Human papilloma virus genotypes in dysplasia and epithelial hyperplasia of oral cavity using the luminex xmap technology. A multicenter study.

BACKGROUND: Oral cancer associated with high risk (HPV-HR) human papilloma virus (HPV) has been increasing. HPV-HR has been associated with epithelial dysplasia, however, little information exists on its frequency in epithelial hyperplasia lesions. The aim of this study is to compare HPV genotypes in dysplastic and hyperplastic lesions of oral cavity. MATERIAL AND METHODS: Two hundred and fifty oral lesions: 131 dysplasia and 119 hyperplasia from two regions of Colombia were evaluated. One hundred seventy-four coming from urban area and 104 from a high risk population to oral cancer from a rural area. HPV was identified by qPCR and Twenty-four HPVs genotypes were evaluated by Luminex® technology. Logistic regressions were performed to establish the associations between HPV infections with oral dysplasia. RESULTS: Twenty-eight percent (70/250) of the samples were positives for any HPV and HPV-HRs were more frequently than low risk HPVs. HPV-16 was the most detected genotype (16%) followed by HPV-31, 53, 18 and 45. HPV, HPV-HRs and HPV-16 were only associated with dysplasia in urban area; OR 3.28 (CI 95% 1.49-7.17), OR 7.94 (CI 95% 2.97-21.2) and OR 5.90 (CI 95% 2.05-17). Individuals in rural area showed more HPV and HPV-HRs infection in hyperplasic lesions than urban population. The majority of HPV+ lesions had multi-type of HPV (52/70) and the urban individuals showed more genotypes than rural population. CONCLUSIONS: HPV-.HRs are frequently found in hyperplastic and dysplastic epithelial lesions. HPV-HRs and HPV-16 were associated with dysplasia in urban population. Rural high risk population and urban population differ in the frequency and variety of HPV genotypes.

Sperm kinematics and subpopulational responses during the cryopreservation process in caprine ejaculates.

Sperm cryopreservation in goats has been a challenge for many years due to the detrimental effects of seminal plasma enzymes produced by the bulbo-urethral glands which catalyse the hydrolysis of lecithins in egg yolk to fatty acids and lysolecithins which are deleterious to spermatozoa. This fact implies to carry out additional processing steps during sperm cryopreservation for seminal plasma removal triggering different sperm responses which may affect sperm functionality. The objective of the present study was to determine specific sperm subpopulation responses in different handling steps during the cryopreservation process by using functional sperm kinematic descriptors in caprine ejaculates. Buck ejaculates (n = 40) were analysed for sperm concentration, viability, morphology and acrosome integrity. Moreover, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system after five different handling steps (fresh sperm, 1st washing, 2nd washing, cooling and frozen-thawed sperm) during a standard cryopreservation protocol for goat semen. The results were analysed using Principal Component Analysis (PCA) and multivariate clustering procedures to establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st and 4th steps, four sperm kinematic subpopulations were observed explaining more than 75% of the variance. Based on velocity and linearity parameters and the subpopulations disclosed, the kinematic response varies among processing steps modifying sperm movement trajectories in a subpopulation-specific and handling step-dependent manner (p < 0.001). The predominant motile subpopulation in freshly ejaculated buck sperm had very fast velocity characteristics and a non-linear trajectory (41.1%). Washing buck sperm twice altered the subpopulation structure as well as cooling which resulted in a dramatic reduction in sperm velocities (p < 0.01). Frozen-thawed spermatozoa showed similar characteristics to cooled sperm except there was a further increase in linearity with a large proportion of sperm attributed to new slow, linear cluster (32.5%). In conclusion, this study confirms the variability and heterogeneity of goat sperm kinematic patterns throughout the cryopreservation process and suggests that the predominant motility pattern (assayed in vitro via CASA) of high quality spermatozoa might be typified by high speed and a non-linear trajectory. The relationships among the number and distribution of sperm subpopulations and the different handling steps were particularlly relevant, specially after the cooling and the post-thawing steps, when effects derived from these critical handling steps were evident and altered drastically the sperm motion patterns.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media.

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.

Prion protein 2 (dublet) gene (PRND): role in ovine semen capacitation, cryopreservation and fertility.

The aim of the present study was to examine the role of Doppel protein in the capacitation process and fertilising ability of both fresh and frozen-thawed (FT) spermatozoa from rams carrying different prion protein 2 (dublet) (PRND) gene polymorphisms. The detection efficacy of new anti-Doppel monoclonal antibodies and PRND mRNA quantification were also explored in ovine spermatozoa. Three different genotypes (AA, GA, GG) were identified for codon 26 of ovine PRND-c.78G>A. Using flow cytometry, a higher fluorescence was detected in fresh compared with FT sperm samples incubated with anti-Doppel primary and fluorescein isothiocyanate-conjugated secondary antibodies (P<0.05). Capacitation was affected by semen treatment (fresh and FT) and male PRND genotype (P<0.05). After IVF, the use of fresh semen resulted in a higher cleavage rate than the use of FT spermatozoa (P=0.004). IVF using spermatozoa from individuals classified as carriers of the AA or GA PRND genotypes resulted in higher cleavage rates than seen using spermatozoa from GG carriers (P≤0.0006). Finally, using semen from rams with the AA PRND genotype resulted in the highest Day 6 and Day 8 embryo rates (P≤0.04). In conclusion, the results of the present study confirm that the identification of different PRND genotypes is important for studying the sperm capacitation process and for improving sperm cryoresistance and embryo production. Furthermore, the detection of Doppel in ejaculated ovine spermatozoa, along with its low expression after cryopreservation, strongly suggests an important physiological function of this protein in male fertility.

The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep.

The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).

Secondary corpora lutea induced by HCG treatment enhanced demi-embryo survival in lactating high-yielding dairy cows.

Using a novel in vivo model considering a low developmental competence embryo (demi-embryo) and a subnormal fertility recipient (lactating high-yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P4 ) and bovine pregnancy-specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi-embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG-treated cows with secondary CL than in hCG-treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG-treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P4 concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non-pregnant cows on days 14-21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P4 concentrations, the survival rate of demi-embryos and the pregnancy rate of high-yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition.

The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 µM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos.

Hepatitis C treatment outcome in former or current intravenous drug users coinfected with HIV, with or without directly observed therapy.

BACKGROUND: Intravenous drug users (IDUs) with hepatitis C virus (HCV)/human immunodeficiency virus (HIV)-coinfection are recognised as a high-risk, vulnerable group. METHODS: Between February 2015 and April 2018, a single-centre, non-interventional cohort study was conducted in an outpatient setting, to evaluate the sustained virologic response (SVR12) and assess treatment uptake models. The study included 385 former or recent IDUs divided into two groups: A-without use of opioid substitution treatment (OST) and B-patients taking opioid substitution; patients in group B received OST and self-administered therapy (B1) or OST and therapy under DOT (B2). Patients were characterised by demographic and clinical features and compared for treatment response. Correlations between SVR12 and independent variables were determined by logistic regression. RESULTS: Patients were mostly males (88.3%) with a mean age of 46 ± 5 years and HCV genotype 1a (63.7%). Approximately 28% were treatment-experienced and 84.9% received sofosbuvir/ledipasvir. The mean CD4+T count was 649 cells/mm3, and most individuals were on antiretroviral therapy with undetectable viral loads (97.4%). SVR12 was achieved in 94.8%, and only eight patients relapsed. No significant differences were found in treatment effect between individuals taking opioid substitutes under different treatment models. Correlations were found between HCV viral response and both HIV suppression and albumin levels. CONCLUSIONS: IDU with HCV/HIV coinfection, including individuals on self-administration of HCV therapy and opioid substitution treatments or in DOT programmes, are no longer considered a difficult-to-treat group, as they achieve high rates of SVR12.

Prion-like Doppel gene polymorphisms and scrapie susceptibility in Portuguese sheep breeds.

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion-like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.

Improvement of fertility in artificially inseminated ewes following vaginal treatment with misoprostol plus terbutaline sulphate.

The effect of vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination (AI) upon the site of AI (vaginal or cervical) and fertility was studied using a total of 87 estrous synchronized Serra da Estrela ewes (control n = 42 and treated n = 45). Artificial insemination was performed using refrigerated semen at 54-55 h after sponge removal. Lambing rate (fertility) and prolificacy were compared between control and treated ewes. The effect of the site of semen deposition on fertility was also evaluated. Prolificacy rate was not different between control (1.5) and treated (1.59) ewes. The proportion of cervical AI achieved in control (45.2%) and treated (37.8%) ewes was not significantly different. Overall, fertility was significantly lower in control than in treated ewes (42.9% vs 64.4%; p < 0.04). Fertility following vaginal AI was significantly lower for control for than treated ewes (30.4% vs 60.7%; p < 0.03) but the difference was smaller and not significant for cervical AI (control 57.9% vs 70.6%). It was concluded that vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination did not affect the proportion of cervical inseminations but significantly improved the fertility of treated ewes. Although needing confirmation, it was hypothesized that drugs might have induced local secretory modifications leading to an increase of cervical ability to retain more viable spermatozoa for fertilization.

Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture.

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day=D0), zygotes were cultured on granulosa cells+M199+10% serum+100microM GSH supplemented with 100microM of t10, c12 CLA (CLA group, n=1394) or without supplementation (control group, n=1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n=24; group B-CLA, n=23). Non-biopsied embryos were also frozen (group NB-Control, n=49; group NB-CLA, n=45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P

Prion protein testis specific (PRNT) gene polymorphisms and transcript level in ovine spermatozoa: Implications in freezability, fertilization and embryo production.

An essential role of prion protein testis specific (PRNT) and prion protein 2 dublet (PRND) genes in the male reproductive function has been highlighted, although a deeper knowledge for the mechanisms involved is still lacking. Our goal was to determine the importance of the PRNT haplotypic variants and mRNA expression levels in ovine spermatozoa freezability and ability for fertilization and embryo developmental processes. Their association with the PRND gene polymorphisms was also analyzed. DNA from rams belonging to three Portuguese sheep breeds (n = 28) was screened by single-strand conformation polymorphism (SSCP) analysis to identify the PRNT and PRND polymorphisms. Semen collected from these rams was cryopreserved and fertility traits evaluated. The SSCP analyses revealed polymorphisms in the codons 6, 38, 43 and 48 of the PRNT coding region - respectively c.17C > T (p.Ser6Phe, which disrupts a consensus arginine-X-X serine/threonine motif); c.112G > C (p.Gly38 > Arg); and synonymous c.129T > C and c.144A > G. The polymorphisms in codons 6, 38 and 48 occur simultaneously while the one in codon 43 occurs independently. Six haplotypes were identified in the PRNT coding region, resulting in three different amino acid polymorphic variants (6S-38G-43C-48V, S6F-G38R-43C-48V and 6F-38R-43C-48V). The PRNT gene mRNA transcript level in spermatozoa was related to the identified haplotypic variants, either considering the codons 6-38-48 (P ≤ 0.0001) or the codon 43 alone (P ≤ 0.0001) or altogether (P ≤ 0.0001). An interaction between PRNT haplotypes and PRND genotypes on PRNT transcript level was also identified (P = 0.0003). Rams carrying the 17C-112G-144A PRNT haplotype had sperm with the highest post-thawed individual motility (P ≤ 0.03). Combined PRNT and PRND polymorphic variation influenced the post-thawed individual motility (P = 0.01). The male PRNT haplotypic, either considering the codons 6-38-48 and 43 altogether or the codon 43 alone, interfered (P ≤ 0.04) in embryo production rates. In conclusion, our data confirm that the PRNT gene is highly polymorphic in sheep and that the PRNT and PRND genotypes are associated. The identified polymorphisms of PRNT coding region seems to interfere on the ram spermatozoa mRNA transcript level and on male fertility, specifically in sperm freezability and ability for embryo development.

Cryosurvival of bovine blastocysts is enhanced by culture with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA).

An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF=day 0). On day 1, presumptive zygotes (n=3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 microM 10t,12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly (P<0.001) lower in SOF (17.9+/-1.6%) than in groups MS (29.8+/-2.5%) and SCLA (27.8+/-2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS (P<0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7+/-4.1%) than in SOCS (50.3+/-4.8%, P=0.0007) or SOF (65.3+/-6.9%, P=0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7+/-3.7%) than in SCLA (63.7+/-5.3%, P=0.0006) or SOF (49.0+/-4.6%, P=0.04). Moreover, re-expanding rates were lower (P=0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.

Increased cervical electrical activity during oestrus in progestagen treated ewes: Possible role in sperm transport.

The aim of this investigation was to characterize the pattern of cervical myoelectrical activity (EMG) in the sheep, during the periovulatory period, after synchronization of oestrus with progestagen and eCG. EMG was measured with a computerised modular system in five ewes previously fitted with a pair of monopolar Teflon needle electrodes in the muscle layers of the cervix. Each ewe was submitted to oestrus synchronization treatment with intravaginal progestagen sponge during 12 days, and the administration of 500 IU of eCG at the withdrawal of sponge. EMG was recorded in each animal during 19 h, starting 44 h after withdrawal of sponge. The number and duration of events were determined every hour during the experiment. Two distinct event durations were identified: one lasting less than 200 s and another lasting between 300 and 500 s. The two types of events analysed (less than 200 s and lasting between 300 and 500 s) had a similar pattern during the period of observation although they were not in synchrony. For events lasting less than 200 s, activity increased between 48 and 50 h after sponge withdrawal, with the peak of activity being observed between 51 and 53 h. For events of 300-500 s duration, the peak of activity was observed between 48 and 50h after sponge withdrawal and activity was maintained until 51-53 h. The increase in cervical motility observed in progestagen-eCG treated ewes is in keeping with the increase in cervical activity observed by others in natural cycling animals, and suggests that exogenous hormones used in synchronization protocols had no deleterious action on cervical motility during periovulatory period. The enhanced activity of cervical muscle layer found around the time of mating and/or AI suggests it may play an important role as a regulatory mechanism of sperm transport. Taking advantage of the cervical responsiveness to various drugs, experimental modulation of cervical activity could be used to facilitate cervical sperm transport and consequent improve of fertility after cervical AI.

Ovarian function after radioiodine therapy in patients with thyroid cancer.

OBJECTIVE: To assess ovarian function in young women treated with radioiodine. METHODS: The study was conducted on 50 women with thyroid carcinoma aged less than 40 years (mean, 29.8 years), with regular menstrual cycles and normal FSH levels prior to radioiodine therapy. FSH determination was repeated 6 and 12 months after radioiodine therapy (mean, 4.24 GBq iodine-131) and menstrual cycles were monitored during this period. RESULTS: Twenty percent of the patients reported amenorrhea during the first year. FSH levels increased after 6 months (from 5.1 +/- 1.8 to 10.6 +/- 2.2 IU/l, p < 0.00 001) and 28 % of the patients showed elevated values, which had returned to normal by the end of the first year. CONCLUSION: Radioiodine causes transitory alterations in ovarian function even in younger women and after a mean activity of 4.2 GBq (115 mCi).

Cryopreservation of in vitro-produced sheep embryos: Effects of different protocols of lipid reduction.

UNLABELLED: The low survival of sheep in vitro-produced (IVP) embryos after cryopreservation is a key limiting step to the widespread of this technology. In the present work, different approaches for enhancing cryosurvival of these embryos were compared: embryo delipidation by centrifugation in the absence or presence of cytochalasin D, a cytoskeleton stabilizer or by embryo culture in the presence of different doses of the trans-10 cis-12-conjugated linoleic acid isomer (CLA). Three experiments were conducted. In experiment 1, IVP blastocysts before vitrification were randomly distributed into four groups: control; centrifuged (cent), cytochalasin D (cyto-D), centrifuged + cytochalasin D (cent + cyto-D). In experiment 2, different doses of CLA (25, 50, and 100 µM) were supplemented during embryo culture before vitrification of blastocysts. A control group ran simultaneously. A third experiment was performed to compare both approaches from the previous ones but without the groups with the worst results (groups: control, cyto-D, cent + cyto-D, CLA25, CLA50). In all experiments, embryos integrity and reexpansion were assessed after warming and after 3 hours of culture. In experiment 1, the postwarming integrity rate was the lowest (P < 0.05) in embryos from the cent group (cent: 50.6 ± 10.3% vs. CONTROL: 74.6 ± 9.2%, cyto-D: 92.3 ± 9.7%, and cent + cyto-D: 90.5 ± 11.2%), whereas the best (P < 0.05) reexpansion scores were obtained in cent + cyto-D embryos (cent + cyto-D: 2.6 ± 0.28 vs. CONTROL: 1.8 ± 0.08, cent: 1.9 ± 0.2, and cyto-D: 1.8 ± 0.31). In experiments 2 and 3, higher (P < 0.05) cleavage rates were observed in CLA25 (50.9 ± 6.2% and 49.2 ± 5.6%, respectively) and CLA50 (48.9 ± 6.2% and 47.6 ± 5.6%, respectively) than those in the control (41.8 ± 6.1% and 40.4 ± 5.4%, respectively) group. In experiment 2, CLA100 presented the lowest (P < 0.002) Day-6 and -7 embryo production rate and quality. After warming, superior (P < 0.02) expansion scores were achieved in CLA25 (3.1 ± 0.29) and CLA50 (3.8 ± 0.17) than in the control (1.9 ± 0.10) group. Similar results were attained in experiment 3. However, although cent + cyto-D embryos showed higher (P = 0.008) postwarming expansion scores than the control (2.8 ± 0.29 vs. 1.9 ± 0.07) group, this score was lower (P = 0.0009) than that in CLA50 embryos (3.8 ± 0.17). In conclusion, our results showed that different protocols of lipid reduction can be successfully applied to improve the cryotolerance of IVP sheep embryos.

Influence of an antiprogestin (onapristone) on in vivo and in vitro fertilization.

The effects of a progesterone antagonist (onapristone) on heat synchronization, luteinizing hormone (LH) surge, ovulation, oocyte maturation and fertilization of superovulated ewes were studied. Its effects on in vitro bovine oocyte maturation and fertilization were also studied. Estrus synchronization and superovulation treatments were applied to 39 adult ewes using an intravaginal sponge with fluorgestone acetate for 9 days with injections of prostaglandin F2 alpha and pregnant mare's serum gonadotrophin given 24 h before sponge withdrawal. The animals were randomly assigned to four different groups; T1 receiving only the synchrony treatment (n = 11); T2 ewes received two injections of onapristone (1 mg kg-1, i.v.) 12 h apart from 3 h after sponge withdrawal (n = 10); T3 ewes received two injections of progesterone 12 h apart from sponge withdrawal (n = 10); and, T4 ewes received both onapristone and progesterone as described (n = 8). Ewes were mated by a fertile male during estrus. Progesterone and LH were measured during the superovulation period in plasma samples taken every 4 h. Uterine flushings for ova recovery were performed at 5 days (n = 25), 48 h (n = 5) and 24 h (n = 5). Non-fertilized oocytes collected at 24 and 48 h were checked for meiosis resumption. The effects of two doses of onapristone (D1 and D2) on in vitro bovine oocyte maturation (control = 100, D1 = 100 and D2 = 100) and fertilization (control = 107, D1 = 40 and D2 = 75) were also studied. The percentage of animals showing heat signs was significantly lower in group T3 (50% vs. 100%). The onset of oestrus (27.6, 24.8, 68.8 and 25.5 h, respectively for T1, T2, T3 and T4) and an LH surge (32.3, 28.8, 76.5 and 30.5 h, respectively for T1, T2, T3 and T4) after sponge withdrawal were significantly delayed in group T3. There were no significant differences in the intervals between estrus and LH surge among groups (4.61 +/- 0.75 h). The response and ovulation rates until 40 h after sponge withdrawal (group T3 excluded) were similar among groups, but the fertilization rates were significantly lower in groups T2 and T4 when compared with T1 (2% and 3% vs. 41%, respectively; P < 0.001) due to sperm arrest in the cervix. Ova recovery rate decreased significantly from 24-48 h to 5 days and was not affected by treatments (76.9% vs. 37.1% respectively). Onapristone did not affect the resumption of meiosis. Fertilization of bovine oocytes in vitro decreased significantly only in group D2 when compared to control (48% vs. 62.6%, respectively). In conclusion, onapristone treatment during the preovulatory period did not interfere with normal synchronization of estrus, ovulation and oocyte maturation but severely compromised fertilization by arresting spermatozoa in the cervix.

Possibility of reducing the luteolytic dose of cloprostenol in cyclic dairy cows.

The administration of cloprostenol by intravulvosubmucous (i.v.s.m.) injection at 1 2 and 1 4 of the dose usually given by intramuscular (i.m.) injection, was tested in dairy cows for luteolysis and estrus synchronization. The i.m. injection was used in ten adult cows at the usual dose of 500 mug/animal. Eleven adult cows and 11 heifers were treated i.v.s.m. with a dose equivalent to 250 mug/animal and 125 mug/animal, respectively. Two injections of cloprostenol were administered 11 days apart to the cows not detected in oestrus after a single injection. Forty-three out of the total 46 animals were detected to be in dioestrus at the time of at least one of the injections, as reflected by the plasma progesterone concentrations at the time of treatments. Three out of the 43 animals injected during dioestrus were refractory to the luteolytic effect of cloprostenol; this appeared to be independent of the dosage and the route of administration (refractory cows were: one adult cow treated i.m. and two treated i.v.s.m. with 125 mug of cloprostenol). The mean time interval from injection to the onset of heat was 82.8 hours with a confidence limit for 95% of probability between 67.9 hours and 92.7 hours. The difference between treatments is not significant. The results suggest that in heifers and adult cows cloprostenol can be given i.v.s.m. route at a reduced dose of 1 4 of the usual 500 mug i.m. dosage without affecting the luteolytic effect of the drug or fertility.

[A case of epilepsy simulating a pheochromocytoma]. / Un caso de epilepsia que simula un feocromocitoma.

INTRODUCTION: Autonomic epilepsy is a rare entity that results from an epileptogenic focus involving certain structures belonging to the autonomous nervous system; it is characterised by the occurrence of convulsive seizures that consist in autonomic phenomena. CASE REPORT: We report the case of a 28-year-old female patient who suffered paroxysmal episodes similar to those accompanying a pheochromocytoma. Following a thorough examination, both the presence of a tumour and the anomalous production of catecholamines were excluded and, although it was not possible to provide electroencephalographic evidence, the patient was diagnosed as suffering from autonomous epilepsy, which was later confirmed by the clinical efficacy of valproic acid therapy. CONCLUSIONS: Autonomic seizures often show characteristics that are singular or similar to those of other clinical entities. This makes the disorder difficult to diagnose and sometimes requires therapeutic testing with anticonvulsive drugs.