Evaluation of Commercial Probes for On-Line Electrical Conductivity Measurements during Goat Gland Milking Process

The measurement of the milk electrical conductivity (EC) during mechanical milking has been widely studied for mastitis detection on cows because its improving of welfare and animal health, although research about small ruminants is scarce. The aim of this study was to evaluate the performance of three commercial conductimeters to be used during mechanical milking of small ruminant halves, especially Murciano-Granadina goats. The objective of this research was to integrate the probes on the milking unit and to check the suitability of the probe selected. The results presented in this research have guided authors to discard the commercial probes and to establish the requirements of a new probe design that is briefly outlined in the conclusions of this contribution

development and test of a portable device to monitor the health status of sarda breed sheep by the measurement of the milk electrical conductivity

The electrical conductivity (EC) of milk is a parameter which is often used for identifying sub-clinical mastitis in dairy animals. It is widely used for cattle, and is measured either by means of probes integrated into the milking machine or by means of portable devices. However this is not the case for small ruminants, where the available devices are few. The aim of this study is to deepen the knowledge of about the relationship between EC and certain constituents of Sarda sheep milk, and thus to develop a portable device specifically designed for on-site measurement of conductivity and to estimate the somatic cell count (SCC) of Sarda sheep milk. Therefore, the device allows a rapid test for checking the acceptability of milk to monitor the effects of udder infection. The receiver operating characteristic (ROC) method was used to evaluate how efficacious EC was in discriminating between animals with a somatic cell level higher or lower of a threshold value previously defined. The cut-off values, sensitivity, specificity and the area under the ROC curve for EC were, respectively, 4.835 mS/cm, 73.08, 75.46 and 0.804, using a threshold of 700 000 cells/ml. Our results gave a positive evaluation of the portable device that we had designed for estimating the SCC in sheep milk. Only 8.8% of the samples were incorrectly identified as negative. A portable device for EC measurement is a useful tool for monitoring the somatic cell level individually, and allows early and efficacious action to contrast new intramammary infections

Evaluation of the fourier frequency spectrum peaks of milk electrical conductivity signals as indexes to monitor the dairy goats’ health status by on-line sensors

The aim of this study is a further characterization of the electrical conductivity (EC) signal of goat milk, acquired on-line by EC sensors, to identify new indexes representative of the EC variations that can be observed during milking, when considering not healthy (NH) glands. Two foremilk gland samples from 42 Saanen goats, were collected for three consecutive weeks and for three different lactation stages (LS: 0\u201360 Days In Milking (DIM); 61\u2013120 DIM; 121\u2013180 DIM), for a total amount of 1512 samples. Bacteriological analyses and somatic cells counts (SCC) were used to define the health status of the glands. With negative bacteriological analyses and SCC 1,000,000 cells/mL, glands were classified as NH. For each milk EC signal, acquired on-line and for each gland considered, the Fourier frequency spectrum of the signal was calculated and three representative frequency peaks were identified. To evaluate data acquired a MIXED procedure was used considering the HS, LS and LS 7 HS as explanatory variables in the statistical model.Results showed that the studied frequency peaks had a significant relationship with the gland\u2019s health status. Results also explained how the milk EC signals\u2019 pattern change in case of NH glands. In fact, it is characterized by slower fluctuations (due to the lower frequencies of the peaks) and by an irregular trend (due to the higher amplitudes of all the main frequency peaks). Therefore, these frequency peaks could be used as new indexes to improve the performances of algorithms based on multivariate models which evaluate the health status of dairy goats through the use of gland milk EC sensors

Improved fuzzy logic system to evaluate milk electrical conductivity signals from on-line sensors to monitor dairy goat mastitis

The aim of this study was to develop and test a new fuzzy logic model for monitoring the udder health status (HS) of goats. The model evaluated, as input variables, the milk electrical conductivity (EC) signal, acquired on-line for each gland by a dedicated sensor, the bandwidth length and the frequency and amplitude of the first main peak of the Fourier frequency spectrum of the recorded milk EC signal. Two foremilk gland samples were collected from eight Saanen goats for six months at morning milking (lactation stages (LS): 0\u201360 Days In Milking (DIM); 61\u2013120 DIM; 121\u2013180 DIM), for a total of 5592 samples. Bacteriological analyses and somatic cell counts (SCC) were used to define the HS of the glands. With negative bacteriological analyses and SCC 1,000,000 cells/mL, glands were classified as not healthy (NH). For each EC signal, an estimated EC value was calculated and a relative deviation was obtained. Furthermore, the Fourier frequency spectrum was evaluated and bandwidth length, frequency and amplitude of the first main peak were identified. Before using these indexes as input variables of the fuzzy logic model a linear mixed-effects model was developed to evaluate the acquired data considering the HS, LS and LS 7 HS as explanatory variables. Results showed that performance of a fuzzy logic model, in the monitoring of mammary gland HS, could be improved by the use of EC indexes derived from the Fourier frequency spectra of gland milk EC signals recorded by on-line EC sensors

Design and development of a novel electronic sensor for detecting mastitis based on conductance

Mastitis, inflammation of the mammary glands of dairy cows, remains one of the costliest diseases in cattle and its early detection remains a major goal of the dairy industry. It has been established that mastitis causes changes in the conductivity of milk, by damaging the mammary membrane leading to alteration of the balance of sodium, potassium and chloride ions. Previous studies have demonstrated that these changes in milk conductivity can be used as a direct indicator o f the presence and degree of mastitis infection but currently available commercial devices do not appear to be sensitive enough to detect mastitis accurately on-line. The objective of this project is to design a novel electrochemical sensor to diagnose mastitis on-line in milking systems for cow herds. The study includes some preliminary experiments to assess the feasibility of developing such a sensor, the fabrication of the sensing system including the probe, the instrumentation and related software and the investigation of integrating the system into milking equipment. A number of primary experiments were carried out in order to investigate what parameters can be used to develop the sensor based on conductivity or impedance measurement. Milk properties such as dielectric properties, conductivity and oxidation behaviour have been investigated using various electrochemical techniques. Cyclic voltammetry was used to characterise the milk initially, electrochemical impedance spectroscopy was carried out to establish the specific electrical properties of milk and basic conductivity measurements were done to calculate the overall conductivity of milk. These methods were cross-referenced against Somatic Cell Count (SCC) measurements which were determined using a bench top instrument in Teagasc National Centre Research laboratories. Electrochemical impedance measurements showed that measurements should be taken above an operating threshold frequency to avoid polarisation effects and to improve the sensitivity o f the conductivity sensor. Above this frequency, a good correlation between Somatic Cell Count (SCC) and impedance in milk has been found

Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milk

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e BiomédicasBovine mastitis is an economic burden for farmers mostly because of decreased milk yield, premature culling and cost of veterinary treatments. The identification of mastitis pathogens is of major importance in order for adequate control measures to be taken, to reduce the risk of appearance of chronic infections, and to target antimicrobial therapy. The aim of this study was to develop and validate a sensitive method for magnetic detection of Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis in raw milk samples. Mastitic milk samples were collected aseptically from 81 cows with subclinical mastitis, from 12 Portuguese dairy farms. Ninety one quarter milk samples were selected based on bacteriological results. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analyzed using a lab-on-a-chip magnetoresistive cytometer, with microfluidics sample handling. This immunological recognition was able to detect bacterial presence above 100 cfu/ ml, depending on antibody and targeted bacteria. Comparison with PCR results showed sensitivities of 73% and 41%, specificity values of 25% and 57%, and PPV values of 35% and 54% for magnetic identification of streptococci species with an anti-S. agalactiae antibody and an anti-GB Streptococcus antibody, respectively. Regarding staphylococci species, the sensitivity values found were of 57.1% and 79.3%, specificities of 75% and 50%, and PPV values of 40% and 95.8% for magnetic identification with an anti-S. aureus antibody and an anti-Staphylococcus spp. antibody, respectively. Both bacterial genus studies translated a fair expectation for a “cow-side” use application, making this integrated platform of potential use after further improvements for fast bacteriological infection screening. Some constraints are described as well as the method´s limitations in bacterial quantification.RESUMO - Plataforma portátil “lab-on-chip” para diagnosticar mastite bovina em leite crú - A mastite bovina representa um custo económico relevante para os produtores de leite principalmente devido ao decréscimo da produção leiteira, abate prematuro e custos associados ao tratamento veterinário. Consequentemente, a identificação atempada dos agentes etiológicos é crítica para a implementação de medidas de controlo adequadas, redução do risco de infecções crónicas e aplicação de uma terapia microbiana específica. O objectivo deste estudo foi desenvolver e validar um método de detecção magnética capaz de identificar Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus e Staphylococcus epidermidis em amostras de leite crú. As amostras de leite mastítico utilizadas foram recolhidas de 81 animais com mastite subclínica, de 12 explorações leiteiras nacionais. As amostras de leite de 91 quartos de úbere foram selecionadas tendo em conta os resultados bacteriológicos. Todas as amostras foram analisadas por PCR e pelo citómetro magnetoresistivo “lab-on-chip”, tendo sido necessário neste caso, adicionar uma solução com partículas magnéticas funcionalizadas com anticorpos específicos. Este reconhecimento imunológico detectou presença bacteriana acima das 100 ufc/ml, dependendo do anticorpo e da bactéria-alvo. Comparando com os resultados da análise por PCR, este método de detecção magnética apresentou sensibilidades de 73% e 41%, valores de especificidade de 25% e 57%, e valores VPP de 35% e 54% para identificar espécies de Streptococcus com os anticorpos anti-S. agalactiae e anti-GB Streptococcus, respectivamente. No que diz respeito às espécies de Staphylococcus, os valores de sensibilidade encontrados foram de 57.1% e 79.3%, de 75% e 50% para a especificidade, e de 40% e 95.8% para VPP com os anticorpos anti-S. aureus e anti-Staphylococcus spp., respectivamente. Os dois estudos apontam para uma potencial utilização do tipo “cow-side”, tornando a plataforma integrada potencialmente utilizável para uma rápida monitorização de infecção bacteriológica, após melhorias futuras. O método desenvolvido apresenta algumas restrições e limitações relativamente à quantificação bacteriana

Rumen-Protected Methionine Supplementation Improves Lactation, Oxidative Status, and Immune Response During a Subclinical Mastitis Challenge In Lactating Dairy Cows.

Mastitis is a significant problem in the dairy industry, posing numerous challenges for dairy farmers worldwide. Mastitis refers to the inflammation of the mammary gland, primarily caused by bacterial infections. This condition affects the health and well-being of dairy cows and has severe economic implications for farmers and overall dairy production. The prevalence of mastitis substantially threatens milk quality, productivity, and profitability, making it a top concern in the dairy industry. Preventing mastitis in cows is crucial for maintaining the herd\u27s health and ensuring highquality milk production. Nutritional strategies have recently gained greater attention due to a direct impact on immune function and a large effect on mastitis prevention. This study evaluated the effects of rumen-protected methionine (RPM) during a subclinical mastitis challenge to enhance the immune system in dairy cows. Thirty-two multiparous Holstein cows were enrolled in a randomized complete block design and assigned to either a basal diet (CON; n=16) or a basal diet supplemented with rumen-protected methionine (SM; n=16, Smartamine M 0.09% DM). The dietary treatment was administered to cows at -21 days relative to a SMC, and data were collected from 0 to 3 d relative to SMC. At 0 d relative to SMC, the mammary gland\u27s rear right quarter was infused with 500,000 cfu of Streptococcus uberis (O140J). RPM did not prevent subclinical mastitis or showed lower SCC during SMC. However, it helped to maintain milk production, improved milk components and showed improvements in oxidative stress status. In addition, RPM supplementation during subclinical mastitis challenge might attenuate inflammation and enhance blood and milk immune cells protein synthesis through the mTOR pathway

Mechatronics applications and prototyping sensors for the precision livestock farming

The study is subdivided into 5 chapters and comprises a review of the main components of Plf, the development of a prototype for EC monitoring in ewe milk, a prototype for monitoring animals body temperature, the optimization of collection rounds of goat milk and the development of a prototype for somatic cell count (SCC) through the measurement of Sodium ions in ewe milk.‬‬‬ The first chapter is a review of the advancements of the main components of Plf, i.e. software, hardware and data transmission, focusing on issues related to hardware modularity and differences between licensed and unlicensed software. From the review it emerges that image processing is one of the most used techniques in Plf systems, in that it allows the detection of behavioral, biological and pathological parameters without interfering with the animals routine activities. In this regard the area occupied by a lamb carcass was calculated by using an image analysis open source software, CellProfiler (Jones et al., 2008). The second chapter deals with the realization of an innovative portable tool for somatic cells count in ewe milk by measuring its electrical conductivity. There are over 15,000 dairy sheep farms in Sardinia, which represent both historically and economically the most important agricultural and livestock sector in the island. Indeed, Sardinia holds more than 40% of the national sheep population thanks to more than 3 million sheep heads that provide about 60% of the total national milk production. One of the most common problems in sheep farms is mastitis, an intramammary infection which may cause a quantitative reduction up to 50% in milk production and a qualitative drop, in particular of lactose and casein. One of the indirect methods for the assessment of somatic cell count (SCC) in ruminants’ milk is through the measurement of its electrical conductivity (EC). In small ruminants, EC has a reasonable correlation R2 = 0.35 with somatic cells but to date there is still not a portable tool that can estimate SCC based on the milk’s EC reading. The prototype was calibrated on Sarda ewe milk. The aim of Chapter 3 was to develop a system using a open source sensors, actuators and micro-controller. The system is able to monitoring the rectal temperature of the animals, sending data via Bluetooth to a smart phone. The micro-controller used was an ATmega32U4, the temperature was read using the LM35 analogic sensor and a Class 1 Bluetooth serial module was connected to Arduino creating a wireless serial link between an Android phone and the Arduino board. The application for receiving data on an android smart phone was created using App Inventor that is an innovative Android application creation software developed by Massachusetts Institute of Technology (MIT). This app is free available on Google Play Store under the name animal_temp. The costs of sheep milk collection rounds in Sardinia have been analysed in chapter fourth. The escalating costs incurred by the dairy processing industries for milk collection from individual farms have focused the attention on the rationalization of milk collection and transport systems. In this regard, the case of the Sardinian goat sector has characteristics that make it unique and not comparable to other logistics optimization realities. The problems of this sector are mainly represented by the particular conditions of the rural road network and the fragmented nature of livestock farms. The aim of the present study was to test a milk collection route optimization software, MilkTour, in the collection rounds of a sample cheese dairy. The software has been developed by the Land Engineering Section of the Agriculture Department of the University of Sassari. A total of 5 routes were analysed and optimized. The results have highlighted the importance of optimizing collection routes as they have a significant impact on business costs. A important contribution that has emerged is the strong correlation between collection density and the cost per litre of collected milk (€cent/l), which allows to detects the cost-effectiveness of a round of collection and its relative optimized around. The objective of chapter 5 was to study the relationship between the ione Na+ and the main components of sheep milk, in particular somatic cells. Moreover, a portable device for estimating SCC in sheep milk was designed. The study was conducted on over 2000 samples. The milk components examined were: fat, proteins, lactose, pH, sodium chloride, urea and the ions Na+. The correlation between Na + and SCC corresponded to R2 = 0.76 (P <0.01). The prototype developed incorporates two containers which receives milk samples taken from each half udder. Each container has integrated inside two sensors, one to detect the level of Na+ in the milk and the other one to compensate the milk temperature. The mathematical model, loaded into the microcontroller by a firmware written in C / C ++, analyze the data and gives back the estimate of SCC level, so it allows farmers to monitor the ewes health status by periodically comparing the somatic cell counts of each half udder. While dealing with different topics the 5 chapters can be enclose in a big new topic, called Precision Livestock Farming (Plf). Plf is the discipline that allows to monitor in real-time the numerous biological and environmental parameters concerning each individual animal of the herd. A Plf system is always made up by three components: a physical element, i.e. the hardware; an element for data processing and presentation, known as the software; and an element for the transmission of data, i.e. the network. The hardware comprises the sensors, the computers and/or microcontrollers, the data transmission and acquisition systems and the actuators. Mathematical models for data processing and the data presentation interface are included in the software loaded into the microcontroller

Proteomic and metabolomic studies on milk during bovine mastitis

The principal objectives of the study presented in this thesis were to study the changes of milk proteomes, peptidomes and metabolomes during the course of bovine mastitis in comparison with normal milk samples and to discover new bovine mastitis biomarkers using various modern and up-to-date methodologies such as proteomics, peptidomics and metabolomics. Bovine mastitis caused by bacterial infection of the mammary gland of dairy cows is often associated with loss of milk production due to a reduction in milk composition and quality which in turns, lead to negative economic impact on dairy industry. Two important acute phase proteins (APPs) which serve as valuable biomarkers in bovine mastitis were investigated in every chapter using developed and validated enzyme linked immunosorbent assay (ELISA) for bovine milk haptoglobin and commercially available ELISA for bovine milk serum amyloid A3 (M-SAA3). These APPs were quantified alongside somatic cell counts (SCC) and California Mastitis Test (CMT) to confirm the disease status of each animal used in this study. Proteomic methodologies were applied including 1D gel electrophoresis, 2D gel electrophoresis, MALDI-TOF analysis and difference gel electrophoresis to investigate the changes of milk proteome in both subclinical and clinical mastitic milk samples in comparison with healthy milk samples. However these investigations did not reveal novel biomarkers for mastitis. Next, peptidomic methodologies were used to study the changes in milk peptidome and to detect the presence of any significant disease biomarkers in the presence of bovine mastitis by using CE-MS and LC-MS/MS. A total of 31 and 14 polypeptides can be used to discriminate control from infected groups and E. coli from S. aureus infected groups respectively. Lastly, metabolomic methodology was applied with an intention to study the changes in milk metabolome and ultimately to detect the presence of novel biomarkers in bovine mastitis. Di- and tri-peptides were found higher in S. aureus than in E. coli infected groups and based on metabolic pathways, arachidonic, arginine and galactose metabolites were seen increased in mastitic milk samples in comparison to healthy milk samples. Overall, the findings detailed in this thesis indicate that the use of advanced proteomic and metabolomic methodologies could deliver on their promise of the discovery of potential significant bovine mastitis biomarkers. Further studies are needed for validation of these proposed biomarkers and it was hoped that better prevention and treatment methods for bovine mastitis can be achieved in the future

Using a GIS technology to plan an agroforestry sustainable system in Sardinia

This study was conducted with the aim to quantify the spread of livestock agroforestry in a Mediterranean ecosystem (island of Sardinia, Italy) and evaluate its sustainability in terms of grazing impact. By using GIS software ArcMap 10.2.2, the map of Sardinia vegetal landscape, obtained by information of Sardinia nature map based on the classification of habitat according to CORINE-Biotopes system, have been overplayed with the map of livestock grazing impact map CAIA developed by INTREGA (spin-off ENEA), to obtain for Meriagos (local agro-silvo-pastoral systems; classified “Dehesa 84.6” according to CORINE-Biotopes system), bushlands and woodlands, the surfaces under grazing and evaluate the extension of overgrazing for each of them