Impairment of caprine oocyte maturation in vitro and alteration of granulosa cells functions by widely used fungicide mancozeb.

Mancozeb is classified as an endocrine disruptor; thus the present study was carried out to investigate the impact of mancozeb on mammalian ovarian functions using in vitro caprine oocyte maturation and granulosa cell culture models. Caprine cumulus oocyte complexes (COCs) and granulosa cells were cultured under standard culture conditions and treated with mancozeb concentrations of 0.3, 3, and 30 µg/ml along with a control for 24 h and assessed. Granulosa cell viability and progesterone concentration in spent culture media after treatments were also assessed. Mancozeb significantly decreased (P < 0.05) the oocytes cumulus expansion and the maturation of caprine oocytes. Marked changes in granulose cell morphology were observed with 30 µg/ml mancozeb and significantly reduced (P < 0.05) cell viability. Interestingly, the same concentrations significantly increased (P < 0.05) the progesterone secretion by the cells. Significant reduction of granulosa cells viability and reduction of cumulus expansion and suppression of metaphase plate formation in oocyte can impair the fertilization ability and developmental potential of the oocytes. High progesterone concentration due to mancozeb treatment may suppress LH surge and suppress ovulation. In conclusion, mancozeb suppresses granulosa cells viability, reduces cumulus expansion, and suppresses metaphase plate formation but induces progesterone secretion from granulosa cells that may inhibit LH surge for ovulation process.

EFFECTS OF GONADOTROPIN-RELEASING HORMONE ANTAGONIST DEGARELIX ON MUSTH AND SERUM TESTOSTERONE CONCENTRATIONS IN ASIAN ELEPHANTS ( ELEPHAS MAXIMUS).

Two male Asian elephants (bulls 1 and 2) in musth were subcutaneously injected with a long-acting gonadotropin-releasing hormone (GnRH) antagonist, degarelix acetate (240 µg/kg; total dose of 960 mg). Musth behavior (MB) and temporal gland secretions (TGS) were monitored and serum testosterone concentrations were determined. In bull 1, MB and TGS ceased on day 1 and reappeared 5.5 mo after the treatment (day 0). During the subsequent musth cycle, MB and TGS ceased on day 1 and did not appear for 4 mo. In bull 2, MB and TGS ceased at day 7 after the treatment. Musth behavior and TGS recurred on Day 11 and continued for 1 wk, then disappeared for 8 mo. Serum testosterone concentrations decreased ( P < 0.05) in all occasions from day 0 (29.8 ± 15.8 ng/ml; mean ± SEM) to day 1 (2.2 ± 1.1 ng/ml), suggesting a sudden drop in circulating testosterone in musth elephants after the GnRH-antagonist treatment.

Anticoccidial effects of Phyllanthus emblica (Indian gooseberry) extracts: Potential for controlling avian coccidiosis.

The protozoan parasite Eimeria causes avian coccidiosis, impacting the poultry industry worldwide. Resistance development to current anticoccidials are a concern and cost effective, environmentally friendly alternatives are needed. Anti-malarial effects of Phyllanthus emblica encouraged us to investigate its anticoccidial effects. Aqueous extracts and dried-powder of P. emblica leaf and fruit were tested for effect(s) on oocyst sporulation in vitro and oocyst infectivity in vivo. Eimeria tenella oocysts were randomly assigned to groups and treated with different concentrations (0.001, 0.1, 1, 5, 25, 50 and 100%) of P. emblica crude extracts in triplicates for three repeats. Sporulated, unsporulated, deformed and lysed oocysts were recorded at 24, 48 and 72 h. Broiler chicks (21 days old) were randomly assigned into four groups with 5 chicks each and experimentally infected on Day 0 with 1 × 104 oocysts/bird: (A) infected and un-supplemented diet, (B) infected and supplemented diet (P. emblica powder 1 g/bird/day), (C) infected with P. emblica-treated oocysts and un-supplemented diet and (D) infected and diet supplemented only from day14. In vivo experiments were terminated on day28. Significant sporulation inhibition and oocyst lysis (p < 0.05) in vitro were observed in a concentration-dependent with P. emblica treatment. In in vivo experiments, group B showed the highest weight gain, lowest fecal oocyst excretion and mildest histopathological lesions. Extracts of P. emblica remarkably inhibited oocyst sporulation, reduced the oocyst infectivity and lowered the fecal oocyst excretion, and reduced the pathogenicity of E. tenella in chickens. Therefore, P. emblica extract demonstrates great potential to be an effective alternative anticoccidial agent.

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway.

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100 ng/ml) for 18 h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100 ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1µM) or INSL3 antagonist (bovine and human; 100 ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100 ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100 ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18 h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

Homozygosity of single nucleotide polymorphisms in the 3' region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40-kb region at the 3' end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3' end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14-17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15-17). A common haplotype block was identified in an approximately 20-kb region encompassing four SNPs (SNPs # 14-17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3' end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.

Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs.

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbecco's Modified Eagle Medium with Ham's nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.

Analysis of single nucleotide polymorphisms in the 3' region of the estrogen receptor 1 gene in normal and cryptorchid Miniature Dachshunds and Chihuahuas.

This study was performed to examine the distribution of single nucleotide polymorphisms (SNPs) and estimated haplotypes in the canine estrogen receptor (ER) alpha gene (ESR1) and the association of them with different phenotypes of cryptorchidism (CO) in Miniature Dachshunds and Chihuahuas. Forty CO and 68 normal dogs were used, and CO was classified into unilateral (UCO; n=33) and bilateral CO (BCO; n=5) or into abdominal (ACO; n=16) and inguinal CO (ICO; n=22). Thirteen DNA fragments located in the 70-kb region at the 3' end of ESR1 were amplified by PCR and sequenced to examine 13 SNPs (#1-#13) reported in a canine SNP database. Ten SNPs (#1-#4, #7, #8, #10-#13) were not polymorphic, and 5 new SNPs (#14-#18) were discovered. A common haplotype block in normal, CO and CO phenotypes was identified for an approximately 20-kb region encompassing 4 SNPs (#14-#17). Allele, genotype and haplotype frequencies in CO without classification by phenotype and also in UCO, ACO and ICO phenotypes were not statistically different from the normal group. Significant differences in genotype frequencies and homozygosity for the estimated GTTG haplotype within the block were observed in BCO compared with the normal group, although the number of BCO animals was small. Our results demonstrate that the examined SNPs and haplotypes in the 3' end of canine ESR1 are not associated with unilateral, abdominal and inguinal CO phenotypes and CO per se in Miniature Dachshunds and Chihuahuas. Further studies are necessary to suggest a clear association between the ESR1 SNPs and bilateral CO in dogs.