Serum anti-Müllerian hormone concentration as a diagnostic tool to identify testicular tissue in canine disorders of sexual development.

Disorders of sexual development (DSD) may have their origin in alterations of the chromosomal, gonadal or phenotypic sex. Affected animals are usually presented because of ambiguous external genitalia, seldom because of reproductive disorders. Anti-Müllerian hormone (AMH) is secreted in the gonads with higher amounts in males than in females and can be used to identify gonadal tissue in sexually normally developed dogs. The aim of this study was to examine the diagnostic potential of serum AMH to identify testicular tissue in 11 dogs with DSD. The diagnostic procedures applied were: determination of the phenotypic sex (n = 11), genital ultrasound (n = 9), determination of the SRY gene (n = 11), karyogram (n = 6), gonadectomy (n = 11), pathohistology of the gonads (n = 10), serum AMH measurement (n = 11). 39 female dogs described in a previous study and 19 male dogs with a normal spermiogram served as controls for the AMH serum concentrations in sexually intact dogs. The 11 dogs with DSD were classified as 7 XY DSD and 4 XX DSD. Presumptive testes were obtained in 10 dogs and 1 dog had an ovotestis combined with a testis. Mean serum AMH values of the dogs with DSD were significantly higher (P < 0.001) than in male and female controls. The upper limit of the AMH test (≥ 23ng/ml) was reached in 6 dogs. High AMH concentrations have been described previously in cryptorchid dogs. 1 dog with a male phenotype and 2 with a female phenotype had AMH values within the range of the male controls, although all of them had cryptorchid testes. A Poodle, in which epididymis were identified but no definitive gonads, had an AMH concentration of the lower limit of the test (≤ 0.01 ng/ml), comparable to previously described castrated dogs. This study indicates that serum AMH levels are a useful diagnostic tool to identify testicular tissue in dogs with DSD and suggests the possible use of AMH to diagnose testicular dysgenesis.

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.

Quantitative determination of progesterone (P4) in canine blood serum using an enzyme-linked fluorescence assay.

Progesterone (P4) measurement in the peripheral blood is an objective parameter for determination of reproductive functions in the bitch. This study evaluates an enzyme-linked fluorescence assay (ELFA) (Biomerieux, France) for the determination of progesterone validated for use in human. The ELFA is to be performed on the MiniVidas automated analyser which provides quantitative results within 45 min. Blood samples from a total of 27 female dogs of different breeds were used. To test the correctness of the ELFA 15 blood samples with a range of 0.3-40.0 ng/ml were compared to a radioimmunoassay (RIA) validated in the dog. The values obtained with the MiniVidas showed a high agreement (mean deviation 15%), deviations were in both directions and the correlation coefficient was 0.989. The coefficient of correlation according to Passing-Bablok test was 0.995. The intra-assay reproducibility in the MiniVidas system was tested on five samples (mean values 61.8, 6.8, 51.4, 43.7 and 1.1 ng/ml). The coefficients of variation (CV; 10-12 replicates) were 3.4%, 6.7%, 2.6%, 3.1% and 25.4%, respectively. Four serum samples (mean value 47.0, 15.1, 49.1 and 4.0 ng/ml) from different bitches were assayed singly in 10 separate series to test the inter-assay variability. The corresponding CV was 2.1%, 2.2%, 3.1% and 4.3% respectively. Samples from three dogs were used to test the accuracy of the assay. These samples were diluted (1/2, 1/4, 1/8 and 1/16) with charcoal-stripped human serum (Biomerieux, France) and tested in three runs. The expected values were met in a range of 60-75%. Measurement of progesterone for the detection of ovulation as well as prediction of parturition provided meaningful results. As a conclusion the use of the MiniVidas system for determination of P4 in peripheral blood of the bitch provides rapid and reliable results.

Estrus induction in Beagle bitches with the GnRH-agonist implant containing 4.7 mg Deslorelin.

Estrogens, gonadotrophins, dopamine agonists, gonadotrophin releasing hormone (GnRH) and its agonists have been used for estrus induction in bitches. A long acting GnRH agonist implant (4.7 mg Deslorelin; Suprelorin®, Virbac) with a continuous hormone release has been developed for suppression of sexual function in male dogs. In this study we administered the Deslorelin implant placed subcutaneously on the medial side of the leg to induce estrus in 11 anestrous Beagle bitches (group A). 6 Beagle bitches (group B) with a spontaneous estrous cycle were used as controls. The progress of pre-estrus and estrus was documented by behaviour, vaginoscopy, vaginal cytology and progesterone concentration. In group A a bloody vaginal discharge was detected on average 4.8 (range 3-10) d after application of the implant. At this moment implants were removed under local anaesthesia. Pre-estrus lasted for an average of 4.5 d (range 1-12). All bitches showed estrous signs and ovulated. The ovulation took place on day 8.2 (range 4-15) after start of pre-estrus. In group B pre-estrus lasted for 7.5 d (range 6-9), and the mean day of ovulation was day 11 (range 9-13). As a consequence of ovulation, progesterone serum concentrations exceeded 10 ng/ml during or after the time of ovulation in all bitches. All bitches were bred to fertile Beagle stud dogs or inseminated with fresh semen intravaginally. Between days nine and 19 after ovulation all bitches underwent ovariohysterectomy. The uterine horns were flushed and flushes were examined for ova or embryos. The pregnancy rate in group A was 63.6% and in group B 66.7%. Despite the significantly shorter period of pre-estrus a fertile estrus could be induced in 7 out of 11 treated bitches. Induction of a fertile estrus can be achieved with a GnRH-implant-already registered for the use in male dogs-placed subcutaneously on the medial side of the leg.

Microarray analysis of equine endometrium at days 8 and 12 of pregnancy.

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Effect of post-IVF developmental kinetics on in vitro survival of vitrified-warmed domestic cat blastocysts.

A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)-derived cat blastocysts. In the present study, IVF-derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post-warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post-warm survival rate was determined by re-expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post-warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF-derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.