C. Valiente

Endocrine effects of the GnRH antagonist, acyline, in domestic dogs

Gonadotrophin-releasing hormone (GnRH) antagonists may have a future role in the control of canine reproductive function. In this study, the effects of a single dose of the potent GnRH antagonist, acyline, on serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were evaluated in male dogs. Blood samples were drawn before (Day -1) and after (30, 60, and 90 min, 3, 6, 9, 12, and 24h, and 3, 6, 9, 14, 22, and 29 d) treatment with acyline (330 microg/kg, sc); serum concentrations of FSH, LH, and T varied throughout the study period (P<0.01, <0.05, and <0.01, respectively). Gonadotrophins decreased below pretreatment concentrations 60 min after injection, whereas T took 90 min to decrease below baseline (P>0.05). Follicle-stimulating hormone, LH and T decreased until Day 9, when they reached their nadir at 2.0 +/-1.1 ng/mL (P<0.01), 1.2+/-0.2 ng/mL (P>0.05), and 0.5+/-0.2 ng/mL (P<0.05), respectively. Both gonadotrophins and T began increasing on Day 14 after treatment, although FSH and T serum concentrations still remained below baseline on that day (P>0.05). Follicle-stimulating hormone and T rebounded above baseline on Day 29, whereas LH reached concentrations were similar to baseline at this time (P>0.05). No local or systemic side effects were detected in any dog following acyline treatment. In conclusion, a single acyline treatment safely and reversibly decreased serum gonadotrophin and T concentrations in dogs for 9 d.

Interruption of the canine estrous cycle with a low and a high dose of the gnrh antagonist, acyline.

To test the efficacy and clinical safety of a low and high dose of the GnRH antagonist, acyline, on estrous cycle interruption and anovulation in female dogs, 20 proestrous (<3d) bitches were randomly assigned to one of the following pharmacological protocols (given sc): acyline 110 microg/kg (ACY-L; n=6); acyline 330 microg/kg (ACY-H; n=8); or placebo (PLACE, n=6). The animals were monitored (clinical and vaginal cytology examinations) daily for 60d. Blood samples for serum progesterone serum concentrations were collected 14d after treatment to determine if ovulation had occurred. Appearance of side effects and days to the onset of the first spontaneous estrous cycle after treatment were also recorded. In both ACY groups, but not the PLACE group, estrous cycles were interrupted after treatment (P<0.05). The interval from treatment to estrus interruption in ACY-L and ACY-H groups was 3.0+/-0.6 and 3.2+/-0.2d, respectively (LSM+/-SEM; P>0.05). In the PLACE bitches, physical, behavioral and cytological proestrus slowly progressed to estrus and diestrus. Ovulation was absent in all ACY, but not in PLACE bitches (P<0.05). None of the females manifested side effects related to the treatments (P>0.05). Spontaneous return to a normal estrous cycle during the study period occurred in all ACY (ACY-L 19.5+/-2.7d vs ACY-H 24.8+/-2.0d; P>0.05), but in none of the PLACE bitches (P<0.05). In conclusion, acyline efficiently, safely and reversibly interrupted an early phase of the estrous cycle in bitches by preventing ovulation.

Comparison of Two Doses of the GnRH Antagonist, Acyline, for Pregnancy Termination in Bitches

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P(4)) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 microg/kg; n = 6) or high (ACY-H; 330 microg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 +/- 1.9 and 6.4 +/- 1.3 days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P(4) serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P(4) diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P(4) reached basal concentrations. In PLACE animals, gestation progressed normally and P(4) did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P(4) serum concentrations.

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat.

Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330 microg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n=17) or a placebo (PLC; n=7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean+/-SEM) 7.0+/-1.3 and 7.0+/-1.7 d after treatment (P>0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P<0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P<0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4+/-1.7 and 120+/-17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n=3), mid pregnancy (from 26 to 45 d; n=4), late pregnancy (> 45 d; n=3), or injection of a placebo in early (n=1), mid (n=2), or late pregnancy (n=1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P(4)) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P(4) concentrations did not differ among treatment groups (P>0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.

Ejaculation training, seminal alkaline phosphatase and semen preservation through cooling in a milk-based extender in domestic cats

The purpose of this report is to describe (1) the training of domestic cats in ejaculation into an artificial vagina (AV), (2) alkaline phosphatase (AP) concentrations in whole ejaculates, and (3) the in vitro effect of a skimmed-milk plus egg yolk (SM-Y) extender on feline spermatozoa incubated at 4ºC. Five post-pubertal cats were trained to ejaculate into an AV three times a week for 20 mins in the presence of a teaser queen. Fifty AV-obtained ejaculates were macro- and microscopically assessed, and the AP therein measured by optimized colorimetry. Eighty AV-obtained ejaculates were pooled, diluted in SM-Y extender [80% (v/v) skimmed milk, 20% (v/v) egg yolk, and antibiotics], stored at 4°C and evaluated daily for 6 days. All the animals could be trained to ejaculate, although the interval up to the first AV ejaculation varied from 1.5 to 5.5 months (mean 3.9 months). The final performance at collection ranged from excellent to poor and was inversely related to the training period required in all cases. The mean AP concentration in whole ejaculates was 20,645.6 ± 4405U/l, which was not correlated with the concentration of spermatozoa. Most seminal parameters [(%); total (77 ± 2.3) and progressive (62.7 ± 3.4) motility, live sperm (91.8 ± 1.2), intact plasmalemma (83.5 ± 2.6), normal acrosomes (83.5 ± 2.6), pH (6.6 ± 0.0) and osmolarity (mOsm/l; 321 ± 5.2)], though decreasing during storage in the cold, remained within values compatible with in vivo fertilization for 2 days.