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.     

Induction of estrus with intramuscular injections of GnRH or PMSG in lactating goats (Capra hircus ) primed with a progestagen during seasonal anestrus

In Experiment 1, goats in seasonal anestrus (n=154) were treated with sponges impregnated with 1 of 2 types of progestagen (MAP or FGA) followed by PMSG (400 IU im) 48 h before sponge removal. The type of progestagen used had no effect on kidding, abortion, pseudogestation, multiple births, stillbirths, number of live births per doe or gestation length. In Experiment 2, lactating goats (n=24) in seasonal anestrus were treated with progestagen sponges (MAP). At sponge removal they received one of the following treatments: 1 injection of PMSG (400 IU im), 1 injection of GnRH (125 mug im; GnRH-1), or 2 injections of GnRH (125 mug/injection im; GnRH-2) at a 48 h interval. Serum samples were taken at 6-h intervals for 96 h, starting 12 h after sponge removal. Heterologous radioimmunoassays were validated for the measurements of goat FSH, LH, E(2) and P(4). The onset of estrus (P=0.004), mean doe receptivity (P=0.0006), maximum preovulatory E(2) concentrations (P=0.0001) and LH peak concentrations (P=0.08) occurred significantly later for GnRH-1 and GnRH-2 than for PMSG treatment. The PMSG treatment induced a preovulatory LH peak in a greater number of goats (P=0.05) and gave a higher gestation rate than GnRH-1 and GnRH-2 treatments (57 vs 0 vs 12%; P=0.03). It is likely that the GnRH treatments administered did not reactivate the hypothalamo-pituitary-gonadal axis. Thus, intramuscular injections of GnRH in lactating goats primed with a progestagen were not as effective in regulating reproductive performance during seasonal anestrus as were injections of PMSG.     

Pharmacokinetics of eCG and induction of fertile estrus in bitches using eCG followed by hCG

The aim was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment 1, three ovariohysterectomized bitches received 10 000 IU of eCG iv, and 15 days later 10 000 IU of eCG im. Blood samples were taken up to 144 h after each injection to measure eCG concentrations. In Experiment 2, 25 healthy, intact late anestrous bitches were assigned to one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Sexual behavior (SB), clinical signs of estrus (CSE) and vaginal cytology (VC) samples were obtained and scored before eCG administration and every other day until onset of estrus, or for 14 days. In Experiment 3, intact late anestrous bitches were assigned to a treatment group (TRT; n = 16) and received eCG (50 IU/kg im) followed by hCG (500 IU im) 7 days later; or to a placebo group (PLA; n = 8) where they received 1 mL saline solution im. All bitches that were induced in estrus were mated or AI with fresh semen. In Experiment 1, maximum observed concentration (C_max) eCG were similar between im and iv routes (6.1 ± 0.9 vs. 8.6 ± 0.5 IU/mL, P > 0.08), whereas time for maximum observed concentration (T_max.) was longer for im compared to iv routes (17.5 ± 0.5 vs. 11.6 ± 0.3 h, P < 0.01). The area under the curve (AUC) was similar for im and iv routes (P > 0.48), and eCG was detectable in serum for at least 144 h for both routes. In Experiment 2, 3 days or 3 to 5 days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, the mean interval from treatment to estrus was 4.0 ± 0.4 days. In Experiment 3, the mean interval from treatment to estrus was shorter in the TRT group compared to the PLA group (4.1 ± 3.3 vs. 68.5 ± 4.4 days, P < 0.01). The previous interestrus interval was similar for TRT and PLA groups (199.6 ± 7.2 vs. 197.5 ± 10.2 days), but the new interestrus interval was shorter for the TRT compared to the PLA group (164.0 ± 7.2 vs. 212.2 ± 10.2 days; treatment by interval interaction, P < 0.007). Serum P₄ concentrations increased on the first day of cytologic diestrus after treatment in bitches in TRT (0.7 ± 0.3 vs. 22.8 ± 4.2 ng/mL; P < 0.01); but did not change in PLA (P > 0.84). Ninety-four percent of animals were bred (15/16; AI, n = 7; natural mating, n = 8), and 80% (12/15) became pregnant. None of the bitches had any side effects from the eCG and hCG therapy. We concluded that 50 IU/kg of eCG combined 7 days later with 500 IU of hCG was effective to induce normal and fertile estrus in bitches at 164 days post estrus, with an 80% pregnancy rate, with no side effects, and with a reduction of 48 days of the interestrus interval.     

Progesterone secretion by induced corpora lutea (CL) of anestrous bitches after administration of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (HOG)

Sixteen bitches were treated with pregnant mare serum gonadotropin (PMSG; 44 IU/kg bwt) intramuscularly (i.m.) for nine consecutive days and each was given 500 IU HOG i.m. on the tenth day or on the first day of induced estrus (Day 0). On Day 12, each bitch was randomly assigned to three groups and treated as follows: Group 1-six bitches, each given 10 mcg GnRH i.m.once every 24 hr; Group 2-six bitches, each given 500 IU HOG i.m. once every 24 hr; and Group 3-four bitches, each given 5 ml 0.9% saline i.m. once every 24 hr. Treatments were continued until Day 55. Blood samples were obtained prior to treatment and every other day until Day 55. Plasma progesterone concentrations were determined by radioimmunoassay (RIA) and data arranged using a split-plot design, with treatment as the main plot and days of sampling as subplots. Analysis of data was by Duncan's Multiple Range Test and treatment-by-day interaction determined by the least-squares method. After treatment, progesterone concentrations from Group 2 were higher (P<0.05) than those of the other groups. A significant effect was seen on Day 20 (P<0.01), while on Day 50 the difference approached significance. These findings suggest that HOG is capable of stimulating the production of progesterone by induced CL in the bitch. However, neither HOG nor GnRH prevented premature regression of these induced CL. Results of this study suggest that premature luteolysis of induced CL is probably not due to lack of gonadotrophic support from either the hypothalamus or the anterior pituitary gland.     

Failure of hCG to support luteal function in bitches after estrus induction using deslorelin implants

Induction of estrus with deslorelin implants was followed by abortions in bitches that conceived during the induced estrus. Lowering the deslorelin dose and choosing a better implantation site prevented the abortions. This study investigated the hypothesis that induction of estrus with deslorelin is followed by reduced serum progesterone concentrations (SPC) during the ensuing diestrus. Assuming that reduced luteal function resulted from reduced LH secretion due to hypophyseal down-regulation of GnRH receptors, the effect of human chorionic gonadotropin (hCG) treatment on the SPC of diestrous bitches was also investigated. In Experiment 1, 10 spontaneously cycling bitches served as controls, whereas estrus was induced with deslorelin implants in 24 others. In Experiment 2, six diestrous bitches were treated with a single dose of hCG between Days 39 and 45 of diestrus. The SPC was lower in deslorelin-induced bitches from Days 35 to 56 of diestrus and hCG increased SPC during the first 24 h after treatment, followed by a dramatic decline thereafter. Although SPC recovered in pregnant bitches, it remained much lower (< or = 1 ng/mL) than in untreated, non-pregnant bitches. The suppression of progesterone secretion after hCG treatment suggested that decreased luteal activity in deslorelin-induced bitches may not be a simple consequence of down-regulation of hypophyseal GnRH receptors.     

Induction of persistent diestrus followed by persistent estrus is indicative of delayed maturation of tonic gonadotropin-releasing systems in rats

The relationship between the amount and duration of administration of estradiol benzoate (EB) to newborn female rats and the induction of sterility was examined in 407 animals. Vaginal smear patterns were classified into 3 types according to the incidence of vaginal proestrus and estrus over a 10-day period: persistent estrous (PE), persistent diestrus (PD), and intermediate (INT), so that the changes in vaginal smear patterns could be analyzed quantitatively. Incidence of the PE pattern was most frequent in the rats that received a single injection of 10 micrograms EB on the day of birth (Day 1). Almost all of the animals receiving 10 daily injections of 10 micrograms EB from Day 1 showed persistent diestrus until at least 100 days of age. In the rats that were given 5 daily injections of 10 micrograms EB Day 1 through Day 5, or a single injection of 100 micrograms EB on Day 3, the incidence of the PD pattern was high at 41-60 days of age, but later the PD-type was replaced by the PE pattern of vaginal smears. In the rats that were treated with 5 daily injections of 10 micrograms EB from Day 1 through Day 5 and were ovariectomized on Day 22, a slight but significant increase in the level of luteinizing hormone in plasma was noted after administration of EB and progesterone on Day 100 but not on Day 50. These results indicated that neonatal injections of EB induce sterility, but the effect is dependent on the amount of EB injected and length of time over which the injections are given.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of persistent estrus by electrolytic lesions placed neonatally in the hypothalamus of the female rat

Electrolytic lesions were placed in the hypothalamus of two-day-old female rats. Destruction of the mediobasal part of the preoptic area resulted in persistent vaginal estrus starting on the day of vaginal opening, while lesions placed laterally in the preoptic-anterior hypothalamic area did not interfere with normal cycles. Therefore, the mediobasal hypothalamus is capable of undergoing maturation without any postnatal influence from at least the mediobasal part of the anterior hypothalamus. Destruction of the anterior wall of the third ventricle also caused persistent or prolonged vaginal estrus preceded by normal cycles. The relationship between the loci of lesions and the occurrence of sexual cyclicity was discussed.     

Induction and synchronization of estrus in dogs

Indications for estrus induction in the bitch include missed breeding opportunities or conception failure, the treatment of primary or secondary anestrus and synchronization of ovulation for embryo transfer programs. Reported methods for canine estrus induction include the use of synthetic estrogens (diethylstilbesterol), dopamine agonists (bromocryptine and cabergoline), GnRH agonists (lutrelin, buserelin, fertirelin, deslorelin, and leuprolide) and exogenous gonadotropins (luteinizing hormone, follicle stimulating hormone, human chorionic gonadotropin, pregnant mare serum gonadotropin, and human menopausal gonadotropin). These methods vary widely in efficacy of inducing estrus, as well as in the fertility of the induced estrus. The applicability of some of these methods for clinical practice is questionable. This review will summarize published reports on estrus induction and synchronization in bitches and summarize preliminary results using a long-acting injectable preparation of deslorelin.     

Induction of estrus in anestrous cows treated with 'Fertivet'

The effectiveness of 'Fertivet' (180 mg. Cisclomiphene Citrate and 120 mg. Transclomiphene citrate mixture) in the induction of estrus and fertility was tested on sixteen anestrous cows. Of all the animals treated 14 (97.5%) expressed estrus and 8 (57.1%) conceived. Among the three breeds of cows treated, 5 out of 5 Brown Swiss, 4 out of 5 Jersey cross breeds and 5 out of 6 Sahiwal (Zebu) cows expressed estrus. These results indicate the usefulness of 'Fertivet' in the induction of fertile estrus in cows.     

Induction of ovulation in gonadotropin treated gilts with synthetic gonadotropin releasing hormone

Four consecutive trials were conducted to investigate the possibility of controlling the time of ovulation in prepuberal gilts pretreated with PMS and HCG. In trial 1 it was shown that the GnRH analog Hoe 766 was superior to other compounds tested. The following trial revealed that 10 mug of that analog is the optimal dose to elicit an ovulatory response. In trial 3 it was found that the majority (73%) of gilts had started ovulating by 39 h after Hoe 766 injection. Individual gilts started ovulating up to 4 h sooner or up to more than 5 h later. Apparently the ovulatory process of an individual gilt extends over a period of 4 - 5 h. Double insemination of 9 gilts at 34 and 41 h after Hoe 766 resulted in fertilization rates and litter sizes that compared favourably with those of corresponding gilts treated with HCG.     

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.     

Reflex ovulation in light-induced persistent estrus (LLPE) rats: Role of sensory stimuli and the adrenals

Penile intromissions have been thought to be the primary stimulus for reflex ovulation in light-induced persistent estrus (LLPE) rats, even though other stimuli also trigger reflex ovulation. To clarify the nature of these noncoital stimuli, intact (nonadrenalectomized) LLPE rats were briefly exposed to a variety of environmental stimuli, other than intromissions, and checked for ova 19–22 hr later. Summary of results (number of rats ovulating/number of rats tested): (A₁) home cage ($\text{3}\text{10}$); (B₁) home cage + vaginal taping ($\text{2}\text{9}$); (C₁) home cage + male-soiled bedding ($\text{15}\text{28}$); (D₁) novel cage ($\text{2}\text{11}$); (E₁) novel cage + vaginal taping ($\text{2}\text{11}$); (F₁) novel cage + vaginal taping + male-soiled bedding ($\text{9}\text{19}$); (G₁) novel cage + vaginal taping + male-soiled bedding + male mounts without intromissions ($\text{14}\text{26}$). The percentage of LLPE rats that ovulated in the last-mentioned test condition was related to the degree of proceptivity/receptivity of the LLPE females. Eight of eight proceptive LLPE females ovulated, but only $\text{6}\text{18}$ nonproceptive females ovulated. To account for reflex ovulation in the absence of intromission it has been suggested that adrenal progesterone (P) stimulates release of an ovulatory quota of luteinizing hormone. This study demonstrates no significant differences in percentage of LLPE females ovulating in corresponding groups of adrenalectomized (ADX) and adrenal-intact females. Summary of results: A₂ = $\text{0}\text{6}$, B₂ = $\text{5}\text{15}$, C₂ = $\text{4}\text{16}$, D₂ = $\text{2}\text{14}$, E₂ = $\text{5}\text{13}$, F₂ = $\text{7}\text{19}$, G₂ = $\text{10}\text{21}$. Conclusion: (a) Exposure to a factor in male-soiled bedding induces reflex ovulation in a significant proportion of adrenal-intact LLPE animals while exposure to a novel cage and/or vaginal taping does not, (b) penile intromissions are not the primary stimulus for reflex ovulation in intact proceptive LLPE rats, and (c) adrenal P is not required for reflex ovulation after tests with noncoital stimuli.     

Continuous subcutaneous infusion of human menopausal gonadotropin in anovulatory women with decreased FSH/LH ratio and androgen excess

Ten women of polycystic ovarian-type (PCO-type) anovulation, having a decreased ratio of FSH to LH and androgen excess, resistant to the previous clomiphene, bromocriptine and/or daily im injections of human menopausal gonadotropine (hMG), were treated with continuous sc infusion of 150 IU/day hMG. The treatment was initiated on cycle day 2-5 and continued until the dominant follicle reached 20 mm or more in diameter, when an im bolus of 10,000 IU human chorionic gonadotropin was given. The treatment elevated the geometric mean of pretreatment serum FSH (8.6 mIU/ml) to 15.9 mIU/ml (p less than 0.001), while serum LH decreased from 29.4 mIU/ml to 20.7 mIU/ml (p less than 0.01). This resulted in a highly significant increase in the FSH/LH ratio from 0.29 to 0.77 (p less than 0.0001). Follicle enlargement was demonstrated in 13 of the 14 treatment cycles, 12 of which were ovulatory. Pregnancy ensued in 4 cases, 1 of which was a quadruplet pregnancy. Continuous infusion of hMG was indicated as an effective way of inducing ovulation in PCO-type anovulation resistant to conventional methods of ovulation induction.     

Preliminary report on induction of estrus with multiple eCG injections in Colored Mohair goats during the anestrus season

This trial was conducted to evaluate the effectiveness of multiple eCG injections in the induction of estrus and pregnancy in Colored Mohair goats during the anestrus season. It was also aimed to determine total dose of eCG required for induction of estrus. Ten multiparous and lactating goats were used. The goats were randomly divided into two groups and treatments were started on May 22. Group eCG (n=5) was treated with eCG intramuscularly for 6 days. Daily dosages of eCG from May 22 to May 27 were 300 IU, 200 IU, 200 IU, 100 IU, 100 IU and 50 IU, respectively. Goats in control group received no treatment. Blood samples were taken from animals in each of the two groups just before and after the beginning of the treatments and serum progesterone concentrations were assayed by RIA. Starting on the fourth day after the first treatments, goats were exposed to fertile bucks twice daily for 30 min to detect standing heat. The estrus goats were allowed to be mated by the bucks. Pregnancies were determined 40 days after mating by real-time ultrasonography. One goat on day 5 and three goats on day 7 exhibited behavioral estrus in eCG group (80%) after the first eCG injection. Three of them (75%) became pregnant. None of the goats in the control group exhibited behavioral estrus. Mean serum progesterone concentrations had prominent elevations indicating ovulation in eCG group, but not in control group, after 20 days from the first treatments. Progesterone concentrations of eCG group were significantly different than those of control group on days 20 and 28 (P<0.05). The results suggest that divided multiple injections of a total 950 IU eCG are effective without progestagen pretreatment in the induction of estrus and obtaining successful pregnancy and live kids in Colored Mohair goats during the anestrus season.     

Effects of cloprostenol, human chorionic gonadotropin and estradiol benzoate treatment on estrus synchronization in dairy cows

Two consecutive experiments were conducted. In Experiment 1, 24 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received intramuscularly (i.m.) 500 mcg of cloprostenol, 1250 IU of human chorionic gonadotropin (hCG) and 5 mg of estradiol benzoate 12 h after cloprostenol treatment. Cows in Group II received 750 IU i.m. of hCG and 3 mg of estradiol benzoate 12 h after cloprostenol treatment. Treatment was given on Day 16 after estrus in both groups. All animals showed estrus within 24 to 48 h after cloprostenol treatment. The average interval from cloprostenol injection to the onset of estrus was not influenced by treatments. Four cows in Group I failed to ovulate and became cystic. In Experiment 2, 71 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received 500 mcg i.m. of cloprostenol after corpus luteum detection by palpation per rectum. Cows in Group II received 500 mcg of cloprostenol plus 750 IU of hCG and 3 mg of estradiol benzoate 12 h after. When estrus ready for service was confirmed by rectal examination, cows were inseminated. The percentage of cows ready for service tended to be lower (P < 0.06) between cows in Group I (88%) and those in Group II (100%). The average interval from cloprostenol treatment to service was longest (P < 0.001) in Group I (78.7 h +/- 14.9, X +/- SD) vs Group II (48 h +/- 2.9). The degree of readiness for service synchrony was lowest (P < 0.001) in Group I (59.3%) vs Group II (94.2%). The pregnancy rates of cows synchronized or treated were not altered by hCG-estradiol benzoate treatment (P > 0.25). These results suggest that in dairy cows treated with cloprostenol following palpation per rectum of a corpus luteum and then with 750 IU of hCG and 3 mg of estradiol benzoate 12 h later, a single fixed-time insemination at 48 h after cloprostenol treatment should be performed.