Histopathologic examination of the genital tract in rabbits treated once or twice with a slow-release deslorelin implant for reversible suppression of ovarian function.

A total of 13 rabbits were treated with a subcutaneous deslorelin long-term release implant (4.7 mg) to study the effect on ovarian function and histologic features of the uterus. Seven rabbits (group 1) were implanted with a slow-release deslorelin implant before onset of puberty for 273 days as a part of a previous study. After resumption of ovarian function had been confirmed, they were implanted again at the age of 430 days. Six adult rabbits (>177 days old; group 2) were implanted with a slow-release deslorelin implant for 273 days. Ovarian function before, during, and after treatment with the implant was assessed by measuring serum progesterone levels 10 days after a challenge injection of a short-acting GnRH (0.8 µg buserelin intramuscularly) on progesterone levels in peripheral blood. Values more than 4 ng/mL progesterone were considered to verify ovarian function. Animals in group 1 underwent ovariohysterectomy during the second treatment with the implant and the uteri, and ovaries were subjected to histopathologic examination. Endometrial hyperplasia and endometritis were observed in 5 of 7 animals. Nonatretic and atretic follicles at different developmental stages, but no active corpora lutea, were present in the ovaries. Ovariohysterectomy of group 2 animals was performed 2 to 12 months after implant removal. The histopathologic examination of the uterus and ovary of four animals neutered during induced pseudopregnancy showed no signs of uterine disorders. In two animals undergoing ovariohysterectomy 12 months after implant removal, endometritis was present. Their ovaries contained follicles at different developmental stages and corpora albicantia. Reversible suppression of ovarian function can be achieved in female rabbits by the use of GnRH slow-release implants administered before or after puberty. The findings of endometrial hyperplasia and endometritis in seven out of 13 rabbits treated once or twice with the implant may indicate that the development of age-related pathologies of the uterus cannot be prevented by the suppression of ovarian function with a long-acting GnRH implant.

Sperm membrane integrity in fresh and frozen-thawed canine semen samples: a comparison of vital stains with the NucleoCounter SP-100.

The objective of this study was to assess sperm membrane integrity in canine semen samples using three different vital stains and the NucleoCounter SP-100 (NC). In addition, the occurrence of half-stained sperm heads, the influence of investigator, and storage-related artifacts using stained smears were examined. Forty fresh (30 dogs) and 40 frozen-thawed (28 dogs) canine semen samples were analyzed. The vital stains eosin (E), eosin-nigrosin (EN), and bromphenolblue-nigrosin (BN) were compared. Two smears per stain were prepared and a total of 200 sperm per slide were classified using bright field microscopy. Each slide was examined twice by three investigators. Spermatozoa with completely red (E, EN) or blue (BN) stained sperm heads were classified as "dead". Half-stained sperm heads were counted separately. Sperm concentration and viability were determined using the NC. The NC works with a built-in fluorescence microscope using propidium iodide as a fluorescence dye. Statistical analysis for comparison of results was made using mean values with standard deviation, Bland-Altman plot and coefficient of variation (CV). Staining with E led to a significant higher percentage of dead sperm compared with EN and BN (P < 0.05), which gave comparable results. Vital stains revealed higher CVs (range 8.8%-32.1%) than the NC (<6.5%). Interobserver viability ranged from 17.5% to 45.4% and was within the same range between stains. If only completely stained sperm heads were considered, best agreement was found between the E and the NC. In case of EN and BN, inclusion of half-stained sperm heads reduced the difference compared with NC. In general, the agreement between methods was better in samples with a low percentage of dead spermatozoa. In smears of fresh semen stored up to 3 months, no increase in the percentage of dead spermatozoa could be observed. In some smears of frozen-thawed samples stained with E (n = 12) or BN (n = 2), all previously unstained spermatozoa were additionally stained during storage. In conclusion, all vital stains examined in this study technically can be used for differentiation between live and dead spermatozoa in canine semen samples, but the relatively high CVs have to be kept in mind. It is recommended to examine smears of frozen-thawed semen soon after preparation. Half-stained sperm heads should be counted as live sperm in E-stained smears. The NC allows assessment of sperm concentration and viability with a reasonable repeatability.

Reversible estrous cycle suppression in prepubertal female rabbits treated with slow-release deslorelin implants.

The aim of this study was to examine the long-term effect of a 4.7-mg deslorelin GnRH analog implant on ovarian function in the prepubertal female rabbit. Seven female rabbits (group 1) were treated with the implant at the age of 60 days. The implant was inserted subcutaneously in the umbilical region. Two animals (group 2) were not treated and served as a control group. The vulva of all 9 animals was examined for the presence of typical cyclical changes, additionally the occurrence of mounting behavior was recorded. Ovarian function was checked by administration of a short-acting GnRH agonist to induce ovulation and pseudopregnancy (0.8 µg of buserelin per animal intramuscularly). Ten days after each treatment with buserelin, blood was collected for progesterone measurement to confirm pseudopregnancy. After implant insertion, the first blood collection (Day 10) was done without preceding induction of ovulation to screen for implant induced ovulation and pseudopregnancy. The implant was in situ for 273 days, and during this time span, 12 attempts of induction of ovulation were carried out in intervals of 21 days, beginning at the age of 81 days. Afterward, it was removed under local anesthesia and 3 further inductions of ovulation by the same scheme were conducted. The insertion of the implant led to the establishment of a pseudopregnancy in 2 of 7 animals; the remaining 5 animals did not show elevated progesterone values. Attempts to induce ovulation by administration of the short-acting GnRH analog while the slow-release GnRH analog implant was in place were not successful in treated animals, and progesterone concentrations were basal. The effect was reversible as ovulation could be induced in 2 subsequent cycles in all animals by the third induction of ovulation after implant removal. Induction of ovulation in control animals at the age of 110 and 131 days resulted in elevated progesterone levels after 10 days. No adverse side effects could be observed in implant-treated animals. The typical red coloration of the vulva could be seen in group 2 and after implant removal in group 1. The results suggest that in 5 of 7 rabbits, puberty was delayed by the treatment with the 4.7-mg deslorelin slow-release analog until the implant had been removed. In the other animals, the treatment induced an initial flare-up phenomenon. Afterward, the treatment could reversibly suppress ovarian function in all 7 treated animals.