Dual testing with QF-PCR and karyotype analysis for prenatal diagnosis of chromosomal abnormalities. Evaluation of 13,500 cases with consideration of using QF-PCR as a stand-alone test according to referral indications.

OBJECTIVE: Evaluate the results obtained from Quantitative Fluorescent (QF)-PCR and conventional karyotype analysis to determine the advantages and disadvantages of dual testing in prenatal diagnosis. METHODS: From 1 June 2006 to 1 June 2010, dual testing by QF-PCR and karyotype analysis was performed in 13,500 prenatal samples. The rates of concordant results between the two methods were evaluated and the rates of clinically significant chromosomal abnormalities undetected by QF-PCR were assessed. RESULTS: Abnormal karyotype was found in 320 out of 13,500 cases (2.37%, 95% confidence interval (CI) 2.11-2.63%). From these, QF-PCR did not detect the abnormality in 70 cases (0.52%, 95% CI 0.4-0.64%), whereas 34 had a high/unknown risk of adverse outcome (0.25%, 95% CI 0.17-0.33%). By selectively applying dual testing only at cases with ultrasound findings and/or genetic history, 13 cases of high/unknown risk would have been missed (0.1%, 95% CI 0.05-0.15%). CONCLUSION: Selective dual testing is expected to achieve a serious beneficial economical outcome and reduce parental anxiety produced by ambiguous cytogenetic findings. However, the percentage of 0.1% undetected clinically significant abnormalities cannot be ignored. A suggestion would include the offering of a choice to the pregnant women, undergoing prenatal screening, by informing them about different approaches and various complications.

Effects of melatonin treatment on follicular development and oocyte quality in Chios ewes - short communication.

Follicular development and oocyte quality were assessed by laparoscopic observation and in vitro fertilisation, respectively, in melatonin-treated (Group M) and control (Group C) anoestrous Chios ewes (n = 10 in each group). Fourteen days after melatonin insertion, all ewes had laparoscopic evaluation of the follicular population followed by oocyte pick-up (OPU); on day 22 intravaginal progestagen sponges were inserted for 14 days. Two days after sponge removal the follicular population was re-evaluated and a second follicular aspiration was performed. Collected oocytes from the second OPU underwent in vitro maturation, fertilisation and culture. The number of large follicles was higher in Group M than in the control ewes during the first OPU and tended to be so (P = 0.06) at the second. Morphologically, oocytes collected from controls were of better quality than those from Group M; however, more oocytes collected from melatonintreated animals fertilised and developed in vitro . These results indicate that melatonin is a potent regulator of follicular development and oocyte competence during the anoestrous period of the ewe.

Synchronisation of lambing with low doses of dexamethasone in Chios ewes--short communication.

The purpose of the present study was to investigate the feasibility of improving the synchronisation of lambing after oestrus synchronisation and artificial insemination (AI). To this end, low doses of dexamethasone 21-isonicotinate (DEX) alone or in combination with prostaglandin F2a (PG) were used in five treated groups (n = 20 each) and one control group (n = 136) of Chios ewes. On day 143 of pregnancy 1.5 mg DEX was given in Group 5, while on day 146 the following treatments were applied: 0.0375 mg PG in Groups 4 and 5, and 1, 1.5 and 2 mg of DEX in ewes of Groups 1, 2 and 3, respectively. The control ewes received no treatment. The 1.5 and 2 mg dose of DEX was more effective in synchronising labour as regards the treatment to lambing interval and the proportion of ewes that gave birth within 3 days. However, obstetrical manipulations were needed, and dead lambs were born when 2 mg DEX was used. It was concluded that lambing can be safely synchronised in Chios ewes with 1.5 mg DEX given on day 146, without affecting the viability of lambs and without parturition complications.

Effect of culture-medium supplementation with alpha-mannosidase and/or beta-N-acetyloglucosaminidase on in vitro bovine embryonic development.

Glycosidases are enzymes with a potential role in embryonic development. The objectives of this study were to assess: (a) whether in vitro bovine embryonic development is affected by the addition of beta-N-acetyloglucosaminidase (beta-NAGASE) and/or alpha-mannosidase to the culture medium and (b) whether these enzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were produced using standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50 microl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 degrees C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) beta-NAGASE, (2) alpha-mannosidase, (3) beta-NAGASE plus alpha-mannosidase, and (4) control (no supplement). Embryos were evaluated and samples of culture medium collected and frozen prior to assay for glycosidases at day 7 of culture. The experimental design was a randomised block arrangement of 4 treatments x 7 replicates with 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean +/- S.E.M. The osmolarity of the control culture medium was 272 mOsm. This was increased to 279 mOsm by the addition of alpha-mannosidase, 424 mOsm by beta-NAGASE and 337 mOsm with a combination of the two enzymes. The beta-NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages (P < 0.002) relative to control medium (35.7 +/- 8.4%). Embryo development was also reduced to 21.9 +/- 3.2 (P< 0.002), relative to control, by alpha-mannosidase supplementation. The reduced embryo development in the beta-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize alpha-mannosidase because its concentration decreased from 600.95 +/- 174.03 IU/l in drops without zygotes/embryos to 211.01 +/- 71.59 IU/l in drops with zygotes/embryos. Other culture media supplementation showed no significant differences between droplets, with or without zygotes/embryos. It was concluded that beta-NAGASE increased medium osmolarity, embryos utilized alpha-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.

Activity of glycosidases (beta-N-acetyloglucosaminidase, alpha-mannosidase, and beta-galactosidase) in the uterine luminal fluid of cows after multiple ovulation.

This study investigated the activity of beta-N-acetyloglucosaminidase (beta-NAGASE), alpha-mannosidase, and beta-galactosidase in the uterine luminal fluid of cows after superovulation treatment, along with the possible associations between the activity of these 3 glycosidases and the superovulatory response. Embryos and a sample of fluid flushed from each uterine horn were collected on day 7 after artificial insemination (on estrus day 0) from 32 cows in which superovulation was induced with porcine follicle-stimulating hormone. Glycosidase activity was assayed colorimetrically. The cows were classified as to superovulatory response according to the number of corpora lutea per ovary (group 1, 1 to 4; group 2, > 4) and according to the total number of embryos per horn (T1, 0; T2, 1 to 2; T3, 3 to 4; T4, > 4) and the number of transferable embryos per horn (TR1, 0; TR2, 1 to 2; TR3, 3 to 4; TR4, > 4). The mean activity of beta-NAGASE was significantly lower (P < 0.05) in group 2 than in group 1, at 95.99 (standard error 20.43) versus 226.72 (46.77) IU/L. It was also significantly lower (P < 0.01) in group T4 compared with groups T1, T2, and T3, at 50.09 (8.21) versus 129.25 (34.60), 222.27 (62.62), and 290.26 (93.77) IU/L, respectively, as well as in group T1 compared with group T3. There was a positive relationship between beta-NAGASE activity and both the total number of embryos (P = 0.047) and the number of transferable embryos per horn (P = 0.013) when 1 to 4 corpora lutea developed per ipsilateral ovary. No difference in alpha-mannosidase or beta-galactosidase activity was detected among the groups.

Relationship between electrical resistance of cervical mucus and ovarian steroid concentration at the time of artificial insemination in ewes.

The purpose of this study was to investigate whether fertile or non-fertile inseminations (AI) in synchronized ewes are correlated with the electrical resistance of cervical mucus (ERCM) and the ovarian steroid concentration. AIs were performed either at fixed-time (group A) or after estrus detection (group B). Retrospective analysis revealed that at AI, pregnant ewes had lower ERCM values and progesterone concentrations than non-pregnant ones (p<0.05). It appears that ERCM may be used as an additional index for fertility enhancement of inseminated ewes.

Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality.

Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the presence of ghrelin were of better quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation.

Association between physical properties of cervical mucus and ovulation rate in superovulated cows.

Possible associations between certain physical properties of cervical mucus (CM) and ovulation rate were studied in 21 superovulated Holstein cows. In CM samples collected at the beginning of estrus (0 h) and in 4 h intervals for the following 24 h, the pH, the spinnbarkeit (spinability), and the crystallization value were measured. Blood samples, collected at the same time points with CM samples, were assessed for progesterone and estradiol concentrations. At 48 h the number of ovulated follicles was counted by transrectal ultrasonography and the animals were allotted into 2 groups according to the occurrence of at least one (group A, n = 16) or no (group B, n = 5) ovulations. The pH was lower (P < 0.05) at 8 h (7.00 + 0.24) in group A compared with group B (7.55 + 0.12). In group A, spinnbarkeit was significantly lower at 0 h and 20 h, and higher at 8 h and 16 h compared with group B (0 h: 2.50 + 0.82 versus 6.95 + 0.41; 20 h: 3.00 + 1.89 versus 5.38 + 0.94; 8 h: 7.00 + 0.87 versus 2.75 + 0.43; 16 h: 7.00 + 1.41 versus 4.30 + 0.71, for groups A versus B, respectively). Crystallization was significantly lower at 4 h (2.00 + 0.63) and 20 h (1.50 + 0.82) in group A compared with group B (3.13 + 0.32 at 4 h and 3.00 + 0.41 at 20 h). Progesterone at all time points, and estradiol at 16 h, 20 h, and 24 h were lower (P < 0.05) in group A than in group B. The pH, crystallization, estradiol, and progesterone differed (P < 0.05) within one group, while sbk differed within both groups.Our results imply that during the periovulatory period, steadily low progesterone concentrations trigger alterations of certain CM characteristics, while extremely high estradiol concentration could prevent the occurrence of these alterations.