Long-term reversible suppression of oestrus in bitches with nafarelin acetate, a potent LHRH agonist

Adult cyclic beagle bitches were treated for up to 18 months with nafarelin acetate via subcutaneously implanted osmotic pumps, starting during the first week of a pro-oestrous vaginal discharge. The imminent ovulation appeared to be unaffected by treatment, but doses of 8 or 32 micrograms analogue/day reduced the integrated luteal progesterone values. No new oestrus was detected in 3 bitches during 18 months of treatment with 32 micrograms/day, which resulted in mean plasma levels of 0.4 ng analogue/ml. A return to oestrus was observed in all 3 bitches between 3 and 18 weeks after cessation of treatment: 2 of the bitches mated at those times and produced normal litters. Another 2 bitches were similarly treated with 32 micrograms analogue/day; they were mated at the oestrus at start of treatment and dosing was continued for about 63 days. One of the bitches conceived and produced a normal litter. Nafarelin acetate treatment begun during anoestrus resulted in an induced heat 1-2 weeks after the start of treatment. The induced heat consisted of pro-oestrous vaginal discharge, oestrous vaginal cytology, and ovulation (judged by increased circulating levels of progesterone). Three bitches mated at the induced heat and treated for the normal duration of gestation did not litter. Nafarelin treatment of 3 bitches before puberty did not induce signs of oestrus and prevented the occurrence of oestrus through 18 months of treatment. The first oestrus in these bitches occurred 3.5-4 months after cessation of treatment, but mating at that time did not result in pregnancy. These studies have established the feasibility of and dosage requirement for the use of the LHRH agonist as a contraceptive in the bitch.     

Induction of fertile oestrus in the bitch using Deslorelin, a GnRH agonist

Oestrus induction in various canine breeds was attempted in 32 bitches. A group of 8 bitches were treated 80–160 d following their previous oestrus (G1) whereas a second group of 24 bitches (G2) were implanted 200–590 d following their previous oestrus. The treatment for each bitch consisted in one Deslorelin implant (Suprelorin®4,7mg, Virbac, France), inserted subcutaneously in the post-umbilical region. Ovulation, pregnancy rate and litter size were recorded. All bitches came in heat 4.3 ± 1.4 d after implantation (2–7 d). Ovulation was reported in 62.5% in G1 and 87.5% in G2. One bitch refused mating and since no AI was performed, she was not considered for further analysis. Pregnancy was obtained in 25% in G1 versus 78.3% in G2. Mean litter size was 6.7 ± 3.5 puppies (1–14). Luteal failure was suspected in 3 bitches, two that remained non-pregnant and one which aborted 58 d post-ovulation since the owner refused progesterone supplementation. Deslorelin implants can therefore be considered as a valuable alternative to induce fertile oestrus in bitches in anoestrus. Follow-up of the luteal phase is recommended, since some bitches might encounter luteal failure.     

Effect of short-term hypothyroidism on reproduction in the bitch

Hypothyroidism in bitches has been reported to cause a variable interestrus interval, infertility, abortion, and stillbirth. The objective of this study was to evaluate the effect of experimentally induced hypothyroidism in bitches on fertility, pregnancy, parturition, and neonatal health. Eighteen healthy multiparous bitches were used; hypothyroidism was induced (by radioiodine administration) in nine bitches and the remaining nine served as untreated controls. After breeding, bitches were evaluated for pregnancy, fetal resorption, gestation length, litter size, duration and strength of uterine contractions (during parturition), interval between delivery of pups, viability of pups at birth, periparturient survival, and weight of pups at birth through 4 weeks of age. Bitches were bred a median of 19 weeks after induction of hypothyroidism. All bitches became pregnant and delivered term litters. There was no difference in the interestrus interval, litter size, or gestation length between hypothyroid and control bitches. Duration of uterine contractions was longer, but contraction strength was weaker in hypothyroid than control bitches; however, the interval between delivery of pups was not affected. Periparturient puppy mortality was significantly higher in litters from hypothyroid bitches. Viability scores and weight at birth were significantly lower in pups from hypothyroid bitches than controls. There was no difference between groups in pup weight gain during the first 4 weeks, in the interval from birth to the eyes opened, or to the onset of walking. Although hypothyroidism of relatively short duration did not affect fertility, it prolonged parturition and reduced pup survival in the periparturient period.     

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.     

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.     

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.     

Effect of maternal treatment with altrenogest on age at puberty, hormone concentrations, pituitary response to exogenous GnRH, oestrous cycle characteristics and fertility of fillies

Puberty was studied using 15 fillies of Quarter Horse phenotype. Fillies were from dams treated daily from Days 20 to 325 of gestation with: (1) 2 ml neobee oil per 50 kg body weight (controls); or (2) 2 ml altrenogest (2.2 mg/ml) per 50 kg body weight. The clitoris was measured at birth and approximately every 12 weeks until 84 weeks of age. Blood samples were collected from 9 fillies (5 treated, 4 controls) every 4 days over a 28-day period at 8-week intervals from 4 to 68 weeks of age; sampling continued every 4 days after 72 weeks of age until first oestrus. Blood samples were collected daily during oestrus (greater than or equal to 35 mm follicle) and on Days 4, 6, 10, and 14 after ovulation for the first 2 oestrous cycles. GnRH challenges (5 micrograms/kg) were administered every 8 weeks from 32 to 96 weeks of age. Puberty was defined as the first oestrus with ovulation. Beginning 1 February 1987, fillies were teased daily and their ovaries were examined by ultrasonography every 3 days (daily during oestrus). Fillies were inseminated with 500 x 10(6) motile spermatozoa from one stallion. Pregnancy was diagnosed by ultrasonography on Days 11, 12, 15 and every 5 days until Day 50 after ovulation. Prenatal altrenogest treatment caused clitoral enlargement (P less than 0.05) and increased serum concentrations of LH from 1 to 7 months of age. The amount of LH released in response to exogenous GnRH was greater (P less than 0.05) in treated fillies at 32, 64, and 72 weeks of age. Treated fillies had higher serum concentrations of FSH from 1 to 4 months (P less than 0.05), but FSH was lower (P less than 0.05) in treated fillies before and during first oestrus. Serum concentrations of LH and FSH peaked transiently at 10 months and LH was depressed from 64 to 88 weeks and began to rise 14 days before first oestrus. Concentrations of FSH began to decline 14 days before first oestrus. The median age at puberty was 90 weeks. Durations of oestrus, dioestrus, and the oestrous cycle were not different between groups and were similar to those for adult mares. First cycle pregnancy rates and overall rates were 100 and 82% and 100 and 91.7% for control and treated fillies, respectively (P greater than 0.05). Maternal treatment with altrenogest did alter gonadotrophin secretion before puberty, but had no effect on functional reproductive performance in fillies.     

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.     

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.     

Decrease of body temperature after aglepristone treatment in bitches

Body temperature responses and the timing of abortions were evaluated in pregnant bitches with the anti-progestin aglepristone. Fifteen purebred and crossbred, 25-45 days pregnant, were included in this study and seven untreated bitches at the same stage of pregnancy served as controls. Treated bitches were administered two applications of aglepristone (10 mg/kg SC) 24 h apart for pregnancy termination. Pregnancy termination was confirmed by ultrasonographic assessment. Body temperature was rectally measured three times a day for 6 days beginning 24 h before treatment or pregnancy diagnosis in the treated and control bitches, respectively. Additionally, serum progesterone concentrations were assessed at time points during the study in the treated bitches. Pregnancy was terminated in 14 treated bitches in a mean+/-S.E.M. of 4.3+/-0.7 days after treatment. Control bitches remained pregnant. In the treated bitches, but not in the controls, body temperature significantly decreased 24 h after the beginning of the treatments (P < 0.01) and then gradually returned to pre-treatment values. Correlation between the day of mean minimum body temperature and the day of pregnancy termination was low (0.07; > 0.05). Progesterone did not show significant change throughout the study. Body temperature does not seem to be a suitable variable to clinically monitor the aborting effect of aglepristone. Decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic effects.     

Synchronization of oestrus in gilts with altrenogest: effects on ovulation rate and foetal survival

Previous studies have shown that use of altrenogest resulted in a high rate of fertility and increased litter size compared with controls under conditions of practical pig production. The present study was designed to evaluate whether ovulation rate and/or foetal survival were increased by altrenogest using crossbred gilts derived from one herd (n = 227) and introduced in the same piggery over 12 months. Each gilt was allocated to a treated group (n = 103) receiving an individual daily dose of 20 mg of altrenogest for 18 days in its feed or a control group (n = 124) after puberty had been diagnosed, (197 ± 1 day; mean ± SEM). They were inseminated (double AI) at the second induced or natural oestrus. Pregnancy was diagnosed by ultrasonography at Days 22 and 42 post-insemination in the absence of return to oestrus. Pregnant gilts were slaughtered at 48 ± 3 days of pregnancy following the second examination. The number of living and dead foetuses were recorded before uterine contents (foetuses and placentae) were weighed and the number of corpora lutea (CL) per ovary counted.Precise synchronization of oestrus was observed after the end of the progestogen administration, with 93% of the gilts in oestrus by Days 5 to 7. For the controls, the interval from first to second oestrus ranged from 17 to 25 days in 93% of the control gilts. The pregnancy rate was 89.3% for treated gilts and 77.4% for controls (P < 0.05). The ovulation rate was increased by the treatment (15.4 ± 0.3 vs 14.6 ± 0.3; mean ± SEM, P < 0.02). Although the altrenogest group had more foetuses (11.1 vs 10.6), this difference was not significant (P > 0.14). The percent of foetal survival was similar in both groups (64.9%; P > 0.27). The foetal and placental weights differed only between dams and increased with stage of gestation. The increase in litter size through feeding altrenogest was associated with an increased ovulation rate.     

Effect of oestradiol benzoate given after prostaglandin at two stages of follicle wave development on oestrus synchronisation,the LH surge and ovulation in heifers

Oestrus synchronization following prostaglandin-induced luteolysis is variable and dependent on follicle wave status in cattle. Oestradiol benzoate (ODB) has been used following prostaglandin to reduce the interval to oestrus and ovulation, but the effect of follicle wave status at the time of ODB administration is not clear. The aim of this study was to characterize the endocrine and follicular responses following ODB after luteolysis at different stages of the follicle wave. Prostaglandin was administered at either emergence or dominance of the second follicle wave. Twenty-four hours later animals received either 0.5mg ODB in oil or a control oil injection. Follicular development was monitored daily by ultrasonography, oestrous behavior was determined and blood samples were collected. In animals treated with ODB at emergence, there was a reduction (P<0.05) in the maximum diameter of the ovulatory follicle (11.7+/-1.2 mm versus 13.1+/-0.1 mm) and in the interval from prostaglandin to oestrus (52.0+/-2.3 h versus 88.0+/-9.6h), to the LH surge (53.3+/-3.5 h versus 89.1+/-6.5 h) and to ovulation (96+/-0.0 h versus 129.6+/-9.6h), compared with controls. In animals treated with ODB at dominance, there was a reduction (P<0.05) in the interval from prostaglandin to the LH surge (54.0+/-3.1 h versus 70.9+/-4.8 h), but not in the interval from prostaglandin to oestrus (53.3+/-2.7 h versus 65.7+/-4.5 h; P=0.11), to ovulation (96.0+/-0.0 h versus 110.4+/-4.8 h; P=0.12) or the maximum diameter of the ovulatory follicle (12.7+/-0.3 mm versus 13.6+/-0.4 mm; P=0.12), compared with controls. Treatment did not affect (P>0.05) the length of the subsequent oestrous cycle or corpus luteum size. In conclusion, the use of ODB advanced, but did not alter the temporal relationships among oestrus, the LH surge and ovulation, regardless of stage of follicle development at treatment.     

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.     

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.     

Treatment with a subcutaneous GnRH agonist containing controlled release device reversibly prevents puberty in bitches

In most species, continuous administration of GnRH agonists desensitizes the pituitary to GnRH, and blocks ovarian function. The aim of this study was to assess the effects of a novel controlled release device containing azagly-nafarelin (Gonazon) to prevent puberty in young Beagle bitches (mean age: 4.88 +/- 0.32 months). Gonazon containing 18.5 mg azagly-nafarelin (n = 10) or a placebo implant (n = 10) was administered subcutaneously. Throughout the 1-year treatment, estrus behaviour was monitored weekly. Plasma progesterone concentrations, as well as body weight and height, were measured monthly. Following implant removal, estrus detection and progesterone measurement were continued until occurrence of puberty in all bitches. Control bitches displayed puberty (estrus, followed by ovulation) at approximately 11.9 +/- 2.7 (range, 8-16) months of age. In contrast, none of the Gonazon treated bitches displayed puberty during the period when Gonazon was present. Following removal of Gonazon, resumption of estrus and ovulation naturally occurred (seven bitches) or was induced (three bitches) approximately 8.5 (1.2-14.3) months later. As a consequence, age of puberty of the Gonazon treated bitches was 25.5 +/- 5 (18-31) months. No clinically detectable side effects were noted in Gonazon treated bitches. Height at withers was unaffected by treatment. Changes in body weight with time were also unaffected by treatment. Implants were well tolerated and generally easy to remove. These data demonstrated that Gonazon safely, efficiently and reversibly prevents reproductive function for 1 year in prepubertal bitches.     

Intratumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy

Summary In two parallel studies, bitches with mammary tumour received single intralesional injections of BCG (1 mg: 10⁷ living bacteria) and Corybacterium parvum (10⁹ killed bacteria) (53 bitches) or C. parvum alone (129 bitches) at the same dosage. Control groups received injections, following the same protocol, of 1 ml BCG suspension medium diluted in saline in the first study (51 bitches) or no injections at all (120 bitches in the second study).A block dissection, including mammary tumours, adjacent mammary glands, and regional lymph nodes, was performed 2 weeks later in all animals. On the basis of histologically confirmed malignant tumours, 48 bitches (25 treated by immunotherapy and 23 controls) in the first study and 67 bitches (30 treated by immunotherapy and 37 controls) in the second study remained for postsurgical follow-up.The clinical tolerance of the treatment was generally good. No significant differences were found in cumulative survival rates between treated and control group in either studies.     

Total unconjugated oestrogen and progesterone concentrations in peripheral blood during the oestrous cycle of the dog.

From the beginning of pro-oestrus to the end of metoestrus, daily peripheral blood samples were withdrawn from six bitches. Further samples were obtained during anoestrus. Oestrogen values rose from the onset of pro-oestrus to attain a peak value of 25-3 +/- 4-8 pg/ml on Day 1 of oestrus. Progesterone concentrations began to rise 2 days before the oestrogen peak, and reached their highest levels of 18-9 +/- 1-0 ng/ml 16 days after the end of oestrus. After oestrus, an oestrogen rise was detected which reached a peak at Day 18 of metoestrus. The duration of oestrus was unrelated to the length of time that oestrogen levels were raised, or the maximum values were attained. No significant oestrogen levels were detected during anoestrus.     

Influence of perioestrous suprabasal progesterone levels on cycle length,oestrous behaviour and ovulation in heifers

In order to induce suprabasal plasma concentrations of progesterone after luteolysis and to determine their effect on oestrous behaviour and ovulation, heifers subcutaneously received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3) or 10 (n = 4) g of progesterone, or an empty implant (controls, n = 5) between days 8 and 25 of the cycle (ovulation designated Day 0). Growth of dominant follicles and time of ovulation were determined by ultrasound, and signs of oestrus were recorded and scored. Blood was collected at 2–4 h intervals from Days 15 to 27 and assayed for progesterone concentration. In all heifers, plasma concentrations of progesterone sharply decreased during Days 16–18. Control heifers had their lowest progesterone levels on Days 20.5 and 21, standing oestrus on Day 19.5 ± 0.4 (mean ± SEM), and ovulated on Day 20.7 ± 0.4. A similar pattern was observed in heifers treated with 2.5 and 5 g progesterone. Heifers treated with 6, 7.5 and 10 g of progesterone showed an extended (P < 0.05) interovulatory interval. Onset of prooestrus and time of maximum expression of signs of oestrus were not significantly different from those in controls. However, there was an absence of standing oestrus in most of the cases, signs of oestrus lasted longer (P < 0.05) and were weaker in intensity when doses increased. In these groups, the lowest progesterone concentrations were attained shortly after implant removal. Some heifers treated with 6 and 7.5 g of progesterone had standing oestrus and post oestrous bleeding as seen in the controls but ovulation occurred from Days 24.5 to 27. When plasma progesterone concentrations were over 1 nmol 1⁻¹, disturbed oestrus and delayed ovulation occurred. The extended period of prooestrus and oestrus and delayed ovulation were similar to that described in cases of repeat breeding. It is suggested that suprabasal plasma concentrations of progesterone, after luteolysis, may lead to asynchrony between onset of oestrus and ovulation and consequently be a cause of repeat breeding in cattle.     

The influence of exogenous steroid hormones on steroid receptors, uterine histological structure and the bacterial flora of the normal bitch.

Oestrogen (ER) and progesterone (PR) receptors have been shown to vary in both concentration and distribution during the oestrous cycle of the bitch, influenced by the normal changes in endogenous reproductive hormones. The influence of exogenous steroid hormones on steroid receptors and the histological structure of the uterus was studied in two groups of parous Beagle bitches. Group A (n = 6) were treated with progesterone (P4) in oil i.m. (3 mg/kg) in late metoestrus on the day that peripheral plasma P4 concentrations were first identified as <10 ng/ml, and subsequently once weekly on three other occasions. Group B (n = 6) were treated with a single i.m. injection of MPA (50 mg, 4.2-5.6 mg/kg) following the same protocol. Full-thickness uterine wall biopsies were obtained from the mid part of one horn 2-7 days after the last (fourth) injection of P4 or MPA. During the subsequent oestrus, when peripheral plasma P4 concentrations were between 8 and 10 ng/ml, each bitch in both groups (n = 12) received a single injection of oestradiol benzoate (ODB) in oil i.m. (7.5 mg, 0.63-0.84 mg/kg). All bitches had an ovariohysterectomy 7 days later. Full-thickness uterine wall samples were obtained from the mid part of the intact horn and other parts of the uterus. Swabs were taken from the uterine lumen for bacteriological examination; all were sterile. Tissue samples were sectioned and examined for evidence of lesions, and stained for ER and PR receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). At the time of ovariohysterectomy, fluid had accumulated in the isolated section of the uterine horn distal to the point of biopsy; the volume was greater in the MPA-treated bitches. There was also evidence in some sections of histological changes in the endometrium. Variations in the expression of both ER and PR were seen between bitches, which may have been due to some not being in mid-metoestrus at the time of treatment. In general, ER scores were low after P4 and MPA treatment, but following ODB there was a significant (P<0.05) increase in ER expression in all parts of the endometrium. PR scores were zero in the glandular epithelium of all 12 bitches after P4, MPA and ODB treatment, whereas in the other parts of the endometrium they were generally moderate to high. Following treatment with ODB, PR generally increased in the three regions of the endometrium where PR were present. The study shows that ER and PR distribution and expression in the endometrium of bitches can be modified by P4, MPA and ODB, with evidence of individual variation.     

Effect of immunization against progesterone on oestrus, cycle length, ovulation rate, luteal regression and LH secretion in the ewe

The effects of active immunization against progesterone on reproductive activity were studied in Merino ewes. Immunization against progesterone caused a shortening (P less than 0.01) of the interval between ovulations from 17-18 days (controls) to between 6 and 10 days (immunized group); this was associated with a corresponding reduction in the interval between LH surges. The immunized ewes also had higher (P less than 0.05) ovulation rates (1.72) than controls (1.25) and exhibited a reduced (P less than 0.01) incidence of oestrus (26% v. 95%). Many immunized ewes continued to ovulate despite the persistence of corpora lutea from earlier ovulations which led to an accumulation on the ovaries of many corpora lutea of different ages. The frequency of LH pulses in ewes immunized against progesterone (1.8 +/- 0.2 pulses/4 h) was significantly (P less than 0.001) higher than that of control ewes (0.3 +/- 0.1 pulses/4 h). This study highlights the importance of progesterone in the control of oestrus, ovulation, ovulation rate, luteal regression and the secretion of LH in the ewe.