Effect of Vitrification of Feline Ovarian Cortex on Follicular and Oocyte Quality and Competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.52 mm3) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

Ovarian remnant syndrome in small animals

The ovarian remnant syndrome (ORS) is an iatrogenic disorder in bitches and queens, which is characterized by recurrence of estrus following surgical spay, due to the presence of a piece of ovarian tissue within the abdominal cavity. In most cases, the remnant ovary is found in the right ovarian pedicle, due to its topographic position, deeper and more cranial than the left ovary. The main clinical signs of ORS in small animals are the heat behavior and the presence of vaginal swelling/secretion, especially in canines. The diagnosis should be performed by means of vaginal cytology when attraction of males is detected, serum estrogen and progesterone levels and/or by challenging test with GnRH or hCG administration. However, vaginal citology is the most suitable and less expensive diagnostic tool. Nowadays the treatment of choice is a new laparotomy or laparoscopy, followed by removal of the remnant ovarian tissue. The surgical treatment has more chances of success if it is performed in the diestrus (i.e., between 15 and 60 days after the detection of attraction of males). Furthermore, a careful and accurate surgical procedure aided by advanced visualization techniques during ovariohysterectomy or ovariectomy is the best way to prevent ORS in companion animals.

Monitoring ovarian cycles and pregnancy in brown brocket deer (Mazama gouazoubira) by measurement of fecal progesterone metabolites

In the present study, pregnancy and the estrous cycle were monitored in captive brown brocket deer (Mazama gouazoubira) by measuring fecal progestagens with a commercial enzyme immunoassay (EIA), along with behavioral data. Fecal samples were collected twice a week during pregnancy and daily during the estrous cycle and post-partum period. It was possible to distinguish between inter-luteal and luteal phases of the estrous cycle. Behavioral estrus corresponded with low concentrations of fecal progestagens. Samples from two consecutive cycles were available from five hinds, and the mean estrous cycle (n = 10) was 26.9 +/- 1.7 d (mean +/- S.E.M.). However, when two extreme cycles (34 and 37 d) were deleted, the mean estrous cycle was 24.7 +/- 1.2 d. Three animals became pregnant (gestation ranged from 208 to 215 d). After fertile breeding, progestagen concentration in these hinds remained among luteal phase concentrations throughout pregnancy, with the exception of a few peaks. Within 4 d post-partum, two hinds reached interluteal phase values, while one hind maintained luteal concentrations for at least 1 week. (C) 2005 Elsevier B.V. All rights reserved.

OVARIAN HISTOLOGY and FOLLICULAR SCORE IN FEMALE RATS TREATED WITH NANDROLONE DECANOATE and SUBMITTED TO PHYSICAL EFFORT

The study was conducted to analyze the histology of the ovaries of adults rats treated with steroids, and submitted or not to physical effort. The control group consisted of females submitted to physical effort and sedentary females, both of which received a physiological solution of 0.9% saline. Treated females, sedentary or not, received 6 mg/kg of body weight of nandrolone decanoate. The steroid and physiological solution were administered intraperitoneally, with a single injection per week for 4 consecutive weeks. The applied physical effort was swimming (20 minutes daily, 5 days/week, for the 4 weeks of treatment). Serial sections (5 mu m) of ovaries were prepared for histological evaluation and follicular score. The weight of ovaries and hypophysis, the number of antral and atretic follicles, and the area of corpus luteum were all affected by the steroids. In the ovaries of the control groups, well-developed corpus luteum was observed. In the treated groups, the cortical stroma was occupied by ovarian interstitial tissue. The females treated with steroids presented estral acyclicity. The use of nandrolone decanoate, whether associated with physical effort or not, affected the morphological pattern of the ovaries.

Recombinant luteinizing hormone supplementation to recombinant follicle-stimulation hormone during induced ovarian stimulation in the GnRH-agonist protocol: A meta-analysis

Purpose: to compare the efficacy of recombinant LH supplementation for controlled ovarian stimulation in recombinant FSH and GnRH-agonist protocol.Methods: Search strategies included on-line surveys of databases. The fixed effects model was used for odds ratio and effect size (weighted mean difference). Four trials fulfilled the inclusion criteria.Results: a fewer days of stimulation (p < 0.0001), a fewer total amount of r-FSH administered (p < 0.0001) and a higher serum estradiol levels on the day of hCG administration (p < 0.0001) were observed for the r-LH supplementation protocol. However, differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates.Conclusions: more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous LH supplementation in ovarian stimulation protocols with FSH and GnRH-agonist for assisted reproduction treatment can be provided.

TRAIL-R3-related apoptosis: Epigenetic and expression analyses in women with ovarian neoplasia

Objective. To assess the expression of TRAIL-R3 and the methylation of a CpG island within the TRAIL-R3 promoter both in cystadenoma tumors and primary and metastatic epithelial ovarian carcinoma (EOC).Methods. RNA was obtained from women with normal ovarian (NO) tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and EOC (n = 16) using Trizol (R). Quantitative PCR (gRT-PCR) was performed to quantify the relative levels of TRAIL-R3. The methylation frequency of the CpG island in the TRAIL-R3 promoter was assessed using the methylation-specific PCR (MSP) assay after DNA bisulfite conversion. The differences between the groups were evaluated using the chi-square, Student's t, ANOVA, Mann-Whitney U, Wilcoxon or Kruskal-Wallis tests as indicated. The survival rates were calculated using the Kaplan-Meier method.Results. Cystadenoma and metastatic EOC tumors expressed significantly more TRAIL-R3 mRNA than primary EOC tumors. Methylation of the TRAIL-R3 promoter was absent in NO tissues, while hemimethylation of the TRAIL-R3 promoter was frequently found in the neoplasia samples with 45.4% of the cystadenoma tumors, 8.3% of the primary EOC samples and 11.1% of the metastatic EOC samples showing at least partial methylation (p = 0.018). Neither the expression of TRAIL-R3 nor alterations in the methylation profile were associated to cumulative progression-free survival or the overall survival in EOC patients.Conclusions. Primary EOC is associated to a lower TRAIL-R3 expression, which leads to a better understanding of the complex disease and highlighting potential therapeutic targets. Promoter DNA methylation was not related to this finding, suggesting the presence of other mechanisms to transcriptional control. (C) 2012 Elsevier B.V. All rights reserved.

Effects of recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-agonist protocol: a matched case-control study

Background: Some studies have suggested that the suppression of endogenous LH secretion does not seem to affect the majority of patients who are undergoing assisted reproduction and stimulation with recombinant FSH (r-FSH). Other studies have indicated that a group of normogonadotrophic women down-regulated and stimulated with pure FSH preparations may experience low LH concentrations that compromise the IVF parameters. The present study aimed to compare the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in r-FSH and GnRH-agonist (GnRH-a) protocol in ICSI cycles.Methods: A total of 244 patients without ovulatory dysfunction, aged < 40 years and at the first ICSI cycle were divided into two groups matched by age according to an ovarian stimulation scheme: Group I (n = 122): Down-regulation with GnRH-a + r-FSH and Group II (n = 122): Downregulation with GnRH-a + r-FSH and r-LH (beginning simultaneously).Result(s): The number of oocytes collected, the number of oocytes in metaphase II and fertilization rate were significantly lower in the Group I than in Group II (P = 0.036, P = 0.0014 and P = 0.017, respectively). In addition, the mean number of embryos produced per cycle and the mean number of frozen embryos per cycle were statistically lower (P = 0.0092 and P = 0.0008, respectively) in Group I than in Group II. Finally the cumulative implantation rate (fresh+thaw ed embryos) was significantly lower (P = 0.04) in Group I than in Group II. The other clinical and laboratory results analyzed did not show difference between groups.Conclusion: These data support r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment.

A new ovarian response prediction index (ORPI): implications for individualised controlled ovarian stimulation

Background: The objective was to present a new ovarian response prediction index (ORPI), which was based on anti-Mullerian hormone (AMH) levels, antral follicle count (AFC) and age, and to verify whether it could be a reliable predictor of the ovarian stimulation response.Methods: A total of 101 patients enrolled in the ICSI programme were included. The ORPI values were calculated by multiplying the AMH level (ng/ml) by the number of antral follicles (2-9 mm), and the result was divided by the age (years) of the patient (ORPI=(AMH x AFC)/Patient age).Results: The regression analysis demonstrated significant (P<0.0001) positive correlations between the ORPI and the total number of oocytes and of MII oocytes collected. The logistic regression revealed that the ORPI values were significantly associated with the likelihood of pregnancy (odds ratio (OR): 1.86; P=0.006) and collecting greater than or equal to 4 oocytes (OR: 49.25; P<0.0001), greater than or equal to 4 MII oocytes (OR: 6.26; P<0.0001) and greater than or equal to 15 oocytes (OR: 6.10; P<0.0001). Regarding the probability of collecting greater than or equal to 4 oocytes according to the ORPI value, the ROC curve showed an area under the curve (AUC) of 0.91 and an efficacy of 88% at a cut-off of 0.2. In relation to the probability of collecting greater than or equal to 4 MII oocytes according to the ORPI value, the ROC curve had an AUC of 0.84 and an efficacy of 81% at a cut-off of 0.3. The ROC curve for the probability of collecting greater than or equal to 15 oocytes resulted in an AUC of 0.89 and an efficacy of 82% at a cut-off of 0.9. Finally, regarding the probability of pregnancy occurrence according to the ORPI value, the ROC curve showed an AUC of 0.74 and an efficacy of 62% at a cut-off of 0.3.Conclusions: The ORPI exhibited an excellent ability to predict a low ovarian response and a good ability to predict a collection of greater than or equal to 4 MII oocytes, an excessive ovarian response and the occurrence of pregnancy in infertile women. The ORPI might be used to improve the cost-benefit ratio of ovarian stimulation regimens by guiding the selection of medications and by modulating the doses and regimens according to the actual needs of the patients.

Ovarian mucinous cystadenoma in a gray brocket deer (Mazama gouazoupira)

An ovarian mucinous cystadenoma is described in a gray brocket deer (Mazama gouazoupira). The tumor was histologically characterized by the presence of cysts and proliferation of papillae, both lined by single- or multi-layered pleomorphic epithelial cells that contained alcian blue-positive mucins.

Ovarian activity and plasma concentrations of progesterone and estradiol during pregnancy in jennies

The objectives of this study were to determine ovarian activity (with ultrasound) and plasma concentrations of progesterone and estradiol during pregnancy in jennies. There was considerable ovarian activity during the second month of pregnancy. Secondary corpora lutea (total of 2 to 7 per jenny) were formed (mainly by luteinization) starting on Day 41.8 +/- 1.0 (range Days 38 to 46; ovulation = Day 0). The echogenicity of the primary and secondary corpora lutea gradually decreased during pregnancy. Plasma progesterone concentrations increased between Days 0 and 10 (0.9 and 19.9 ng/mL, respectively), gradually decreased to Day 30 (12.1 ng/mL), increased between Days 30 and 40 (plateau, at approximately 17 ng/mL), gradually declined from Days 110 to 160 (nadir of approximately 6 ng/mL), and remained nearly constant until increasing again just before parturition. Plasma estradiol concentrations increased gradually from Day 65, peaked (1.2 ng/mL) on Day 165 (greater than or equal to 1 ng/mL on Days 150 to 210), and decreased thereafter, with very low concentrations during the last 20 d before parturition. Ovarian function and hormone profiles were generally similar to those previously reported during pregnancy in mares. (C) 1998 by Elsevier B.V.

Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries - partial results

Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36 degrees C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5 degrees C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 mu l of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

Structural and ultrastructural characterization of buffalo fetus (Bubalus bubalis) ovarian germinative cells

The objective of the present study was to characterize ovogones, primary oocytes and preantral follicles of buffalo fetus in different ages of gestation. For this, 29 fetuses were collected from a slaughterhouse (Frigol, Brazil) and crown-rump lengths were measured to estimate the fetal age (0-3, 4-6, 7-10 months of gestation). The ovaries were removed and ovarian tissue was processed for classic histology and transmission eletron microscopy examination. The structural evaluation demonstrated that in the first period of the gestation (0-3 months) the buffalo fetus showed ovogones (in mitotic division) and in some cases, the primary oocytes surrounded by somatic cells. In the second period (4-6 months), it was verified that the preantral follicles were completely formed. In the last period (70 month to the end of gestation) the ovaries contained a large amount of preantral follicles, and in some fetuses, antral follicles were observed. The ultrastructural analysis of the ovogones, primary oocytes and preantral follicles showed that these cells have few organelles and the quantity of mitochondria, endoplasmatic reticulum and apparatus Golgi complex is increased as the germinative cells passing from one stage to another.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation (3) administration of EPE twice daily in successively decreasing doses. In the first experiment. 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of greater than or equal to35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation. or two doses of a GnRH-a agonist upon detection of a follicle greater than or equal to35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles greater than or equal to35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF(2gamma) and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF(2alpha), but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles :35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose. improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates. (C) 2002 Elsevier B.V. All rights reserved.