A Brazilian regional basic diet-induced chronic malnutrition drives liver inflammation with higher ApoA-I activity in C57BL6J mice.

Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1ß immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.

A Brazilian regional basic diet-induced chronic malnutrition drives liver inflammation with higher ApoA-I activity in C57BL6J mice

Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1β immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats.

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.

Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

Evidence of left atrial remodeling and left ventricular diastolic dysfunction in type 2 diabetes mellitus with preserved systolic function.

BACKGROUND AND AIMS: Prognosis of type 2 diabetes is associated with the occurrence of cardiovascular diseases. Left atrial (LA) size is a predictor of outcome in several diseases, including diabetes. Long duration of diabetes is an established risk factor of poor prognosis. No data are available on the relationship between LA size and duration of diabetes. The present study was aimed to investigate the relationship between LA volume index (LAVI) and the duration of diabetes to test the hypothesis that LA volume will increase as a function of diabetes duration. METHODS AND RESULTS: Forty-four male patients with newly diagnosed and 172 male patients with established type 2 diabetes were recruited for this cross-sectional study. All patients were evaluated with a transthoracic echocardiographic Doppler. About 28.2% of patients had increased LAVI. Indices of both diastolic and systolic function were significantly lower in patients with larger left atrium. The values of LAVI increased across classes of duration of diabetes. In multivariable analysis, longer duration was a predictor of LAVI ≥34 ml/m2 (odds ratio 1.65, 95% CI 1.11-2.46, p = 0.014) after adjusting for age, hemoglobin A1c, hypertension, microvascular complication status, and relevant echocardiographic parameters of systolic and diastolic function. CONCLUSIONS: These results indicate that duration of diabetes is strongly and positively associated with larger LAVI in type 2 diabetic men with preserved systolic function. Future studies are needed to better elucidate the biological mechanisms underlying linking type 2 diabetes with abnormally increased LAVI in subjects with type 2 diabetes.

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos.

Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

The transgenic animal platform for biopharmaceutical production.

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice

Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE-/-) and wild-type (APOE+/+) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE-/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE-/--challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge.

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice.

Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE-/-) and wild-type (APOE+/+) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE-/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE-/- -challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge.

Comparative expression profiles of genes related to oocyte development in goats after long-term feeding with biodiesel castor industry residues.

The aim of this study was to determine whether the consumption of detoxified castor meal (DCM) by goats over a long period of time affects mRNA levels in oocytes, and in mural granulosa and cumulus cells. A total of 41 adult does were supplemented (DCM group, n=21) or not (control group, n=20) with detoxified castor meal (DCM) for a period of 500 days. Then, 13 and 12 does were randomly selected for slaughter from the DCM and control treatments groups, respectively, for the determination of the number of visible ovarian follicles, retrieved cumulus-oocyte complexes (COCs), and viable and non-viable oocytes. The relative expression levels for distinct genes were determined by quantitative PCR in viable immature oocytes prior to in vitro maturation (IVM), in oocytes attaining or not the metaphase stage after IVM, as well as in granulosa cells obtained upon oocyte collection, and in cumulus cells obtained after IVM. The number of follicles ≥4 mm did not differ between treatments (overall mean 23.3 ± 2.0) and no significant differences were observed in the recovery of viable, non-viable, or total mean numbers of oocytes (control group: 44.7 ± 4.6, DCM group: 54.9 ± 5.9, respectively) between control and DCM fed goats. The maturation rate was significantly higher for control than DCM oocytes (58.0% vs. 45.3%; P<0.05). The mRNA levels in immature COC for controls were significantly higher for GLUT1 and lower for HSP70 (P<0.05) than for DCM. Following maturation, MII oocytes from both treatments had mRNA levels that were significantly higher for GDF9 and lower for BMP15 than for NC oocytes (P<0.05). In cumulus cells, the mRNA levels were significantly higher for LHR, FSHR, LeptinR, and IGF1, and lower for MnSOD in the control group compared with the DCM group (P<0.05). In conclusion, the inclusion of DCM in goat feed for long periods of time changed gene expression in immature oocytes and in cumulus cells. This was reflected by a decrease in the in vitro oocyte maturation rate.

Systemic immunosuppression by methylprednisolone and pregnancy rates in goats undergoing the transfer of cloned embryos.

The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.

Evidence for epigenetic abnormalities of the androgen receptor gene in foreskin from children with hypospadias.

CONTEXT: Hypospadias is a malformation of the penis due to an incomplete development of the male urethra, the exact etiology of which in the majority of cases remains unknown. OBJECTIVE: The objective of the study was to assess whether defects of the androgen receptor (AR) gene (CAG repeats and methylation pattern) and DNA methyltransferases (DNMT) family are present in hypospadic patients. DESIGN: CAG repeats length, methylation status, and expression of the AR gene were analyzed. The DNMT family was studied at the protein level and the DNMT3A sequenced. SETTING: The study was performed at a pediatric endocrinology referral clinic. PATIENTS OR OTHER PARTICIPANTS: Twenty boys with isolated glandular hypospadias and 20 age-matched control children undergoing a surgical procedure for circumcision were studied. MAIN OUTCOME MEASURE(S): CAG repeats length and AR methylation pattern in PBLs and foreskin tissue, DNMT expression and sequencing in patients and controls, and in vitro studies in cultured fibroblasts were measured. RESULTS: AR gene methylation in foreskin tissues from patients with hypospadias was higher than in normal children. AR expression in foreskin tissue of hypospadic patients was lower than in controls, whereas the DNMT3A protein level was significantly higher in patients than controls. In cultured fibroblasts, both dihydrotestosterone and testosterone significantly reduced AR gene methylation and DNMT3A expression in a dose-dependent fashion and increased AR expression. CONCLUSION: The AR gene in target tissues from patients with hypospadias is more methylated than in control children, resulting in a decreased expression of the AR. The mechanism underlying the modulation of the AR gene expression seems to be mediated by DNMT3A. This epigenetic alteration of the AR gene might be involved in the pathogenesis of hypospadias.

Subclinical interstitial lung abnormalities in stable renal allograft recipients in the era of modern immunosuppression.

BACKGROUND: Interstitial lung abnormalities have been detected in up to 24% of kidney transplant patients receiving traditional immunosuppressive therapies (eg, cyclosporine, azathioprine); they usually occur early after transplantation and tend to resolve over time. Newer immunosuppressants such as mycophenolic acid and, particularly, mammalian target of rapamycin (mTOR) inhibitors (eg, sirolimus) may cause significant lung toxicity. However, the prevalence and severity of interstitial lung lesions in long-term, stable kidney transplant patients receiving either traditional or newer immunosuppressants is not known. METHODS: We conducted a prospective, cross-sectional study examining high-resolution lung computed tomography (CT) scans in 63 stable kidney transplant recipients whose immunosuppressive therapy had remained unchanged for over 24 months. We compared CT findings of patients taking newer (mycophenolic acid and mTOR inhibitors) and traditional (calcineurin inhibitors and azathioprine) immunosuppressive drugs. RESULTS: Interstitial lung alterations were observed in only 3/63 patients (4.8%); the prevalence was 11.5% (3/26) versus 0% (0/37) among the newer versus traditional immunosuppressive therapy groups, respectively (P = .065). The CT patterns were usual interstitial pneumonia and nonspecific interstitial pneumonia-like. The median time between transplant and CT was 49 months in the three patients with CT alterations and 95 months in the remaining 23 patients on newer immunosuppressants. It was 75 months for all patients on newer immunosuppressive drugs and 133 months for those on traditional therapies (P = .0015). A follow-up CT, performed in 2/3 patients with interstitial abnormalities, showed that the lesions were stable in one, while they had disappeared in the other. CONCLUSIONS: Interstitial lung abnormalities are infrequent and mild in stable kidney transplant patients treated with newer as well as traditional immunosuppressive drugs. As such abnormalities were detected in patients screened earlier after transplantation, the time since transplantation rather than the drug type is probably the major determinant.

Anticoccidial effects of coumestans from Eclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production.

The aim of this study was to evaluate the anticoccidial efficacy of a product containing coumestans from Eclipta alba. Experimental conditions were set up as to reproduce the environment conditions for husbandry adopted in commercial broiler farms. Broilers were raised in broiler chicken shed provided with feeders, drinkers, illumination and temperature control systems and floor covering to afford an adequate nourishing environment. Male Cobb broilers (240) were assigned to four experimental groups being each experimental group set apart in rice straw-covered shed isolated with wire mesh. One-day-old broilers were reared in a coccidian-free environment with ad libitum supply of filtered water and freely available standard feed, from the 1st to the 35th day of life. The T1 group received standard feed (negative control); T2 was treated with standard feed supplemented with 66 ppm of salinomycin (positive control); groups T3 and T4 had standard feed supplemented with the ethyl acetate fraction from methanolic extract of E. alba aerial parts, which contains the coumestans WL and DWL (120 and 180 ppm, respectively). The chicken broilers were individually infected with 2 × 104 oocysts of Eimeria tenella when they were 14 days old and were monitored weekly to evaluate zootechnical parameters such as weight gain and food conversion ratio. Counting of coccidial oocyst in chicken feces was assessed from random samples, from the 21st to 28th days of life, which corresponded to 7-14 days after the infection. Five chickens selected at random from each experimental group were subsequently euthanized at 21, 28 or 35 days of life to determine the lesion score in the cecal region and to excise a cecum portion for histopathological evaluation. The group treated with coumestans from E. alba presented an average weight gain and food conversion ratio higher than the negative control group and similar to the mean value of the positive control group. Coumestan-treated groups showed a significant decrease in the oocyst counting since the 21 th day of life and displayed a reduced number of macroscopic lesions. Histopathological evaluations of cecum fragments showed that both treatments induced the migration of defense cells at the site of infection. A severe destruction of the cecal lining was found in the intestinal tract of broilers fed with a coumestans dose of 180 ppm. Overall, our results validate the use of a phytotherapy containing E. alba coumestans at a dose of 120 ppm as a therapeutic or prophylactic agent against avian coccidiosis.

Real-time qRT-PCR analysis of EGF receptor in cumulus-oocyte complexes recovered by laparoscopy in hormonally treated goats.

Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus-oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.

Non-alcoholic fatty liver disease is independently associated with an increased prevalence of chronic kidney disease and retinopathy in type 1 diabetic patients.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) is associated with an increased prevalence of chronic kidney disease (CKD) and retinopathy in patients with type 2 diabetes. Information on this issue is lacking for type 1 diabetes. We evaluated whether NAFLD is associated with increased prevalence of retinopathy and CKD in type 1 diabetic patients. METHODS: All type 1 diabetic patients (n = 202) who regularly attended our diabetes clinic and did not have any clinical evidence of cirrhosis or other secondary causes of chronic liver disease were studied. Main study measures were detection of NAFLD (by patient history and liver ultrasound), diabetic retinopathy (diagnosed by ophthalmoscopy) and CKD (defined as abnormal albuminuria or estimated GFR of < or =60 ml min(-1) 1.73 m(-2)). RESULTS: The age- and sex-adjusted prevalence of diabetic retinopathy (53.2 vs 19.8%) and CKD (37.8 vs 9.9%) was markedly higher in patients with NAFLD than in those without (p < 0.0001). In multivariate logistic regression analysis, NAFLD was associated with prevalent retinopathy (adjusted OR 3.31, 95% CI 1.4-7.6, p = 0.005) or CKD (adjusted OR 3.90, 95% CI 1.5-10.1, p = 0.005). These associations were independent of age, sex, diabetes duration, HbA(1c), medication use and presence of the metabolic syndrome. CONCLUSIONS/INTERPRETATION: Our findings suggest that ultrasound-diagnosed NAFLD is associated, independently of several confounding factors, with a higher prevalence of CKD and retinopathy in type 1 diabetic individuals.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Advances in reproductive technologies in cattle: from artificial insemination to cloning: [review] / Avances en biotecnología reproductiva en bovinos: de la inseminación artificial a la clonación: [revisión]

The urge for the control of reproductive processes in animals has propelled a great gain in knowledge, also setting off the development of four generations of assisted reproductive technologies (AR T) for humans and animals. The use of assisted reproductive techniques has been of great importance in livestock production. In general terms, the main first three generations of ARTs, including 1) artificial insemination (AI) and gamete and embryo freezing, 2) multiple ovulation and embryo transfer (MOET) and 3) in vitro fertilization(IV F) procedures, have matured into successful commercial applications, facilitating the increase in production through genetics, the reduction in generation intervals, the control ofdiseases, and the cutback in production costs. The fourth generation of AR T encompasses processes that are still more experimental, comprising cloning by nuclear transfer (NT) of embryonic or somatic cells, transgenesis, and stem cell biology. Such technologies are intertwined with one another and with currently available molecular tools, being completely dependent upon the previous generations of technologies. However, many reproductive challenges still hinder maximal livestock reproductive performance, affecting productivity and profitability. It is clear that the application of such technologies as lucrative activities will remain questionable if not associated with other components of animal production,such as animal health, nutrition and adequate animal husbandry practices.

El afán por controlar los procesos reproductivos en animales ha llevado a una gran ganancia en conocimiento, impulsando el desarrollo de cuatro tecnologías reproductivas asistidas(AR Ts) para animales y humanos. El uso de AR Ts ha sido de gran importancia en la producciónganadera. En términos generales, las tres principales generaciones de AR T, incluyendo 1) inseminación artificial (AI) y congelación de gametos y embriones, 2) superovulación y transferencia de embriones (MOET) y 3) procedimientos de fertilización in Vitro, han madurado en aplicaciones comerciales exitosas, facilitando el incremento en la producción a través de la genética, reducción del intervalo generacional, control de enfermedades, y reducción de costos de producción. La cuarta generación de AR Ts incluye procesos que aún son muy experimentales, como transferencia de núcleos (NT) de células somáticas, transanigénesis, y biología de células madre. Estas tecnologías se intercalan las unas con las otras y con las herramientas moleculares actuales, dependen completamente de las generaciones de tecnologías previas. Sin embargo, hay muchos retos reproductivos que no permiten alcanzarel potencial reproductivo máximo, afectando la productividad y la rentabilidad. Es claro que la aplicación de tales tecnologías como actividades lucrativas se mantendrán cuestionadassi no se asocian a otros componentes de la producción pecuaria, como la salud animal, nutrición, y prácticas de manejo adecuadas.

Diabetic retinopathy is associated with an increased incidence of cardiovascular events in Type 2 diabetic patients.

AIMS: We investigated the association of diabetic retinopathy with the risk of incident cardiovascular disease (CVD) events in a large cohort of Type 2 diabetic adults. METHODS: Our study cohort comprised 2103 Type 2 diabetic outpatients who were free of diagnosed CVD at baseline. Retinal findings were classified based on fundoscopy (by a single ophthalmologist) to categories of no retinopathy, non-proliferative retinopathy and proliferative/laser-treated retinopathy. Outcomes measures were incident CVD events (i.e. non-fatal myocardial infarction, non-fatal ischaemic stroke, coronary revascularization procedures or cardiovascular death). RESULTS: During approximately 7 years of follow-up, 406 participants subsequently developed incident CVD events, whereas 1697 participants remained free of diagnosed CVD. After adjustment for age, body mass index, waist circumference, smoking, lipids, glycated haemoglobin, diabetes duration and medications use, patients with non-proliferative or proliferative/laser-treated retinopathy had a greater risk (P < 0.001 for all) of incident CVD events than those without retinopathy [hazard ratio 1.61 (95% confidence interval 1.2-2.6) and 3.75 (2.0-7.4) for men, and 1.67 (1.3-2.8) and 3.81 (2.2-7.3) for women, respectively]. After additional adjustment for hypertension and advanced nephropathy (defined as overt proteinuria and/or estimated glomerular filtration rate < or = 60 ml/min/1.73 m(2)), the risk of incident CVD remained markedly increased in those with proliferative/laser-treated retinopathy [hazard ratio 2.08 (1.02-3.7) for men and 2.41 (1.05-3.9) for women], but not in those with non-proliferative retinopathy. CONCLUSIONS: Diabetic retinopathy (especially in its more advanced stages) is associated with an increased CVD incidence independent of other known cardiovascular risk factors.

Non-alcoholic fatty liver disease is independently associated with an increased prevalence of chronic kidney disease and proliferative/laser-treated retinopathy in type 2 diabetic patients.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease in type 2 diabetes. Currently, there is a lack of information on associations between NAFLD and microvascular complications of diabetes. We assessed the associations between NAFLD and both chronic kidney disease (CKD) and retinopathy in a large cohort of type 2 diabetic individuals using a cross-sectional design. METHODS: Prevalence rates of retinopathy (by ophthalmoscopy) and CKD (defined as overt proteinuria and/or estimated GFR