The aim of the research reported in this Research Communication was to identify differentially expressed proteins in dairy cows with normal and lutein diet and to elucidate the mechanisms of lutein-induced effects on bovine mammary gland metabolism using a comparative proteomic approach. Thirty-three differentially expressed proteins were identified from mammary gland of control diet-fed and lutein diet-fed dairy cows. Among these proteins, 15 were upregulated and 18 were downregulated in the lutein group. Functional analysis of the differentially expressed proteins showed that increased blood flow, depressed glycolysis, enhanced lactose anabolism, decreased fatty acid oxidation and up-regulated beta lactoglobulin expression were connected with lutein addition. These results suggested that the increased blood flow, reduced glucose catabolism, enhanced capacity for milk lactose synthesis, depressed fatty acid catabolism and increased expression of antioxidantion related protein may be the prime factors contributing to the increased milk production and enhanced immune status in lutein-fed dairy cows. This study provides molecular mechanism of dietary lutein in regulating lactation of dairy cows.

High temperature is a major stress that negatively affects welfare, health, and productivity of dairy animals. Heat-stressed animals are more prone to disease, suggesting that their immunity is hindered. Although productive and physiologic responses of dairy animals to heat stress are well known, there is still limited information on the response at the transcriptome level. Our objective was to evaluate the changes in performance and blood transcriptomics of dairy goats under heat stress. Eight multiparous Murciano-Granadina dairy goats in mid-lactation were assigned to 1 of 2 climatic treatments for 35 d. Treatments and temperature-humidity index (THI) were: (1) thermal neutral (TN: n = 4; 15–20 °C, 40–45%, THI = 59–65), and (2) heat stress (HS: n = 4; 12 h at 37 °C–40%, THI = 86; 12 h at 30 °C–40%, THI = 77). Rectal temperature, respiratory rate, feed intake and milk yield were recorded daily. Additionally, milk composition was evaluated weekly. Blood samples were collected at d 35 and RNA was extracted for microarray analyses (Affymetrix GeneChip Bovine Genome Array). Differences in rectal temperature and respiratory rate between HS and TN goats were maximal during the first 3 d of the experiment, reduced thereafter, but remained significant throughout the 35-d experimental period. Heat stress reduced feed intake, milk yield, milk protein and milk fat contents by 29, 8, 12, and 13%, respectively. Microarray analysis of blood revealed that 55 genes were up-regulated, whereas 88 were down-regulated by HS. Bioinformatics analysis using the Dynamic Impact Approach revealed that 31 biological pathways were impacted by HS. Pathways associated with leukocyte transendothelial migration, cell adhesion, hematopoietic cell lineage, calcium signaling, and PPAR signaling were negatively impacted by HS, whereas nucleotide metabolism was activated. In conclusion, heat stress not only negatively affected milk production in dairy goats, but also resulted in alterations in the functionality of immune cells, which would make the immune system of heat-stressed goats less capable of fending-off diseases.

The temperature and pH of the contents of the reticulorumen of cattle can be monitored using sensors placed in a rumen bolus. In this research communication we test the hypothesis that these measurements can be used as a predictor of the likelihood of reproductive success. The study was performed on 20 Lithuanian Black and White dairy cows. The pH and temperature of the contents of their reticulorumens were measured using boluses manufactured for animal care (smaXtec animal care technology®). The data from reticulorumen were recording at 1 to 7 and 7 to 56 d post calving and then post insemination (on average 60 ± 12) until 170 d after calving. The pregnancies were tested with ‘Easy scan’ ultrasound 30–35 d after insemination. Those cows which were pregnant were assigned to the PD+ group (n = 12), and those which were not pregnant – assigned to the PD− group (n = 8). Rumen pH was initially lower in the PD+ group but then higher than in PD−, but temperature did not differ between the groups until after conception. We can conclude that cows with disturbed rumen metabolism (with low rumen pH) are less likely to conceive. The measurements of reticuloruminal pH may be useful as a predictor of the likelihood of reproductive success, but more research is needed before practical application.

This study examined the hypothesis that xanthosine (XS) treatment would promote mammary-specific gene expression and stem cell transcripts and have a positive influence on milk yield of dairy goats. Seven primiparous Beetal goats were assigned to the study. Five days after kidding, one gland (either left or right) was infused with XS (TRT) twice daily for 3 d and the other gland with no XS infusion served as a control (CON). Mammary biopsies were collected at 10 d and RNA was isolated. Gene expression analysis of milk synthesis genes, mammary stem/progenitor cell markers, cell proliferation and differentiation markers were performed using real time quantitative PCR (RT-qPCR). Results showed that the transcripts of milk synthesis genes (BLG4, CSN2, LALBA, FABP3, CD36) and mammary stem/progenitor cell markers (ALDH1 and NR5A2) were increased in as a result of XS treatment. Average milk yield in TRT glands was increased marginally (approximately ~2% P = 0·05, paired t-test) per gland relative to CON gland until 7 wk. After 7 wk, milk yield of TRT and CON glands did not differ. Analysis of milk composition revealed that protein, lactose, fat and solids-not-fat percentages remained the same in TRT and CON glands. These results suggest that XS increases expression of milk synthesis genes, mammary stem/progenitor cells and has a small effect on milk yield.

The aim of the present study was to evaluate the potential for diagnosis of fatty liver by means of ultrasonographic measurement of liver and perivisceral adipose tissue as an alternative to blood indicators of lipomobilization and liver biopsy in periparturient high-yielding dairy cows. Thirty cows were enrolled and divided into two groups. The evaluation of body condition score (BCS), non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), liver and perivisceral adipose tissue ultrasonographic measurement and histological liver lipid content (GdL) was performed at 15 ± 5 d prepartum (T0), 10 ± 2 d postpartum (T1), 30 ± 2 d postpartum (T2) and 50 ± 2 d postpartum (T3). Mesenteric fat thickness (the thickness of the perivascular adipose tissue) measured on ultrasound was shown to be an independent determinant of fatty liver. The cut-off of the ultrasonographic evaluation of the liver may be useful as a first and practical approach in identifying fatty liver. In conclusion, a non-invasive and reliable diagnostic method for predicting the risk of fatty liver in high yielding dairy cows has been demonstrated.

Dairy cows with type II ketosis display hepatic fat accumulation and hyperinsulinemia, but the underlying mechanism is not completely clear. This study aimed to clarify the regulation of lipid metabolism by insulin in cow hepatocytes. In vitro, cow hepatocytes were treated with 0, 1, 10, or 100 nm insulin in the presence or absence of AICAR (an AMP-activated protein kinase alpha (AMPKα) activator). The results showed that insulin decreased AMPKα phosphorylation. This inactivation of AMPKα increased the gene and protein expression levels of carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c), which downregulated the expression of lipogenic genes, thereby decreasing lipid biosynthesis. Furthermore, AMPKα inactivation decreased the gene and protein expression levels of peroxisome proliferator-activated receptor-α (PPARα), which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation. In addition, insulin decreased the very low density lipoprotein (VLDL) assembly. Consequently, triglyceride content was significantly increased in insulin treated hepatocytes. Activation of AMPKα induced by AICAR could reverse the effect of insulin on PPARα, SREBP-1c, and ChREBP, thereby decreasing triglyceride content. These results indicate that insulin inhibits the AMPKα signaling pathway to increase lipid synthesis and decrease lipid oxidation and VLDL assembly in cow hepatocytes, thereby inducing TG accumulation. This mechanism could partly explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type II ketosis.

This Research Communication describes the effect of post-operative pain and non-steroidal anti-inflammatory drug (NSAID) treatment on heart rate variability (HRV) of dairy cows. Postoperative pain in farm animals is often left untreated, and HRV could be a promising tool for assessing pain. The aim of this study was to assess if postoperative state after subcutaneous surgery affects HRV in dairy cows and to determine whether this could be modulated by NSAID. Nine cows were inserted with an implantable electrocardiograph logger. Cows were divided into the NSAID treatment group and the control group. The cows in the NSAID group had higher HRV than the control group, indicating a higher sympathetic activity in control animals, most likely due to untreated post-operative pain. Besides the ethical need for treating pain in production animals, ongoing pain has an adverse effect on animal productivity. Thus post-operative pain alleviation is recommended.

The hypothesis of the study was that inhibition of PPARβ/δ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPARβ/δ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPARβ/δ using GSK-3787, the synthetic PPARβ/δ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPARβ/δ in the presence or absence of fetal bovine serum was achieved with ≥ 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPARβ/δ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPARβ/δ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPARβ/δ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPARβ/δ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells.

The possible association between teat morphometric traits and subclinical mastitis (SCM) in dairy buffaloes was studied. Teat morphometric parameters, i.e. teat shape (bottle, conical, cylindrical, and others), teat-end shape (flat, round, and pointed), teat length (TL), teat diameter (TD), and teat-end to floor distance were measured before milking, but after proper milk let-down, in clinically healthy buffaloes (47 Murrah and 34 Nili-Ravi breeds). Subclinical mastitis was defined on the basis of bacteriology and somatic cell count (SCC) of quarter foremilk samples. A high proportion of cylindrical teats (40%) and pointed teat-ends (64·4%) was observed. Hind teats were longer and thicker than fore teats (P < 0·05). A significant breed effect was found with respect to teat shape, length and diameter (P < 0·05). Teats were mostly cylindrical (43·3 vs. 35·4%) and conical (34·2 vs. 30·8%) shaped, smaller (mean 8·2 vs. 9·5 cm) and thinner (mean 3·3 vs. 3·6 cm) in the Murrah breed compared with the Nili-Ravi breed. Teats that had ‘other’ shapes and were longer, wider, and placed closer to the floor were more associated with SCM (P < 0·05). Mean SCC was significantly higher (P < 0·05) in Nili-Ravi buffaloes, teat shapes classified as ‘others’, and quarters with SCM. Teat morphometric traits seem to be associated with indicators of udder health in buffaloes, thus, their inclusion in breeding programmes for selection against undesirable dairy type traits may be of value in reducing susceptibility to intramammary infections in Indian buffaloes.

This Research Paper aimed to investigate donkey welfare in dairy husbandry systems and to identify the potential factors affecting it at animal level. In 2015, twelve dairy donkey farms (19–170 donkeys per farm, mean = 55 ± 48), distributed throughout Italy, were visited. On each farm, the Animal Welfare Indicators (AWIN) welfare assessment protocol for donkeys was used by two trained assessors to evaluate the welfare of animals for a total of 257 donkeys assessed. The protocol includes animal-based indicators that were entered in a digitalised system. Prevalence of different scores at individual, farm and category level were calculated. Farmers were asked to fill out a questionnaire including information regarding the management of donkeys and their final destination. Answers to the questionnaire were then considered as effects in the risk factor analysis whereas the scores of the animal-based indicators were considered as response variables. Most of the donkeys (80·2%) enjoyed a good nutritional status (BCS = 3). 18·7% of donkeys showed signs of hoof neglect such as overgrowth and/or incorrect trimming (Min = 0% Max = 54·5%). Belonging to a given farm or production group influenced many of the welfare indicators. The absence of pasture affected the likelihood of having skin lesions, alopecia, low BCS scores and a less positive emotional state. Lack of routine veterinary visits (P < 0·001) and having neglected hooves (P < 0·001) affected the likelihood of being thin (BCS < 3). Belonging to specific production groups, lack of access to pasture and showing an avoidance reaction to an approaching human (AD) resulted in risk factors associated with a higher prevalence of signs of hoof neglect. Our results support the idea that lack of knowledge of proper donkey care among owners was behind many welfare issues found.

Sterol regulatory element binding protein 1 (SREBP1) has a central regulatory effect on milk fat synthesis. Lipopolysaccharides (LPS) can induce mastitis and cause milk fat depression in cows. SREBP1 is also known to be associated with inflammatory regulation. Thus, in the current study, we hypothesized that LPS-induced milk fat depression in dairy cow mammary epithelial cells (DCMECs) operates via decreased SREBP1 expression and activity. To examine the hypothesis, DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS (10 µg/ml). LPS treatment of DCMECs suppressed lipid-metabolism-related transcription factor SREBP1 mRNA expression, nuclear translocation and protein expression, leading to reduced triglyceride content. The transcription levels of acetyl-CoA carboxylase-1 and fatty acid synthetase were significantly down-regulated in DCMECs after LPS treatment, suggesting that acetyl-CoA carboxylase-1 and fatty acid synthetase involved in de novo milk fat synthesis was regulated by SREBP1. In summary, these results suggest that LPS induces milk fat depression in dairy cow mammary epithelial cells via decreased expression of SREBP1 in a time-dependent manner.

The experiments reported in this research communication aimed to plot the expression pattern of Sirt3, a master regulator of energy metabolism and antioxidation defence, in the liver of dairy goats during perinatal period. Ten healthy dairy goats in late pregnancy were chosen, and needle biopsy was applied to collect liver samples at 1-week intervals. Protein levels of hepatic Sirt3 were analysed by western-blotting. Serum enzyme activities of manganese superoxide dismutase (Mn-SOD) and non-esterified fatty acids (NEFA) levels were measured, and their correlation with Sirt3 mRNA levels was also estimated. Compared with >3-week before parturition (BP), Sirt3 proteins were significantly reduced at 1-week after parturition (AP) and 2-week AP (P < 0·05), but increased on the day of parturition (P < 0·01). Correlation analysis revealed a positive association between hepatic Sirt3 mRNA levels and serum enzyme activity of Mn-SOD (r = 0·46), but a negative association between that and serum NEFA levels (r = −0·41). These data indicate that the decreased hepatic expression of Sirt3 might be one of the reasons that dairy goats undergo oxidative stress after parturition.

Until now there are only few data on the effects of thermal treatments on the nutritional and hygienic characteristics of donkey milk. This Research Communication aims to provide information on the effects of pasteurization (at +65 °C for 30 min) and prolonged storage at refrigeration and freezing temperatures (21 d at + 3 °C ± 2 °C and up to 90 d at −20 °C ± 5 °C) on some nutritional and hygienic characteristics of Amiata donkey milk. The milk was monitored by chemical and microbiological analysis. Pasteurization ensured compliance with EC Regulation No 1441/2007, as Enterobacteriaceae were never found in the milk, or during storage at refrigeration and freezing temperatures. Colony count at 30 °C in pasteurized milk never went beyond 1 log CFU/ml. The heat treatment and the storage did not result in any variations in the main constituents of the milk. Only a decrease in lactose and few variations in some fatty acids at 90 d of freezing were observed. In conclusion, pasteurization was able to achieve and maintain a high hygienic-sanitary quality over time; storage at refrigeration or freezing temperatures did not alter the nutritional quality of fat and the gross composition of the product. These findings are useful to improve knowledge on the milk shelf life in order to guarantee safety and nutritional quality for infants who need small quantities of daily milk.

The aim of this study was to estimate the associations of the first occurrence of pathogen-specific clinical mastitis (CM) with milk yield and milk composition (somatic cell count (SCC), lactose, fat, protein content in milk and milk urea nitrogen (MUN)). We studied 3149 dairy cows in 31 Hokkaido dairy farms in Japan. Five pathogen groups were studied: Streptococcus spp.; Staphylococcus aureus (S. aureus); coagulase-negative staphylococci (CNS); coliforms; and fungi. Test-day milk data and clinical records were collected from June 2011 until February 2014. Mixed models with an autoregressive correlation structure were fitted to quantify the effects of CM and several other control variables (herd, calving season, parity, week of lactation, and other diseases). Primipara (first lactation) and multipara (second and later lactations) were analysed separately. All pathogens, particularly S. aureus and fungi, were associated with significant milk losses in multipara. In this study, S. aureus and CNS infections were not associated with significant milk loss in primipara. All pathogens, in particular S. aureus and fungi, significantly increased SCC in both parity groups. All pathogens, especially CNS (in primipara) and S. aureus (in multipara), decreased lactose content. All pathogen groups except for fungi were associated with significant changes in fat, protein and MUN. Some pathogens such as Streptococcus spp. and coliforms seemed to be associated with long-term fat, protein and MUN changes. These findings provide estimates that could be used to calculate precise costs of CM, and also provide better indicators of pathogen-specific mastitis.

This research communication describes the use of contrast-enhanced ultrasonographic examination (CEUS) in mammary glands of ewes for diagnosis of chronic mastitis; this is the first report of the use of this modality in diagnostic imaging of mammary glands of ruminants. For this purpose, a convex transducer was used, with the following settings: frequency: 2·0/4·0 MHz, mechanical index: 0·09, power: 22 dB, scanning depth: 70 mm, and sulphur hexafluoride in microbubbles at a dose of 20 µl as the contrast agent. In four healthy mammary glands (2 ewes), CEUS examination revealed a steady biphasic pattern of contrast agent kinetics characterised by initial uptake within 15–40 s post-injection, at which time intensity peaked with strong enhancement (130–200 AEU) followed by a gradual wash-out phase. In three mammary glands with history of clinical mastitis (2 ewes), the pattern was particularly inconsistent and unclear, with weak enhancement (<100 AEU) (P < 0·01) lasting for a short period. Notwithstanding issues regarding cost and withdrawal period of contrast-agent, this imaging modality may contribute to improved diagnosis of mastitis cases, especially on occasions when abnormalities cannot be easily confirmed by more conventional methods.

During drying off and transition period, cows are subject to changes in endocrine status, metabolic stressors and altered immune functions, which could lead to an increased risk of disease. To expand our knowledge on the immune/inflammatory status and to identify markers to define cow status during this interval, the pattern of 9 different cellular parameters, 5 cytokines, 2 enzymes and 3 cellular ratios in blood samples were assessed in 15 primiparous cows belonging to three different dairy herds in Lombardy. Our data showed that the variation of almost all parameters was influenced by the physiological period in which the samples were collected, except for apoptosis, IL-1β, IL-6, lysozyme and granulocyte/monocyte ratio. Several markers were directly correlated either to the herd alone (IL-1β, IL-6, lysozyme, granulocyte/lymphocyte ratio and granulocyte/monocyte ratio) or in association with the sampling time (white blood cell count, necrosis, lymphocytes count, CD4+ lymphocytes proportion). Hierarchical cluster analysis identified three herd-associated sample clusters showing different frequency along the follow-up period. The results of this field study highlight the importance of the herd factor in the immune/inflammatory response. Furthermore, these results suggest that cellular parameters are probably the most suitable markers to define cow status during drying-off and the peripartum period.

This Research Communication describes the relationship between casein, free fatty acids (FFAs) and the storage period of ultra-high temperature-treated (UHT) whole milk observed for a period of 120 d of labelled shelf-life. Moreover, we aim to estimate the daily rate of casein degradation in UHT whole milk, and the total length of time estimated for its full degradation. With this aim, ten sets of samples were evaluated from batches of UHT milk manufactured by a dairy processing plant in Parana State, Brazil on 10 different days. Each set was comprised of one liter of raw milk and 12 units of 1 litre cartons of UHT milk, and represented one batch of production. Total mesophilic (TMC), psychrotrophic (TPC), and somatic cell counts (SCC) of raw milk were assessed. UHT milk was assessed for fat (%), sialic acid (mg/l), casein (%), and FFA contents. TMC ranged from 3·5 × 106 to 3·1 × 107 CFU/ml; TPC, from 106 UFC/ml and higher; and SCC, from 18 × 104 SC/ml to 4·83 × 105 CS/ml. Casein (r = −0·991; R2 = 0·9822) and FFA (r = 0·962; R2 = 0·9245) contents, and storage time of UHT milk were correlated (P < 0·05). The rate of casein hydrolysis was estimated as 0·021 g/100 g UHT whole milk/day. A complete breakdown of casein was estimated to occur by the 560th day post-manufacture. Although age gelation was not observed in our study, the report herein corroborates the understanding that the microbiological quality and SCC of raw milk are important components involving the integrity of casein and lipids of UHT milk during shelf-life.

This Research Communication describes the effects of a synthetic analogue of the Bovine Appeasing Pheromone (BAP) on milk parameters in Valdostana dairy cows during the first turning out from tie-stalls to confined lowland pastures around the farms. Thirty healthy lactating Valdostana cows were enroled in the study and randomly divided into 2 groups: experimental group (EG, n = 15) and control group (CG, n = 15). The two groups were separately housed in the same farm and managed outside in two different pens. Treatment (BAP and solution) and control (solution only) were poured on the nuchal skin area between the horns when the animals were inside the farm at the feeding rack every 7 d for 28 d (T0–T4). Milk samples were evaluated at the same time points (T0–T4). Daily milk production (kg/day) was higher in the EG than in the CG, particularly during the first day after the turning out to pasture (T1). Somatic Cell Count (103 cells/ml) was higher in the placebo group than in the EG, especially at T1. Proteins, fat, fat-free dry matter and casein (g/100 g) were not affected by the treatment. In T1 urea (mg/dl) content was higher in CG vs. EG, suggesting a more correct metabolic balance in the group treated with BAP. The use of BAP appears to modulate adaptation in ways that may improve dairy cow performance in the context of changes in management routines.

This study tested the hypotheses that machine milked dairy sheep have a high prevalence of teat-end hyperkeratosis (TEH), which contributes to udder health problems. A random sample of 1360 milking ewes from 28 dairy sheep farms was monitored. Milking procedures, milking parlour characteristics and maintenance were recorded during a designated on farm audit; records were obtained through observations and interviews with farmers. Number of ewes/milker, ewes/milking unit and milkings/milking unit were calculated. Vacuum level, pulsation rate and ratio were measured. Four combinations of vacuum level and pulsation rate were defined; <40 kPa and <150 cycles/min (VP1), <40 kPa and ≥150 cycles/min (VP2), ≥40 kPa and <150 cycles/min (VP3), ≥40 kPa and ≥150 cycles/min (VP4). California Mastitis Test (CMT; scores 0–4) was done on all ewes. Then the udder of each ewe was assessed for TEH (scores 1–4) and ewes were assigned into three groups according to TEH severity (no or mild, medium and severe TEH). Severe TEH (scores 3 and 4) prevalence at teat-level was ca. 13%. TEH severity was associated with the combination of vacuum level and pulsation rate; ewes milked with VP4 combination were more likely to have a one-level increase on TEH severity compared to ewes milked with VP2 and VP3 combinations. More ewes/milker and less ewes/milking unit increased the likelihood of a one-level increase on TEH severity. Finally, ewes with severe TEH were more likely to have a one-level increase on CMT score. Therefore, our hypotheses that TEH is prevalent in dairy ewes and contributes to udder health problems were confirmed. Additionally, farmers can reduce TEH prevalence by optimising the way they milk and their milking parlour.

The objective of the research described in this Research Communication was to describe potential associations of subclinical mastitis with sheep breeds in Greece. A countrywide survey (2198 ewes in 111 farms) was performed. Prevalence of subclinical mastitis was 0·260. Results did not indicate any difference in the prevalence of subclinical mastitis between farms with pure-bred and farms with cross-bred animals, nor any difference in prevalence between farms with Greek pure-bred animals and farms with imported pure-bred animals. Results indicated that prevalence of subclinical mastitis was smaller in farms with Assaf-breed (0·100) and higher in farms with Frisarta-breed (0·625) (P < 0·02). Prevalence of mastitis was smaller in farms with Greek traditional indigenous breeds (0·221) (P = 0·007). In a model that included sheep breed and management system in farm, breed emerged as a significant factor for prevalence of subclinical mastitis (P = 0·003).