Romana Turk

Animal board invited review: advances in proteomics for animal and food sciences

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid – i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 – Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East–West and North–South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Farm animal milk proteomics

Milk is one of the most important nutrients for humans during lifetime. Farm animal milk in all its products like cheese and other fermentation and transformation products is a widespread nutrient for the entire life of humans. Proteins are key molecules of the milk functional component repertoire and their investigation represents a major challenge. Proteins in milk, such as caseins, contribute to the formation of micelles that are different from species to species in dimension and casein-type composition; they are an integral part of the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recent years. Milk proteins can act as enzymes or have an antimicrobial activity; they could act as hormones and, last but not least, they have a latent physiological activity encoded in their primary structure that turns active when the protein is cleaved by fermentation or digestion processes. In this review we report the last progress in proteomics, peptidomics and bioinformatics. These new approaches allow us to better characterize the milk proteome of farm animal species, to highlight specific PTMs, the peptidomic profile and even to predict the potential nutraceutical properties of the analyzed proteins.

Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis

Cow serum proteome was evaluated by three different complementary approaches in the control group, subclinical and clinical mastitis in order to possibly find differential protein expression useful for a better understanding of the pathophysiology of mastitis as well as for an early diagnosis of the disease. The systemic inflammatory and oxidative stress response in cows with subclinical and clinical mastitis were observed. The collected evidence shows a differential protein expression of serpin A3-1, vitronectin-like protein and complement factor H in subclinical mastitis in comparison with the control. It was also found a differential protein expression of inter-alpha-trypsin inhibitor heavy chain H4, serpin A3-1, C4b-binding protein alpha chain, haptoglobin and apolipoprotein A-I in clinical mastitis compared to the control. Among the inflammatory proteins up-regulated in clinical mastitis, vitronectin is over-expressed in both subclinical and clinical mastitis indicating a strong bacterial infection. This suggests vitronectin as an important mediator in the pathogenesis of the onset of mastitis as well as a valuable marker for diagnosis of the subclinical form of the disease. Obtained data could be useful for the detection of mastitis during the subclinical phase and for a better comprehension of the pathophysiological mechanisms involved in the onset of the disease.

Lipid mobilisation and oxidative stress as metabolic adaptation processes in dairy heifers during transition period

The objective of this study was to evaluate metabolic disorders and oxidative stress in dairy heifers during the transition period. Possible relationships between lipid mobilisation indicators and oxidative stress markers were investigated as well. Nineteen dairy heifers were included in the study. Blood samples were collected at the time of estrus synchronisation in heifers, at insemination, three weeks after insemination, one week before calving, at calving and 1, 2, 4 and 8 weeks postpartum. Common metabolic parameters, beta-hydroxybutyrate (BHB), free fatty acids (FFA), paraoxonase-1 (PON1) activity and total antioxidative status (TAS) were analysed. Around insemination, no significant difference was observed in the majority of tested parameters (P>0.05). However, the transition period markedly affected the concentration of triglycerides, total cholesterol, HDL-C, BHB, FFA, TAS and PON1activity. Positive correlations between PON1 activity and total cholesterol, HDL-C and triglycerides were noted but inverse correlations with FFA, BHB and bilirubin were found indicating that PON1 activity changed with lipid metabolism and was influenced by negative energy balance. These findings suggest that lipid mobilisation and oxidative stress are part of a complex metabolic adaptation to low energy balance which reaches equilibrium later in advanced lactation.

A Proteomics Perspective: From Animal Welfare to Food Safety

A fundamental issue of farm animal welfare is to keep animals clinically healthy, without disease or stress, particularly in intensive breeding, in order to produce safe and quality food. This issue is highly relevant for the food industry worldwide as they are directly linked to public health and welfare. The aim of this review is to explore how proteomics can assess and improve the knowledge useful for the strategic management of products of animal origin. Useful indications are provided about the latest proteomics tools for the development of novel biotechnologies serving the public health. The multivariate proteomics approach provides the bases for the discovery of biomarkers useful to investigate adaptation syndromes and oxidative stress. These two responses represent the milestones for the study of animal welfare. Moreover their implementation in the characterization and standardization of raw materials, process development, and quality and safety control of the final product of animal origin represents the current frontier in official surveillance and tests development.

Lipid status, paraoxonase-1 activity and metabolic parameters in serum of heifers and lactating cows related to oxidative stress

The objective of this study was to investigate serum lipids, metabolic parameters and activity of the anti-oxidative enzyme paraoxonase-1 (PON1). The study was conducted on non-pregnant heifers with optimal health status and on healthy dairy cows in the period of intensive lactation, assuming that the energy and metabolic demands during lactation reduce anti-oxidative protection. Total cholesterol and HDL-cholesterol concentrations were significantly higher (P<0.05) in lactating cows than in heifers. Bilirubin concentration and γ-glutamyltransferase (GGT) activity were also significantly higher in lactating cows (P<0.05), indicating increased hepatic efforts of cows to meet energy requirements for lactation. Significantly lower PON1 activity and PON1/HDL ratio in lactating cows compared to heifers (P<0.05) showed that metabolic efforts during pregnancy, parturition and lactation influence PON1 activity due to oxidative stress. Concurrent increase in total and HDL-cholesterol during lactation indicated that the HDL particle is a major carrier of cholesterol in cows.

The influence of sex on biochemical markers of bone turnover in dogs

Biochemical markers of bone turnover have been shown to be useful as inexpensive and noninvasive tools for monitoring skeletal health. The reference range for bone markers in dogs has been set by different age groups. However, other sources of biological variations were not fully investigated in dogs. To explore whether sex influences the interpretation of bone marker data we examined serum bone markers in 33 male and 25 female dogs. The bone markers selected for this study were: bone alkaline phosphatase (BALP) and osteocalcin (OC) as indicators of bone formation, and C-terminal telopeptide (CTx) of type I collagen as marker of bone resorption. All concentrations of bone markers were lower, but still within the reference range reported for dogs. We found statistically significant differences of the median OC and CTx serum concentrations between males and females. The results of this study suggest that there are sex differences in biochemical markers of bone turnover in dogs which should be considered in interpretation of bone marker data.

The role of fat dormouse ( Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein-Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase-1 (PON1) and apolipoprotein A-I (ApoA-I) were analysed in sera taken on days -30, -10, -2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL-C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA-I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL-C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.

Progesterone and Insulin-Like Growth Factor I Levels in Blood of Boer Goats During Puerperium Out-of-Season in a Mild Climate Region

The aims of this study were to compare insulin-like growth factor-I (IGF-I) and progesterone concentrations as indicators of cycle activity in the blood of Boer goats during puerperium out-of-season in mild climate conditions and to investigate the influence of parity and litter size on the concentration of IGF-I and progesterone in serum during the study period. Twenty Boer (8 primiparous and 12 pluriparous) goats were examined in this study. Blood samples were taken every 4 days starting on the 3rd day and up to the 40th day of puerperium. IGF-I and progesterone (P4) blood levels were determined using radio immune assay (RIA). Results indicated cyclic ovarian activity outside the breeding season in 18 of 20 Boer goats. IGF-I levels in blood sera followed oestrus and cyclic ovarian activity. IGF-I concentrations correlated significantly with P4 concentrations. The IGF-I peak preceded the P4 peak by approximately 8 days in cycling goats. IGF-I and P4 blood levels were not influenced by parity or litter size. Changes in blood serum P4 and IGF-I concentrations levels during puerperal period can assist in the evaluation of reproductive status of goats.