Genomewide analysis of bull sperm quality and fertility traits.

Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein-Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high-density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD-adjusted Bonferroni <0.05) with the non-compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non-compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.

High-resolution melt analysis does not reveal mutagenic risk in sexed sperm and in vitro-derived bovine embryos.

The objectives of the present work were to verify whether simultaneous exposure to Hoechst 33342 and UV irradiation during sorting by flow cytometry may induce gene point mutations in bovine sperm and to assess whether the dye incorporated in the sperm may imply a mutagenic effect during the embryonic development. To this aim, high-resolution melt analysis (HRMA) was used to discriminate variations of single nucleotides in sexed vs. non-sexed control samples. Three batches of sorted and non-sorted commercial semen of seven bulls (42 samples) were subjected to HRMA. A set of 139 genes located on all the chromosomes was selected, and 407 regions of the genome covering a total of 83 907 bases were analyzed. Thereafter, sperm of one sexed and one non-sexed batch of each bull was used in in vitro fertilization, and the derived embryos were analyzed (n = 560). One hundred and thirty-three regions of the bovine genome, located in 40 genes, were screened for a total coverage of 23 397 bases. The comparison between the frequencies of variations, with respect to the sequences deposited, observed in the sexed and non-sexed sperm (843 vs. 770) and embryos (246 vs. 212) showed no significant differences (P > 0.05), as measured by chi-square tests. It can be concluded that staining with Hoechst 33342 and exposure to UV during sorting does not lead to significant changes in the frequencies of variants in the commercial sexed semen and in embryos produced in vitro with the same treated sperm.

Identification of bovine sperm head for morphometry analysis in quantitative phase-contrast holographic microscopy.

An investigation is reported of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps of biological cells by digital holographic microscopy. In particular, two different methods have been developed for in vitro bull sperm head morphometry analysis. We show that semen analysis can be accomplished by means of the proposed techniques . Extraction and measurement of various parameters are performed. It is demonstrated that both proposed methods are efficient to skim the data set in a preselective analysis for discarding anomalous data.

Digital self-referencing quantitative phase microscopy by wavefront folding in holographic image reconstruction.

A completely numerical method, named digital self-referencing holography, is described to easily accomplish a quantitative phase microscopy for microfluidic devices by a digital holographic microscope. The approach works through an appropriate numerical manipulation of the retrieved complex wavefront. The self-referencing is obtained by folding the retrieved wavefront in the image plane. The folding operation allows us to obtain the correct phase map by subtracting from the complex region of interest a flat area outside the microfluidic channel. To demonstrate the effectiveness of the method, quantitative phase maps of bovine spermatozoa and in vitro cells are retrieved.

In vitro competitive binding index using fluorochrome-labelled spermatozoa for predicting bull fertility.

This work evaluated if an in vitro test, with the combined power of the statistical evaluation of spermatozoa and zona pellucida (ZP) competitive binding ability and a rapid method for accessory sperm counts, could predict the bull fertility. Ten Holstein Friesian bulls of known field fertility (five of high and five of low fertility) were selected. An in vitro heterospermic insemination approach, based on differential staining, was tested on 45 possible pairs of bulls (two batches per bull). Motility and quality (abnormalities and membrane status) seminal characteristics and estimated relative conception rates (ERCR) highlighted only one association between membrane integrity and ERCR (p = 0.007). Differences in ZP binding allowed us to rank bulls into two categories based on low and high binding ability. For eight bulls, this classification reflected the ERCR. Differences between batches were reported for two bulls, in which the effect of heterospermic insemination (the number of sperm binding to ZP from different bulls not in a 1:1 ratio) showed a significant bull-related effect (p < 0.001) in the first batch and no effect (p > 0.05) in the second batch for both bulls. Reduction of the number of oocytes per assay from 25 to 5 had no effect (p > 0.5) on the bulls' ranking. Our results suggest that in vitro competitive binding is a promising approach for estimating bull fertility and support concepts for further implementation, e.g. drastic reduction of oocyte number in a single pair assay and larger scale testing for batches.

[Retroperitoneal abscess due to asymptomatic duodenal perforation by foreign body. A case report]. / Ascesso retroperitoneale da corpo estraneo dopo perforazione duodenale asintomatica. Case report.

We present a case of retroperitoneal abscess due to asymptomatic duodenal perforation by foreign body. Patient has been admitted for lumbar pain and subocclusive crisis, with a medical history negative for acute symptomatology. After both clinical and radiologic evaluation, an abscess-like mass was detected in the context of right psoas muscle. Patient underwent surgical operation and a lumbar abscess has been found containing a foreign body (toothpick). There has been a transduodenal migration of the foreign body, without clinical signs of duodenal perforation.

An approach to address Candida rugosa lipase regioselectivity in the acylation reactions of trytilated glucosides.

Candida rugosa lipase crude preparations (CRL) catalyse the regioselective acylation of methyl 6-O-trytil beta-d-glucopyranoside in organic solvents, using vinyl acetate as acyl donor. The ratio of the two products formed, namely methyl 2-O acetyl 6-O-trytil beta-d-glucopyranoside and methyl 3-O acetyl 6-O-trytil beta-d-glucopyranoside was found to be markedly affected by the nature of the reaction medium. In hydrophobic solvents values up to 80% of the monoacetylated product in position C-3 were obtained compared to less than 30% in solvents with low hydrophobicity. Computational studies were carried out to simulate the interactions between methyl 6-O-trytil beta-d-glucopyranoside and both CRL and the solvents, in order to rationalize the experimental results.

[Exercise deep venous thrombosis: myth or reality? About three cases of pulmonary embolism in long-distance runners]. / Maladie thromboembolique veineuse d'effort: mythe ou réalité? A propos de trois cas d'embolies pulmonaires chez des coureurs de fond.

The most frequent manifestation of exercise deep vein thrombosis (DVT) is the one occurring in upper limbs, in some specific and stereotypic conditions, sometimes complicated by pulmonary embolisms. A few cases of lower limbs DVT are reported in athletes. Some pathophysiologic arguments (rheological modifications, parietal lesions and coagulation abnormalities) suggest a link between DVT and exertion, but the causality is sometimes difficult to establish. We report three cases of pulmonary embolism occurring after a prolonged effort of running in trained marathon athletes. To our knowledge, very few similar cases have ever been reported. The possible responsibility of such physical efforts is discussed, as well as other potential cofactors such as coagulation abnormalities and hormonal contraception.

AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression.

Accumulating evidences have shown the association between aberrantly expressed microRNAs (miRs) and cancer, where these small regulatory RNAs appear to dictate the cell fate by regulating all the main biological processes. We demonstrated the responsibility of the circuitry connecting the oncomiR-221&222 with the tumor suppressors miR-126&126* in melanoma development and progression. According to the inverse correlation between endogenous miR-221&222 and miR-126&126*, respectively increasing or decreasing with malignancy, their enforced expression or silencing was sufficient for a reciprocal regulation. In line with the opposite roles of these miRs, protein analyses confirmed the reverse expression pattern of miR-126&126*-targeted genes that were induced by miR-221&222. Looking for a central player in this complex network, we revealed the dual regulation of AP2α, on one side directly targeted by miR-221&222 and on the other a transcriptional activator of miR-126&126*. We showed the chance of restoring miR-126&126* expression in metastatic melanoma to reduce the amount of mature intracellular heparin-binding EGF like growth factor, thus preventing promyelocytic leukemia zinc finger delocalization and maintaining its repression on miR-221&222 promoter. Thus, the low-residual quantity of these two miRs assures the release of AP2α expression, which in turn binds to and induces miR-126&126* transcription. All together these results point to an unbalanced ratio functional to melanoma malignancy between these two couples of miRs. During progression this balance gradually moves from miR-126&126* toward miR-221&222. This circuitry, besides confirming the central role of AP2α in orchestrating melanoma development and/or progression, further displays the significance of these miRs in cancer and the option of utilizing them for novel therapeutics.

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.

The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.

Comparison between primary sex ratio in spermatozoa of bulls and secondary sex ratio in the deriving offspring.

The objectives of the present work were to compare the primary sex ratio in sperm with the secondary sex ratio recorded in the offspring produced by artificial insemination (AI) with the same sperm and assess whether the primary sex ratio is influenced by sperm survival and motility after thawing. Calving data of 98 Holstein Friesian bulls used in AI were collected during 4 years, and commercial semen of the same bulls was analyzed immediately after thawing and after swim-up using a real-time polymerase chain reaction method developed and validated in our laboratory. Calving data relative to single bulls did not reveal any significant deviation between genders from the theoretical 1:1 for none of the bulls, being the mean values of male and female calves born 52.1 ± 2.80% and 47.9 ± 2.71%, respectively. Thereafter, calving events of bulls were classified and analyzed according to four classes of years: 2009 (n = 13,261), 2010 (n = 21,551), 2011 (n = 24,218), and 2012 (n = 41,726), and seasons categorized as winter, spring, summer, and fall. When data aggregated per years were analyzed, the difference between the two sexes was significant (P < 0.005) in favor of the male gender, whereas no influence of the season was evidenced. Real-time polymerase chain reaction did not evidence any difference between the mean values of frequency of Y chromosome-bearing sperm detected in three sperm batches of the same bulls analyzed immediately after thawing (51.1 ± 2.1), nor a difference with respect to the theoretical 1:1 ratio was reported after sperm analysis of one batch of sperm of the bulls analyzed after swim-up and immediately after thawing (50.1 ± 2.1 and 49.8 ± 1.8, respectively). The results are consistent with the observation of the farmers who often report a skewed sex ratio of the calves being born with AI in favor of the male gender. However, we have not evidenced differences in the primary sex ratio with respect to the theoretical 1:1 ratio both at thawing and after swim-up, thus demonstrating that the freezing procedure itself does not impact selectively on the survival of the X or Y chromosome-bearing sperm. Therefore, we hypothesize that the difference between genders observed after AI is more likely due to the events occurring after fertilization, which can comprise an impaired function of the X- or Y-bearing sperm with consequences on embryo development or a maternal influence.

The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes.

Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189-196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino-terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid-late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.

Cardiovascular-reflex testing and single-fiber electromyography in botulism. A longitudinal study.

Four patients with botulism were studied on admission and at different times after intoxication, using a battery of cardiovascular autonomic tests. The results were compared with clinical status and single-fiber electromyographic findings. In the early stage of intoxication, the control of heart-rate and blood-pressure responsivity was markedly impaired, as was the neuromuscular transmission. At follow-up, results of sympathetic tests normalized earlier than those of parasympathetic tests. The recovery of autonomic function was slower than that of neuromuscular transmission in three patients. Monitoring autonomic derangement in botulism adds further information on the course of the disease and may identify patients at risk for cardiac or respiratory arrest. Further clinical investigation can help in determining more precisely the autonomic sites where the toxin acts.

The cardiovascular hemodynamics and leukotriene kinetics during prostacyclin and anti-prostacyclin antibody infusions in septic shock.

This study evaluated whether or not prostacyclin (PGI2) was necessary or sufficient by itself in a pathophysiologic concentration to mediate the cardiovascular dysfunction of septic shock. Anesthetized adult swine received anesthesia only (ANESTHESIA CONTROL, n = 6); graded Aeromonas hydrophila, 10(10)/mL, infusion at 0.2 mL/kg/h that increased to 4.0 mL/kg/h over 3 h (SEPTIC SHOCK CONTROL, n = 6); pathophysiologic prostacyclin infusion to match septic shock control plasma levels without bacteremia (PGI2 INFUSION, n = 6), or graded Aeromonas hydrophila plus anti-prostacyclin antibody infusion (ANTI-PGI2-Ab INFUSION, n = 5). This graded porcine bacteremia model was 100% lethal after 4 h. Cardiovascular hemodynamics, arterial blood gases, and plasma levels of arachidonate metabolites were measured at baseline and hourly over a 4-h period. The results showed that PGI2 was not a necessary mediator of impaired cardiovascular hemodynamics in graded bacteremia, as anti-PGI2 antibody infusion did not improve the cardiac index, systemic vascular resistance, or peripheral oxygen balance in septic animals. Also, PGI2 was not sufficient alone to cause the cardiovascular dysfunction of sepsis, as pathophysiologic infusion of PGI2 did not reproduce such changes in normal animals. PGI2 blockade during bacteremia significantly increased LTC4D4E4, and LTB4 whereas PGI2 infusion suppressed LTC4D4E4 concentration, suggesting that endogenous PGI2 may blunt leukotriene release during septic shock. These results indicate a complex dynamic equilibrium among prostacyclin and leukotrienes in septic shock.

Left axis deviation in healthy infants and children.

In a series of 1036 routine electrocardiograms, 14 children, without clinical and echocardiographic evidence of heart disease, showed a left axis deviation in the standard electrocardiogram. Six children also had an incomplete right bundle branch block. Left axis deviation is defined as a mean frontal QRS complex axis being -30 degrees or less. The age varied from three months to 10 years, with a clear predominance of males (13/14). The unusual finding of left QRS axis deviation in normal children is emphasized.

Uremic autonomic dysfunction evaluated by pattern recognition analysis.

A pattern recognition analysis of six cardiovascular reflex tests was performed with the aim to increase their diagnostic efficiency. A normal model was defined by SIMCA method using log-transformed values of 85 healthy subjects. The model was tested with data from 60 patients with chronic renal failure. Specificity resulted 94% and sensitivity 96%. 55% of the uremics were recognized affected by autonomic damage. The method allowed a better definition of autonomic function in those patients defined as "borderline" on conventional evaluation of the tests. Pattern recognition analysis of autonomic tests provides a global assessment of all the tests performed. Its use should be encouraged as a diagnostic tool.

Computer analyzed histology of ischemic injury to the gut.

Histopathologic alterations in the intestinal mucosa after ischemic injury have been extensively described in the literature, but these descriptions have primarily been qualitative in nature. The purpose of this study was twofold: (1) to establish parameters obtained by computerized digital image analysis that would be useful in identifying ischemic injury, and (2) to use these parameters to identify the critical period of intestinal ischemia producing measurable histopathologic change. Seventy male Sprague-Dawley rats weighing 80 to 150 g underwent various times of gut ischemic injury by vascular occlusion of the superior mesenteric artery and vein with a microaneurysm clamp. The clamp times were 0, 1, 20, 30, 40, 50, 60, 70, 80, and 90 minutes. Histological sections of the terminal ileum were quantitatively analyzed using Jandel Scientific's computerized morphometric image analysis system. Parameters studied were surface index (SI, surface length per linear unit of mucosa), average villous thickness (AVT), average villous height (AVH), and the number of villous cells/100 microns length (VC). Ischemic times of 1, 20, 30, and 40 minutes produced no measurable injury as compared with baseline (P[40 minutes versus baseline] = SI, .60; AVT, .84; AVH, .93; VC, .09). At 50 minutes, SI and AVH showed a measurable change from baseline (P[50 minutes versus baseline] = SI, .01; AVH, .02). Sixty minutes of ischemic time produced measurable change in all parameters (P[60 minutes versus baseline] = SI, .007; AVT, .001; AVH, .002; VC, .007).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cyclosporine in gut ischemic injury: a computerized morphometric and enzymatic analysis.

PURPOSE: Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A (CSA), a potent immunosuppressant, attenuates immune/inflammatory cellular reactions. CSA also might be useful for inhibiting cellular immune responses involved in tissue ischemia/reperfusion injury. The authors hypothesized that CSA would attenuate inflammatory cellular changes associated with gut ischemic injury and that these effects could be quantified by computerized morphometry. METHODS: Twenty Sprague-Dawley rats underwent 60 minutes of gut ischemia by vascular occlusion of the superior mesenteric vessels. After 1 hour of reperfusion, the ischemic small bowel was harvested for histopathological examination and computerized morphometry, as well as xanthine oxidase (XO, U/mg protein) and maltase (MALT, mmol/L substrate degraded/min/mg protein) assays. CSA (5 mg/kg/d subcutaneously) was given to experimental animals (CSA, n = 10) for 5 days before ischemia, and vehicle was given to controls (CON, n = 10). The computer morphometric parameters studied were: surface index (SI, mucosal surface length per linear unit of intestine), average villous thickness (AVT), and average villous height (AVH). RESULTS: Results are provided in Table 1. CONCLUSION: The results of this study show that CSA may play a role in attenuating ischemia/reperfusion injury in the gut. Enzymatic analysis showed a beneficial role in the preservation of mucosal cell function after gut ischemia/reperfusion injury, as demonstrated by an elevated maltose level. Computerized morphometry demonstrated significant differences in all parameters in the experimental group, showing that CSA does confer gut mucosal protection during ischemia.

[Endoscopic and pharmacologic treatment with omeprazole of upper digestive hemorrhage]. / Trattamento endoscopico e farmacologico con omeprazolo delle emorragie digestive alte.

Upper gastrointestinal bleeding is still a subject much discussed as much for the diagnostic approach and as by the therapeutic decisions read. The authors present their experience of the treatment of upper gastrointestinal bleeding not caused by portal hypertension or by neoplasm. The patients undergo emergency endoscopy by haemostatic treatment if necessary and pharmacological therapy by omeprazole. The evaluation criteria are: stopped bleeding, the need of blood transfusion, the healing of the bleeding site. Stopped bleeding has been watched at first endoscopic check in 85% of patients; only 26 blood units has been necessary; the complete healing of bleeding injury happened not later than 30 days.

Pattern recognition analysis of cardiovascular reflexes: its application in the diagnosis of autonomic neuropathy.

We describe pattern recognition analysis of six cardiovascular reflex tests and its application in the diagnosis of autonomic neuropathy. A discriminating model was performed by SIMCA method using the results of 85 healthy subjects. The prediction ability of the model was then verified with data from 95 patients at risk for autonomic neuropathy. Specificity resulted 93% and sensitivity 97%. The method increases the diagnostic efficiency of the tests, being particularly useful in patients with borderline autonomic involvement.