Genotype by environment interaction for Holstein cattle populations using autoregressive and within- and across-country multi-trait reaction norms test-day models.

The progenies of international bulls in diverse climatic conditions and management levels may lead to different expressions of their genetic potential resulting in a re-ranking of these bulls. Therefore, evaluate the presence of genotype by environment interaction (G×E) within and across countries is important to guide the decision-making on alternative selection strategies. Thus, a two-step reaction norm (RN) approach was used to investigate the presence of G×E in Portuguese and Brazilian Holstein cattle. In step 1, we performed a within-country genetic evaluation using an autoregressive model to obtain precorrected phenotypes and environmental gradients (herd test-day solutions, HTD levels). In step 2, the precorrected phenotypes were considered as two distinct traits in a bi-trait RN model to estimate variance components across HTD levels, genetic correlation between HTD levels in Portugal and Brazil, and RN of the estimated breeding values. Additionally, the genetic correlation between countries using a bi-trait random regression (RR) sire model was obtained. In step 1, genetic additive variance for milk yield (MY) in Portugal was 14.1% higher than in Brazil. For somatic cell score (SCS), the genetic additive variance in Portugal was 12.7% lower than in Brazil. Although similar heritability estimates for SCS were observed in both countries, MY heritabilities were 0.31 for Portugal and 0.23 for Brazil. Genetic correlations (SD) between both countries obtained using RR sire model were 0.78 (0.051) for MY and 0.75 (0.062) for SCS. In step 2, MY genetic correlations among HTD levels within countries were higher than 0.94 for Portugal and 0.98 for Brazil. Somatic cell score genetic correlations among HTD levels ranged from 0.70 to 0.99 for Portugal and from 0.84 to 0.99 for Brazil. The average (SD) of genetic correlation estimates between Portuguese and Brazilian HTD levels were 0.74 (0.009) for MY and 0.57 (0.060) for SCS. These results suggest the presence of G×E for MY and SCS of Holstein cattle between both countries. Although there was no indication of G×E between Brazilian herd environments, the low genetic correlation for SCS indicates potential re-ranking of bulls between extreme environmental gradient in Portugal. Overall, the results of this study evidence the importance of national and international genetic evaluation systems to assist dairy farmers in the selection of the best genotypes to obtain the expected returns from investments in imported semen and to realize genetic progress in dairy populations under local environmental conditions.

Autoregressive single-step test-day model for genomic evaluations of Portuguese Holstein cattle.

The multiple-lactation autoregressive test-day (AR) model is the adopted model for the national genetic evaluation of dairy cattle in Portugal. Under this model, animals' permanent environment effects are assumed to follow a first-order autoregressive process over the long (auto-correlations between parities) and short (auto-correlations between test-days within lactation) terms. Given the relevance of genomic prediction in dairy cattle, it is essential to include marker information in national genetic evaluations. In this context, we aimed to evaluate the feasibility of applying the single-step genomic (G)BLUP to analyze milk yield using the AR model in Portuguese Holstein cattle. In total, 11,434,294 test-day records from the first 3 lactations collected between 1994 and 2017 and 1,071 genotyped bulls were used in this study. Rank correlations and differences in reliability among bulls were used to compare the performance of the traditional (A-AR) and single-step (H-AR) models. These 2 modeling approaches were also applied to reduced data sets with records truncated after 2012 (deleting daughters of tested bulls) to evaluate the predictive ability of the H-AR. Validation scenarios were proposed, taking into account young and proven bulls. Average EBV reliabilities, empirical reliabilities, and genetic trends predicted from the complete and reduced data sets were used to validate the genomic evaluation. Average EBV reliabilities for H-AR (A-AR) using the complete data set were 0.52 (0.16) and 0.72 (0.62) for genotyped bulls with no daughters and bulls with 1 to 9 daughters, respectively. These results showed an increase in EBV reliabilities of 0.10 to 0.36 when genomic information was included, corresponding to a reduction of up to 43% in prediction error variance. Considering the 3 validation scenarios, the inclusion of genomic information improved the average EBV reliability in the reduced data set, which ranged, on average, from 0.16 to 0.26, indicating an increase in the predictive ability. Similarly, empirical reliability increased by up to 0.08 between validation tests. The H-AR outperformed A-AR in terms of genetic trends when unproven genotyped bulls were included. The results suggest that the single-step GBLUP AR model is feasible and may be applied to national Portuguese genetic evaluations for milk yield.

Effects of two freezing methods and two cryopreservation media on post-thaw quality of stallion spermatozoa.

Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio® ). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.

Retraction Note to: L-glutamine supplementation induces insulin resistance in adipose tissue and improves insulin signalling in liver and muscle of rats with diet-induced obesity.

In light of forensic evidence indicating duplication and/or manipulation of western blot images the Editor-in-Chief is retracting the article cited above.

Genomic differentiation between swamp and river buffalo using a cattle high-density single nucleotide polymorphisms panel.

Buffalo (Bubalus bubalis) is an important livestock species in many tropical and subtropical regions. In recent decades, the interest in buffalo's milk have expanded and intensive buffalo farms start to emerge. However, breeding programs and population genetics information for this species is scarce or inexistent. The present study aims to test the suitability of the commercial high-density single nucleotide polymorphisms (SNP) genotyping panel, the Illumina BovineHD BeadChip, to estimate population genetics parameters, pedigree control and identification of common variants in major production candidate genes. From a total of 777 962 SNPs included in the panel, 20 479 were polymorphic in water buffalo at a call rate of 86% and an average expected heterozygosity (HE) of 0.306. From these, 357 were mapped within or around the flanking regions of several major candidate genes. A principal components analysis identified three different clusters, each representing pure swamp buffalo type, pure river buffalo type and admixed river buffalo. The hybrids between swamp and river buffalo were clearly identified as an intermediary cluster. The suitability of these SNPs data set for parentage and identity testing demonstrated that the combination of just 30 to 50 SNPs were enough to attain high probabilities of parentage exclusion (0.9999) in both types and identity (2.3×10-5 and 2.0×10-7) for river and swamp buffalo, respectively. Our analysis confirms the suitability of the BovineHD BeadChip to assess population structure, hybridization and identity of the water buffalo populations.

Genetic Analysis of Feline Panleukopenia Virus Full-length VP2 Gene in Domestic Cats Between 2006-2008 and 2012-2014, Portugal.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are two closely related viruses, which cause acute gastroenteritis in carnivores, and cats may be infected by strains of both viruses. The viruses are found worldwide and may have changing host ranges and genetic variation that can be found around the world in some cases. Here, we screened a Portuguese population of cats by a conventional PCR assay for the presence of FPV/CPV viruses in faecal samples and tissues between 2006-2008 and 2012-2014. The sequence analysis of the complete VP2 gene showed that 18 of 31 animals tested were positive for FPV DNA, and no case of CPV infection was detected. The analysis of specific DNA detected three new non-synonymous substitutions in the VP2 gene that were found in single groups and were related to viruses reported elsewhere by phylogenetic analysis - some were related to Italian isolates, one was closely related to isolates from Vietnam and China, and two were related with older strains from the USA. The results of our study show that FPV circulated in the Portuguese cat population and as expected the found strains are slightly divergent from those reported previously.

Genetic parameters for test day somatic cell score in Brazilian Holstein cattle.

Selection for lower somatic cell count has been included in the breeding objectives of several countries in order to increase resistance to mastitis. Genetic parameters of somatic cell scores (SCS) were estimated from the first lactation test day records of Brazilian Holstein cows using random-regression models with Legendre polynomials (LP) of the order 3-5. Data consisted of 87,711 TD produced by 10,084 cows, sired by 619 bulls calved from 1993 to 2007. Heritability estimates varied from 0.06 to 0.14 and decreased from the beginning of the lactation up to 60 days in milk (DIM) and increased thereafter to the end of lactation. Genetic correlations between adjacent DIM were very high (>0.83) but decreased to negative values, obtained with LP of order four, between DIM in the extremes of lactation. Despite the favorable trend, genetic changes in SCS were not significant and did not differ among LP. There was little benefit of fitting an LP of an order >3 to model animal genetic and permanent environment effects for SCS. Estimates of variance components found in this study may be used for breeding value estimation for SCS and selection for mastitis resistance in Holstein cattle in Brazil.

Management practices associated with the bulk tank milk prevalence of Mycoplasma spp. in dairy herds in Northwestern Portugal.

The objective of this study was to evaluate the effect of some management practices on the prevalence of Mycoplasma spp. in Northwestern Portuguese dairy farms from bulk tank milk (BTM) samples. Additionally, the within-herd prevalence of Mycoplasma spp. was also determined, but only in BTM positive herds. From May 2007 to November 2008, 492 BTM samples from 164 dairies randomly chosen in a population of 1234 dairy farms were analyzed. Five herds (3.0%) had positive mycoplasmal culture results, from which 4 out of 164 (2.4%) were Mycoplasma bovis, with simultaneous presence of Mycoplasma bovigenitalium or Mycoplasma canadense in two of those samples. In one out of 164 (0.6%) herds Mycoplasma capricolum subsp. capricolum was also found. In BTM positive Mycoplasma spp. herds, the apparent intra-herd prevalence was low and varied between 2.5% and 4.5%. Multiple locus variable-number of tandem-repeat analysis was conducted in order to compare the genetic relationship between the isolates. Mycoplasma spp. was found to be present in cows with subclinical mastitis with or without California Mastitis Test positive results, hence all cows should be tested when the agent is isolated from bulk tank rather than selecting suspected cows. A multivariable logistic regression using the Firth's penalized maximum likelihood estimation was performed showing that increasing number of lactating cows (OR=1.05; P<0.01) was associated with a higher probability of isolating Mycoplasma spp. On the other hand, identifying problem cows was associated with a lower probability (OR=0.06; P<0.05). Particular importance was given to the prevalence of M. bovis, and the results obtained highlight the need to include this agent in mastitis control protocols in national dairies and in sanitary controls of transitioned animals between European countries.

Double-stranded RNA-activated protein kinase is a key modulator of insulin sensitivity in physiological conditions and in obesity in mice.

The molecular integration of nutrient- and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of κB kinase ß. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of κB kinase ß phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity.

Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice.

AIMS/HYPOTHESIS: A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration. METHODS: Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry. RESULTS: Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase ß (IKKß) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5'-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80(+) macrophage cells in the adipose tissue of antibiotic-treated mice. CONCLUSIONS/INTERPRETATION: These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.

Mucin 6 and Tn antigen expression in canine mammary tumours: correlation with pathological features.

Overexpression of mucins is known to decrease cell-to-cell adhesion and thus to facilitate the invasion of cancer cells through the extracellular matrix. Mucin 6 (MUC6) is overexpressed and aberrantly O-glycosylated in human breast cancer, serving as a carrier for one of the most specific cancer-associated antigens, Tn antigen. Despite its relevance in breast cancer, MUC6 expression has not yet been characterized in canine mammary tumours (CMTs). The aims of this study were to assess the expression of MUC6 and Tn antigen in 55 benign and 77 malignant CMTs of different histological types and to investigate possible correlations with pathological features. MUC6 and Tn antigen were found to be significantly overexpressed in malignant compared with benign CMTs. MUC6 was significantly overexpressed in simple and complex carcinomas compared with simple and complex adenomas, respectively. When considering only the epithelial population, significant MUC6 overexpression was observed in carcinosarcomas when compared with benign mixed tumours. In addition, MUC6 was significantly overexpressed in simple compared with complex carcinomas. Finally, double-labelling immunofluorescence performed on seven malignant CMTs showed MUC6 and Tn co-expression. Therefore, MUC6 and Tn antigen overexpression is associated with malignant phenotypes of CMTs.

Cytotoxic drugs for patients with breast cancer in the era of targeted treatment: back to the future?

BACKGROUND: Despite current trend of targeted therapy development, cytotoxic agents are a mainstay of treatment of patients with breast cancer. We reviewed recent advances in cytotoxic therapy for patients with metastatic breast cancer (MBC). MATERIALS AND METHODS: Medline searches were conducted for English language studies using the term 'MBC' and 'cytotoxic drugs'. The data search was restricted to the period 2000-2011. RESULTS: Several novel cytotoxic compounds, all microtubule inhibitors, have been approved for clinical use in MBC: (i) nab-paclitaxel, reported to improve tumour response and decrease hypersensitivity reactions in comparison with other taxanes; (ii) ixabepilone, shown to have clinical benefit in taxane- and anthracycline-resistant disease and (iii) eribulin, shown to improve overall survival in heavily pre-treated patients, when compared with best available standard treatment. Agents, such as larotaxel, vinflunine, trabectidin and formulations, including cationic liposomal paclitaxel or paclitaxel poliglumex, are currently under evaluation in phase II/III trials. CONCLUSION: Toxicity and chemotherapy resistance are still major limitations in the treatment of patients with MBC. Further research into new cytotoxic compounds is needed in order to maximise benefit, whilst minimising toxicity.

Multidisciplinary approach in the treatment of patients with small cell bladder carcinoma.

Small cell carcinoma of the urinary bladder (SCCUB) is considered to be a tumor with a neuroendocrine phenotype characterised by aggressive behaviour and poor prognosis. Small cell carcinoma of the urinary bladder comprises 0.35 to 1% of all bladder cancers and is frequently observed in combination with other histological subtypes of carcinoma. Clinical presentation is characterized by advanced stage at diagnosis and rapidly progressive disease. In daily clinical practice there is no gold standard for the management of patients affected by this disease. Treatment of patients with limited disease combines neoadjuvant platinum-based chemotherapy followed by specific local treatment of the primary tumour. Cystectomy or radiotherapy should be proposed on an individual basis. In the metastatic setting, prognosis remains poor with a potential benefit from chemotherapy containing platinum compounds. Treatment of small cell carcinoma of the urinary bladder is based on evidence obtained from case reports and retrospective analyses. Due to low disease frequency there is a lack of randomized trials to provide guidance as to optimal therapy. Thus, systemic and local approaches are extrapolated from the literature available for the treatment of small cell carcinomas at other (non-urological) sites. We provide an overview of the currently available literature with it's main focus on the treatment of either locally advanced or metastatic small cell carcinoma of the urinary bladder.

Short communication: Temperature sensibility of Prototheca blaschkeae strains isolated from bovine mastitic milk.

Dairy cow mastitis associated with microalgae of the genus Prototheca has been reported worldwide. This alga is extremely resistant to most antimicrobials commonly used in mastitis therapy. In milk processing, different thermal treatments are generally efficient at inactivating and eliminating microorganisms. Until recently, no reports on Prototheca blaschkeae susceptibility to heat treatment have been described. Thus, considering the potential zoonotic risk that Prototheca may represent, the objective of this study was to test the susceptibility of P. blaschkeae field isolates retrieved from bovine mastitis to different temperature/time ratios that are generally used in the milk processing industry: 62°C/15 min and 30 min; 70°C/20s, 15 min, and 30 min; 75°C/20s; 90°C/1s; and 100°C/1s. The results showed a growth reduction of all isolates after the heat treatments, but only at 100°C was a total growth inhibition observed.

Histologic characterization of dilated cardiomyopathy in Estrela mountain dogs.

Dilated cardiomyopathy (DCM) is the second-most-important acquired cardiovascular disease in dogs (excluding heartworm disease in some geographic regions) and a major cause of morbidity and mortality in Estrela Mountain Dogs. The objective of this study is to describe the histologic features of DCM in Estrela Mountain Dogs, with special attention to the localization and quantification of attenuated wavy fibers (AWFs), fibrosis, and fatty infiltration. Myocardial samples from 10 areas were collected from the hearts of 10 dogs with DCM and 7 dogs without signs of cardiac disease-namely, the basal, middle, and apical portions of the free wall of both cardiac ventricles and the interventricular septum, as well as the left ventricular papillary muscle. In each sample, the presence or absence of AWFs was noted, and fatty infiltration and fibrosis were quantified. Fatty infiltration, fibrosis, and AWFs were observed in the myocardium of all dogs with DCM, in contrast to what has been described in other breeds. The left ventricular myocardium was the best tissue for diagnosis of DCM, based on these histologic features. The authors concluded that quantification of fibrosis and observation of AWFs in the left ventricular myocardium are useful in the histologic diagnosis of DCM in Estrela Mountain dogs.

Fertility time trends in dairy herds in northern Portugal.

The economics of dairy production are in great part dictated by the reproductive efficiency of the herds. Many studies have reported a widespread decrease in fertility of dairy cows. In a previous work (Rocha et al. 2001), we found a very poor oestrus detection rate (38%), and consequently a delayed calving to 1st AI and calving to conception intervals. However, a good conception rate at 1st AI was noted (51%) resulting in a low number of inseminations per pregnancy (IAP) (1.4). Here, results from a subsequent fertility time trend assessment study carried out in the same region for cows born from 1992 to 2002 are reported. Statistical linear models were used to analyse the data. Estimate linear contrasts of least square means were computed from each model. The number of observations per studied index varied from 12,130 (culling rate) to 57,589 (non-return rate). Mean age at first calving was 28.9 ± 0.14 months, without (p > 0.05) variation over time. There was a small, but significant (p < 0.05), deterioration of all other parameters. Non-return rates at 90 days and calving rate at 1st AI decreased 0.3% per trimester, with a consequent increase of 0.04 IA/parturition. Oestrus detection rate decreased 0.13% per year, and calving at 1st AI and calving-conception intervals increased 0.17 and 0.07 days/year respectively, while intercalving interval increased 1.7 days per year. From 12,130 cows calving, only 1,816 had a 4th lactation (85% culling/losses). The data was not meant to draw conclusions on the causes for the decreased fertility over time, but an increase of milk production from 6537 kg to 8590 kg (305 days) from 1996 to 2002 is probably one factor to take into consideration. Specific measures to revert or slow down this trend of decreasing fertility are warranted. Available strategies are discussed.