3+/-0.1 ng/mL) and at the onset of proestrus shortly before the end of treatment (0.4+/-0.1 ng/mL) were lower (P<0.05) than those present in anestrus prior to treatment (1.7+/-0.6 ng/mL) or in control bitches. Prolactin was also low at the onset of proestrus in control bitches (0.5+/-0.2 ng/mL). The results demonstrate that prolactin-lowering doses of the dopamine agonist cabergoline can terminate the normal obligate anestrus in dogs, and that the effect occurs more slowly in early anestrus than in mid or late anestrus.     

Follicular development and plasma concentrations of LH and prolactin in anestrous female dogs treated with the dopamine agonist cabergoline

The effect of a daily administration of a dopamine agonist (cabergoline, 5 microg/kg) for 4 weeks, starting about 95 days after the end of estrus on follicular development and its relationship with LH and prolactin secretion has been investigated in two groups of anestrous bitches (Beagles and Greyhounds). Pro-estrus was detected in 80% (8/10) of beagles and 50% (3/6) of treated greyhounds. The mean inter-estrus interval of treated animals was 132+/-5.0 and 169+/-7.0 days for beagles and greyhounds, respectively, and in both this differed significantly from the cycle preceding treatment (192+/-9.0 and 198+/-12.0 days) and from that in untreated bitches (194+/-11.0 and 196+/-11.0 days for beagles and greyhounds, respectively (all comparisons at P<0.001). The interval from the beginning of treatment to pro-estrus in responding animals was 13.3+/-1.90 days in beagles and 20.3+/-1.70 days in greyhounds. Cabergoline increased (P<0.001) the length of pro-estrus (10.6+/-0.50 and 11.7+/-0.50 days) in the treated estrus cycle compared to the previous estrus cycle (8.4+/-0.30 and 8.8+/-0.40 days for in beagles and greyhound, respectively). Ovarian enlargement and follicle development was detected by ultrasound in 90% of treated beagles and in 83% of greyhound between the second and third weeks of treatment, but only 80% of beagles and 66% of treated greyhound displayed pro-estrus and estrus. In the treated bitches, mean plasma LH increased (P<0.001) before pro-estrus. There was high variability in mean plasma prolactin levels between animals. These data indicate that the administration of the dopamine agonist cabergoline to anestrous bitches increases mean LH plasma levels and induces follicular development shortly before pro-estrus but this activity is not always followed by pro-estrus and estrus. Finally, prolactin per se does not have a prominent role in the control of folliculogenesis in the bitch.     

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.     

Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes

Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes.     

Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic (n = 353) or imported (n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (+/- S.E.M.) litter size 5.7 +/- 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 +/- 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 +/- 0.5, n = 61). Two inseminations (n = 384) yielded a higher whelping rate (P < 0.05) and mean litter size (P < 0.01) than one insemination (n = 241), 78.1% and 6.0 +/- 0.2 and 70.5% and 5.1 +/- 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 +/- 0.2 and 5.3 +/- 0.3). Semen classified as of poor quality (progressive motility < 50% or percentage abnormal sperm > 20%) resulted in a lower whelping rate (P < 0.01) than semen classified as of good quality (progressive motility > or = 50% and percentage abnormal sperm < or = 20%), 61 and 77%, respectively. Small breeds (n = 50) had a smaller litter size (3.9 +/- 0.3; P < 0.01) than larger breeds (medium [5.7 +/- 0.3, n = 94], large [5.9 +/- 0.2, n = 295] or giant breeds [6.1 +/- 0.5, n = 62] [P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer (P < 0.01) for bitches with a litter size of < 3 pups (61.7 +/- 0. 4 days, n = 30) than for bitches with larger litters (60.5 +/- 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches < or = 6 years old 2 and 3 days after ovulation with semen of good quality from males < or = 8 years old.     

Inability of progestogen pretreatment to prevent premature luteolysis of induced corpora lutea in the anestrous bitch

Fourteen mature anestrous bitches were used to determine the effectiveness of pretreatment with an orally active progestogen to prevent premature luteolysis of induced corpora lutea (CL) in the anestrous bitch. In Group 1, seven bitches were treated orally with megestrol acetate (Ovaban((R))) at the rate of 2.2 mg/kg body weight for eight days. Three days later, the bitches were treated daily with pregnant mare's serum gonadotropin (PMSG) (44 IU/kg body weight) administered intramuscularly for nine consecutive days, and each bitch was given 500 IU human chorionic gonadotropin (HCG) on day 10, or on the first day of induced estrus if the bitches exhibited estrus while being treated with PMSG. A control group (Group 2) of seven bitches was not treated with Ovaban((R)) but was similarly given PMSG and HCG. Estrus was detected twice daily using a vasectomized male dog and verified by vaginal cytology. Blood samples were obtained on the first day of induced estrus (day 0) and every other day until day 90 post-estrus. Plasma progesterone (P(4)) concentrations were determined by a non-extraction solid phase radioimmunoassay (RIA), and data were analyzed by Student's t-test. There was no significant difference between the progesterone profiles of both groups of bitches. In addition, P(4) values were less than 1 ng/ml by day 50 post-estrus. Results of this study suggested that pretreatment with an orally active progestogen was not effective in preventing premature luteolysis of induced CL in the anestrous bitch.     

A model for cystic endometrial hyperplasia/pyometra complex in the bitch

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.