C57BL/6J mouse superovulation: schedule and age optimization to increase oocyte yield and reduce animal use.

Superovulation protocols have been described for different mouse strains, however the numbers of animals used are still high and still little information is known about hormone administration schedules and estrous cycle phases. In this study, we aimed to optimize a superovulation protocol by injecting 5 IU of pregnant mare serum gonadotropin followed by 5 IU of hCG 48 h later, using three different schedules related to the beginning of the dark cycle (3, 5 and 7 pm) in a light cycle of 7 am to 7 pm, with light on at 7 am. C57BL/6J mice at 3, 4 and 5 weeks of age were used and the estrous cycle phase for times of PMSG and hCG injections was also analyzed. Total oocyte number was counted in the morning after hCG injection. Hormones given at 3 weeks of age at 3 pm (59 ± 15 oocytes) and 7 pm (61 ± 10 oocytes) produced a significantly higher oocyte number compared with oocytes numbers collected from females at the same age at 5 pm (P = 0.0004 and <0.0001 respectively). Females at 4 and 5 weeks of age produced higher numbers of oocytes when superovulated at 7 pm. No statistical differences between females at different phases of the estrous cycle were found. These results showed that in C57BL/6J mice, hormones should be given at 3 or 7 pm for females at 3 weeks of age, however older females should be superovulated closer to the beginning of the dark cycle to reduce female mouse use and increase the numbers of oocytes produced per female.

Autoregressive model for genetic evaluation of longitudinal reproductive traits in Brazilian Holstein cattle.

Reproductive efficiency is major determinant of the dairy herd profitability. Thus, reproductive traits have been widely used as selection objectives in the current dairy cattle breeding programs. We aimed to evaluate strategies to model days open (DO), calving interval (CI) and daughter pregnancy rate (DPR) in Brazilian Holstein cattle. These reproductive traits were analysed by the autoregressive (AR) model and compared with classical repeatability (REP) model using 127,280, 173,092 and 127,280 phenotypic records, respectively. The first three calving orders of cows from 1,469 Holstein herds were used here. The AR model reported lower values for Akaike Information Criteria and Mean Square Errors, as well as larger model probabilities, for all evaluated traits. Similarly, larger additive genetic and lower residual variances were estimated from AR model. Heritability and repeatability estimates were similar for both models. Heritabilities for DO, CI and DPR were 0.04, 0.07 and 0.04; and 0.05, 0.06 and 0.04 for AR and REP models, respectively. Individual EBV reliabilities estimated from AR for DO, CI and DPR were, in average, 0.29, 0.30 and 0.29 units higher than those obtained from REP model. Rank correlation between EBVs obtained from AR and REP models considering the top 10 bulls ranged from 0.72 to 0.76; and increased from 0.98 to 0.99 for the top 100 bulls. The percentage of coincidence between selected bulls from both methods increased over the number of bulls included in the top groups. Overall, the results of model-fitting criteria, genetic parameters estimates and EBV predictions were favourable to the AR model, indicating that it may be applied for genetic evaluation of longitudinal reproductive traits in Brazilian Holstein cattle.

Autoregressive single-step model for genomic evaluation of longitudinal reproductive traits in portuguese holstein cattle.

We investigated the applicability of ssGBLUP methodology under the autoregressive model (H-AR) for genomic evaluation of longitudinal reproductive traits in Portuguese Holstein cattle. The genotype data of 1,230 bulls and 1,645 cows were considered in our study. The reproductive traits evaluated were interval from calving to first service (ICF), calving interval (CI) and daughter pregnancy rate (DPR) measured during the first four parities. Reliability and rank correlation were used to compare the H-AR with the traditional pedigree-based autoregressive models (A-AR). In addition, a validation study was performed considering different scenarios. Higher genomic estimated breeding values (GEBV) reliabilities were obtained for genotyped bulls when evaluated under the H-AR model, with emphasis on bulls with less than 9 daughters. For this group, the averages of GEBV reliabilities corresponded to 0.62, 0.69 and 0.62 for ICF, CI and DPR, respectively, while the averages obtained by the A-AR model were 0.27, 0.15 and 0.16. The validation study was favourable to H-AR. The best results were observed in the scenario where genotyped cows were combined with contributing bulls (genotyped bulls with daughter or relationship information in the population). Overall, the results suggest that ssGBLUP methodology under the autoregressive model is a feasible and applicable approach to be used in genomic analyses of longitudinal reproductive traits in Portuguese Holstein cattle.

Autoregressive repeatability model for genetic evaluation of longitudinal reproductive traits in dairy cattle.

We investigated the efficiency of the autoregressive repeatability model (AR) for genetic evaluation of longitudinal reproductive traits in Portuguese Holstein cattle and compared the results with those from the conventional repeatability model (REP). The data set comprised records taken during the first four calving orders, corresponding to a total of 416, 766, 872 and 766 thousand records for interval between calving to first service, days open, calving interval and daughter pregnancy rate, respectively. Both models included fixed (month and age classes associated to each calving order) and random (herd-year-season, animal and permanent environmental) effects. For AR model, a first-order autoregressive (co)variance structure was fitted for the herd-year-season and permanent environmental effects. The AR outperformed the REP model, with lower Akaike Information Criteria, lower Mean Square Error and Akaike Weights close to unity. Rank correlations between estimated breeding values (EBV) with AR and REP models ranged from 0.95 to 0.97 for all studied reproductive traits, when the total bulls were considered. When considering only the top-100 selected bulls, the rank correlation ranged from 0.72 to 0.88. These results indicate that the re-ranking observed at the top level will provide more opportunities for selecting the best bulls. The EBV reliabilities provided by AR model was larger for all traits, but the magnitudes of the annual genetic progress were similar between two models. Overall, the proposed AR model was suitable for genetic evaluations of longitudinal reproductive traits in dairy cattle, outperforming the REP model.

Autoregressive and random regression test-day models for multiple lactations in genetic evaluation of Brazilian Holstein cattle.

Autoregressive (AR) and random regression (RR) models were fitted to test-day records from the first three lactations of Brazilian Holstein cattle with the objective of comparing their efficiency for national genetic evaluations. The data comprised 4,142,740 records of milk yield (MY) and somatic cell score (SCS) from 274,335 cows belonging to 2,322 herds. Although heritabilities were similar between models and traits, additive genetic variance estimates using AR were 7.0 (MY) and 22.2% (SCS) higher than those obtained from RR model. On the other hand, residual variances were lower in both traits when estimated through AR model. The rank correlation between EBV obtained from AR and RR models was 0.96 and 0.94 (MY) and 0.97 and 0.95 (SCS), respectively, for bulls (with 10 or more daughters) and cows. Estimated annual genetic gains for bulls (cows) obtained using AR were 46.11 (49.50) kg for MY and -0.019 (-0.025) score for SCS; whereas using RR these values were 47.70 (55.56) kg and -0.022 (-0.028) score. Akaike information criterion was lower for AR in both traits. Although AR model is more parsimonious, RR model assumes genetic correlations different from the unity within and across lactations. Thus, when these correlations are relatively high, these models tend to yield to similar predictions; otherwise, they will differ more and RR model would be theoretically sounder.

Aberrant P-cadherin expression is associated to aggressive feline mammary carcinomas.

BACKGROUND: Cadherins are calcium-dependent cell-to-cell adhesion glycoproteins playing a critical role in the formation and maintenance of normal tissue architecture. In normal mammary gland, E-cadherin is expressed by luminal epithelial cells, while P-cadherin is restricted to myoepithelial cells. Changes in the expression of classical E- and P-cadherins have been observed in mammary lesions and related to mammary carcinogenesis. P-cadherin and E-cadherin expressions were studied in a series of feline normal mammary glands, hyperplastic/dysplastic lesions, benign and malignant tumours by immunohistochemistry and double-label immunofluorescence. RESULTS: In normal tissue and in the majority of hyperplastic/dysplastic lesions and benign tumours, P-cadherin was restricted to myoepithelial cells, while 80% of the malignant tumours expressed P-cadherin in luminal epithelial cells. P-cadherin expression was significantly related to high histological grade of carcinomas (p <0.0001), tumour necrosis (p = 0.001), infiltrative growth (p = 0.0051), and presence of neoplastic emboli (p = 0.0401). Moreover, P-cadherin positive carcinomas had an eightfold likelihood of developing neoplastic emboli than negative tumours. Cadherins expression profile in high grade and in infiltrative tumours was similar, the majority expressing P-cadherin, regardless of E-cadherin expression status. The two cadherins were found to be co-expressed in carcinomas with aberrant P-cadherin expression and preserved E-cadherin. CONCLUSIONS: The results demonstrate a relationship between P-cadherin expression and aggressive biological behaviour of feline mammary carcinomas, suggesting that P-cadherin may be considered an indicator of poor prognosis in this animal species. Moreover, it indicates that, in queens, the aberrant expression of P-cadherin is a better marker of mammary carcinomas aggressive behaviour than the reduction of E-cadherin expression. Further investigation with follow-up studies in feline species should be conducted in order to evaluate the prognostic value of P-cadherin expression in E-cadherin positive carcinomas.

Gut Microbiome Studies in Livestock: Achievements, Challenges, and Perspectives.

The variety and makeup of the gut microbiome are frequently regarded as the primary determinants of health and production performances in domestic animals. High-throughput DNA/RNA sequencing techniques (NGS) have recently gained popularity and permitted previously unheard-of advancements in the study of gut microbiota, particularly for determining the taxonomic composition of such complex communities. Here, we summarize the existing body of knowledge on livestock gut microbiome, discuss the state-of-the-art in sequencing techniques, and offer predictions for next research. We found that the enormous volumes of available data are biased toward a small number of globally distributed and carefully chosen varieties, while local breeds (or populations) are frequently overlooked despite their demonstrated resistance to harsh environmental circumstances. Furthermore, the bulk of this research has mostly focused on bacteria, whereas other microbial components such as protists, fungi, and viruses have received far less attention. The majority of these data were gathered utilizing traditional metabarcoding techniques that taxonomically identify the gut microbiota by analyzing small portions of their genome (less than 1000 base pairs). However, to extend the coverage of microbial genomes for a more precise and thorough characterization of microbial communities, a variety of increasingly practical and economical shotgun techniques are currently available.

In vitro susceptibility of Prototheca to pH and salt concentration.

Prototheca sp. can assume high economic significance in the dairy industry and pose a potential risk for the public health. We investigated the in vitro susceptibility of Prototheca isolates retrieved from mastitic milk (P. zopfii and P. blaschkeae) to different pH buffers and salt concentrations using a microbroth assay adapted from the Clinical Laboratory Standards Institute guidelines. Different pH buffer solutions ranging from pH 1 to pH 12 and different sodium chloride concentrations, 4.5, 9 and 18%, were tested. P. zopfii strains presented an optimal growth between pH 5 and 9, a complete growth inhibition at pH 3, and limited growth at pH 1 and 12, whereas P. blaschkeae strains showed higher susceptibility to all pH values except for pH 3 where it demonstrated a moderate growth when compared to P. zopfii strains. When salinity was incremented, P. blaschkeae was more resistant than P. zopfii, although a reduction in growth for all strains of Prototheca was observed. This study demonstrated differences in the in vitro susceptibilities of P. zopfii and P. blaschkeae to different pH and salt concentrations and intend to be a contribution on the understanding of some of the physiologic features that can be associated with the survival of these microalgae in the environment.

α-catenin, ß-catenin and P-120-catenin immunoexpression in canine mammary tissues and their relationship with E-cadherin.

Mammary tumors represent the second most common neoplasia in the canine species, where more than 50% of the cases are classified as malignant. The histological classification is used as a prognostic tool. Cadherins and catenins are responsible for cell adhesion and are intrinsic connected with the process of metastasis. E-cadherin expression in canine mammary tissues have been extensively studied. However, the studies with catenins are still scarce in the canine species. This study evaluated 74 canine mammary tissues by assessing the expression of E-cadherin and α, ß and P-120 catenin molecules using the immunohistochemistry technique and their relationship with clinicopathological parameters. Three patterns of expression were identified in this study: membranous, cytoplasmic and both (membranous and cytoplasmic). In benign tumors, more than 80% of the cases had preserved expression and in malignant tumors 20% of the cases had reduced expression. A correlation between E-cadherin and P-120-catenin expression was found as well as a significant relationship between the histological type and the expression of α-catenin in malignant tumors.

GWAS and gene networks for milk-related traits from test-day multiple lactations in Portuguese Holstein cattle.

This study focused on the identification of QTL regions, candidate genes, and network related genes based on the first 3 lactations (LAC3) of milk, fat, and protein yields, and somatic cell score (SCS) in Portuguese Holstein cattle. Additionally, the results were compared with those from only first lactation (LAC1) data. The analyses were performed using the weighted single-step GWAS under an autoregressive test-day (TD) multiple lactations model. A total of 11,434,294 and 4,725,673 TD records from LAC3 and LAC1, respectively, including 38,323 autosomal SNPs and 1338 genotyped animals were used in GWAS analyses. A total of 51 (milk), 5 (fat), 24 (protein), and 4 (SCS) genes were associated to previously annotated relevant QTL regions for LAC3. The CACNA2D1 at BTA4 explained the highest proportion of genetic variance respectively for milk, fat, and protein yields. For SCS, the TRNAG-CCC at BTA14, MAPK10, and PTPN3 genes, both at BTA6 were considered important candidate genes. The accessed network refined the importance of the reported genes. CACNA2D1 regulates calcium density and activation/inactivation kinetics of calcium transport in the mammary gland; whereas TRNAG-CCC, MAPK10, and PTPN3 are directly involved with inflammatory processes widely derived from mastitis. In conclusion, potential candidate genes (TRNAG-CCC, MAPK10, and PTPN3) associated with somatic cell were highlighted, which further validation studies are needed to clarify its mechanism action in response to mastitis. Moreover, most of the candidate genes identified were present in both (LAC3 and LAC1) for milk, fat and protein yields, except for SCS, in which no candidate genes were shared between LAC3 and LAC1. The larger phenotypic information provided by LAC3 dataset was more effective to identify relevant genes, providing a better understanding of the genetic architecture of these traits over all lactations simultaneously.

Algaemia in a dairy cow by Prototheca blaschkeae.

We describe the first known case of an algaemia by Prototheca blaschkeae in a dairy cow, which occurred after a chronic episode of mastitis caused by this pathogen. The organism was isolated from milk, joint fluid and blood samples, and microbiologic and molecular methods were performed to obtain a definitive identification of the algae. The affected cow was culled only after confirmation of a systemic infection by Prototheca.

Bornaviruses in naturally infected Psittacus erithacus in Portugal: insights of molecular epidemiology and ecology.

Background: The genus Orthobornavirus comprises non-segmented, negative-stranded RNA viruses able to infect humans, mammals, reptiles and various birds. Parrot bornavirus 1 to 8 (PaBV-1 to 8) causes neurological and/or gastrointestinal syndromes and death on psittacines. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a death of two female Psittacus erithacus (grey parrot). Methods and Results: Both parrots were submitted for a complete standardised necropsy. Tissue samples were analysed by PCR. The findings in necropsy were compatible with bornavirus infection. Analysis revealed PaBV-4 related with genotypes detected in captive and in wild birds. The N and X proteins of PaBV-4 were more related to avian bornaviruses, while phosphoprotein was more related to variegated squirrel bornavirus 1 (VSBV-1). Within the P gene/phosphoprotein a highly conserved region between and within bornavirus species was found. Conclusions: Portugal is on the routes of the intensive world trade of psittacines. Broad screening studies are required to help understanding the role of wild birds in the emergence and spread of pathogenic bornaviruses. PaBV-4 phosphoprotein is closer to VSBV-1 associated with lethal encephalitis in humans than with some of the avian bornaviruses. The highly conserved P gene/phosphoprotein region is a good target for molecular diagnostics screenings.

Detection and phylogenetic analysis of parrot bornavirus 4 identified from a Swedish Blue-winged macaw (Primolius maracana) with unusual nonsuppurative myositis.

Background: The genus Orthobornavirus comprises RNA viruses infecting humans, mammals, birds and reptiles, where parrot bornavirus 1 to 8 causes fatal neurological and/or gastrointestinal syndromes in psittacines. There is, to the best of our knowledge, no publication describing avian bornaviruses in pet parrots in Sweden. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a history of severe weight loss and death of a Primolius maracana.Methods and results: The results of histopathology, immunohistochemistry and real-time RT-PCR were compatible with avian bornavirus infection. Sequencing indicated infection by parrot bornavirus 4 (PaBV-4). The genotype reported shared high identity with PaBV-4 identified from pet psittacines and from wild birds in several countries. The N gene and X protein showed genotype clusters formation. P protein revealed to be more conserved within and between species of bornaviruses. Findings suggest horizontal transmission within and between avian orders and species.Conclusion: There seems to be a worldwide trading without biosafety measures, hence, further disease transmission could be avoided. For screening purposes, the P gene is a good candidate as a universal target in molecular diagnostics. Wild birds may be key pieces in the puzzle of bornavirus epidemiology.

Bovine mastitis associated with Prototheca blaschkeae.

Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.

Detection and quantitative analysis of human herpesvirus in pilocytic astrocytoma.

We investigated the hypothetical role of human herpesviruses (HHVs) in tumour formation of the cerebellum. Thirty-five samples of pilocytic astrocytoma and 10 control samples of cerebellum from patients who died of unrelated diseases were examined. Presence of the 8 known HHVs was first studied using specific real-time quantitative Polymerase Chain Reaction (qPCR) targeting viral DNA polymerase. HHV's DNA polymerase was found present in 20 samples (7 controls, 13 astrocytomas) and was absent in 25 samples (3 controls, 22 astrocytomas). DNA polymerase of Epstein-Barr Virus (EBV) was present in 16 samples, 7/10 controls (70%) and 9/35 astrocytomas (26%). HHV-1 and Varicella-Zoster virus were detected only twice and HHV-2, Cytomegalovirus, HHV-7 and HHV-8, only once. HHV-6 was not detected. In all cases, the gene copy numbers of DNA polymerase were low (<100/100 ng DNA). A second approach was to search for novel HHVs, using consensus-degenerated hybrid oligonucleotide primers (CODEHOP) PCR: no sequence indicative of a new HHV was detected. In summary, EBV was the most frequent HHV detected in pilocytic astrocytoma, but at very low levels. According to the actually accepted threshold the results suggest that EBV cannot be considered responsible for tumorigenesis of pilocytic astrocytoma.

Canine parvovirus 2c infection in central Portugal.

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.

Transthoracic echocardiography in Estrela Mountain dogs: reference values for the breed.

Of 100 Estrela Mountain dogs, 74 were examined to obtain echocardiographic reference values for the breed. The influence of bodyweight, age and sex on different echocardiographic parameters was studied using either analysis of variance or regression analysis. Statistically significant differences were found between sex and heart rate as well as interventricular septal thickness at end-systole and left ventricular internal dimension at end-diastole. A statistically significant linear correlation was also found between several parameters and (1) age (mean arterial pressure, left ventricular internal dimension at end-systole, fractional shortening, circularity index in systole, aortic valve velocity time integral, pulmonic valve velocity time integral and mitral valve E wave peak velocity), (2) weight (left ventricular posterior wall thickness at end-diastole and end-systole, end-diastolic volume index, left atrium diameter, aortic valve peak velocity and mitral valve E wave deceleration time), (3) sex and age (left ventricle end-diastolic volume), and (4) sex and weight (aortic root diameter and pulmonic valve peak velocity). Reference values for the breed are presented, as well as dispersion graphics for selected parameters, based on a regression equation.

E-cadherin, beta-catenin, invasion and lymph node metastases in canine malignant mammary tumours.

Recent studies of canine malignant mammary tumours suggest that reduction of E-cadherin and/or beta-catenin correlates with invasive behaviour and lymph node metastasis. The aims of this study were to examine the interrelationships between the expression of E-cadherin and beta-catenin, and the relationship between the expression of E-cadherin and/or beta-catenin and the mode of growth and metastatic capacity of canine malignant mammary tumours. 90 spontaneous malignant tumours and local and regional lymph nodes were studied. A significant relationship was evidenced between membranous expression of E-cadherin and beta-catenin (p=0.0027), but not between E-cadherin and cytoplasmic beta-catenin. Only E-cadherin as a separate factor was significantly related to tumour invasion (p=0.0072) and lymph node metastasis (p=0.0001). Neither membranous nor cytoplasmic beta-catenin expression was significantly related to either of these phenomena.

Sialyl Lewis x expression in canine malignant mammary tumours: correlation with clinicopathological features and E-Cadherin expression.

BACKGROUND: Sialyl Lewis x (sLex) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLex is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels.E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer. The aim of this study was to analyse the sLex expression in canine malignant mammary tumours and to evaluate if the presence of sLex correlates with the expression of E-Cadherin and with clinicopathological features. METHODS: Fifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLex (IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLex tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLex and E-Cadherin. RESULTS: sLex expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLex expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLex was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship. CONCLUSION: In the present study we demonstrate the importance of sLex in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLex as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLex and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLex and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.

MIB-1 labelling indices according to clinico-pathological variables in canine mammary tumours: a multivariate study.

The relationship between MIB-1 labelling indices (LI), as detected by immunohistochemical methods, and other clinico-pathological characteristics was studied in a series of 77 malignant mammary tumours surgically removed from 47 female dogs. The immunostaining was assessed on the basis of the estimated percentage of positive cells in the areas of highest labelling. Multivariate logistic regression demonstrated no influence of breed, age, previous pregnancies, previous progestin administration, histological type or location of the tumour on MIB-1 LI. MIB-1 LI was significantly related to the size of the tumour, necrosis, invasive growth and histological grade, but not with ulceration, lymph node metastasis, skin fixation or E-cadherin expression. The significant relationship between MIB-1 LI and other known factors of poor prognosis suggests that a high LI may have prognostic value in canine malignant mammary tumours.