All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless specified otherwise. Male Wistar rats (60 days old; ≈350 g; Charles River, Calco, Lecco, Italy) were caged in a temperature-controlled room and exposed to a daily light–dark cycle (12/12 h) with free access to standard chow diet (15.88 kJ/g) and drinking water. After a week of acclimatization, the rats were divided into two experimental groups (n = 8 each), one that kept eating only standard chow diet (CTR) and the other received, in addition to the diet, a daily intake of raw goat milk (GM, 82 kj, 21 mL/day), at an energetic dose used in previous experiments with milk-fed rats, for 4 weeks [27 (link)]. GM was obtained from a dairy farm located in the province of Foggia (Apulia region, Italy). The flock, consisting of about 200 mid-lactating Garganica goats, grazed in fenced paddocks in the morning and was housed in shaded open pens at night. The pasture composition consisted predominantly of Graminaceae, and also of Papilionaceae, Compositae, and Leguminosae, and animals were supplemented with oat hay, and oat and barley grains. Lactating goats were in parity 1–6 and were machine-milked twice daily with an average milk yield of about 780 g/day. Before milk collection, goats were examined by a veterinarian to confirm the absence of any sign of clinical mastitis. Bulk milk from the morning milking was sampled, immediately transported to the laboratory under refrigeration, and analyzed for gross composition (Milko Scan 133B; Foss Electric, Hillerød, Denmark), pH (GLP 21 Crison, Spain), and somatic cell count (Fossomatic Minor; Foss Electric, Hillerød, Denmark). Mean values were 4.3 ± 0.09%, 4.5 ± 0.07%, and 3.4 ± 0.06%, 2.5 ± 0.06% for fat, lactose, protein, and casein, respectively; 6.63 ± 0.06 for pH; and 383 ± 57 cells × 103/mL for milk somatic cells. Milk was heat-treated in a stainless-steel container, double jacketed with an internal propeller (CASARO, Philips, Netherlands) at 75 °C for 15 s and immediately cooled down in an ice bath (0 ± 2 °C) and frozen for further use. Standard rodent diet (4RF21) was purchased from Mucedola (Mucedola srl, Settimo Milanese, Milan, Italy). At the end of the treatments, the animals were anesthetized by intra-peritoneal injection of chloral hydrate (40 mg/100 g body weight), and blood was taken from the inferior cava vein. The liver and skeletal muscle were removed and immediately processed to obtain the mitochondrial fraction, and the samples not immediately used for mitochondrial preparation were stored at −80 °C. Animal experiments were carried out following the Directive 2010/63/UE, enforced by Italian D.L. 26/2014, and approved by the animal care and the Committee of the University of Naples ”Federico II” (OPBA), Naples, Italy, and the Italian Ministry of Health, Rome, Italy (authorization n. 97/2019-PR).
Milkoscan 133b
Manufactured by Foss
86 citations
Sourced in Denmark, Japan, Italy
About the product
The MilkoScan 133B is a compact, automated milk analyzer designed for the dairy industry. It provides rapid and precise analysis of various milk components, including fat, protein, lactose, and solids-non-fat, without the need for complex sample preparation. The device utilizes Fourier Transform Infrared (FTIR) technology to deliver accurate results in a timely manner.
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86 protocols using «milkoscan 133b»
1
Goat Milk Modulates Mitochondrial Function
2
Milk Yield and Composition Evaluation
Immediately after calving, the individual MY was measured daily (by TDM software version 4.1), while individual milk samples were collected two times a week for 4 weeks and analyzed for the chemical composition, urea, and caseins (Milko Scan 133B, Foss Matic, Hillerod, Denmark). Buffalo standard milk (fat- and protein-corrected milk (FPCM) = 740 kcal) was calculated by the following equation [30 ]:
Finally, the mozzarella cheese yield (MCY) was calculated by the formula of Altiero et al. [31 ]:
Finally, the mozzarella cheese yield (MCY) was calculated by the formula of Altiero et al. [31 ]:
3
Milk Yield and Fatty Acid Profile Analysis
From May to July 2021, individual milk yield was daily recorded and individual milk samples, obtained by weighting the morning and the evening milkings, were collected every 20 days (a total of five sampling). Milk chemical composition was evaluated using Milko Scan 133B (Foss Matic, Hillerod, Denmark) standardized for goat milk.
Milk FA profile was determined by extraction of total fat with a hexanopropanol and isopropanol (3/2 v/v) (Hara and Radin 1978 (link)) mixture and subsequent trans methylation (Christie 1982 (link)) modified by Chouinard et al. (1999 (link)).
The methyl esters were quantified by GC (ThermoQuest 8000TOP gas chromatograph, Thermo Electron Corporation, Rodano, Milan) with flame ionization detector and with capillary column (CP-SIL 88 fused silica capillary column, 100 m × 0.25 mm internal diameter with 0.2-μm film thickness; Varian, Inc., Walnut Creek, CA, USA) adopting the following temperature ramp:
The temperature of injector and detector were at 250 and 260 °C, respectively. Gas flows were: carrier gas (helium) 1 ml/min; hydrogen 30 ml/min; air 350 ml/min; make-up gas (helium) 45 ml/min.
FA peaks were identified by comparing with a standard mixture of fatty acid methyl esters (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö, Sweden). CLA isomers were identified by comparing samples chromatograms with those of single purified isomers (CLA cis-9, trans-11; CLA trans-10, cis-12; CLA cis-9, trans-11; CLA trans −9, trans-11) (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö, Sweden).
Milk FA profile was determined by extraction of total fat with a hexanopropanol and isopropanol (3/2 v/v) (Hara and Radin 1978 (link)) mixture and subsequent trans methylation (Christie 1982 (link)) modified by Chouinard et al. (1999 (link)).
The methyl esters were quantified by GC (ThermoQuest 8000TOP gas chromatograph, Thermo Electron Corporation, Rodano, Milan) with flame ionization detector and with capillary column (CP-SIL 88 fused silica capillary column, 100 m × 0.25 mm internal diameter with 0.2-μm film thickness; Varian, Inc., Walnut Creek, CA, USA) adopting the following temperature ramp:
The temperature of injector and detector were at 250 and 260 °C, respectively. Gas flows were: carrier gas (helium) 1 ml/min; hydrogen 30 ml/min; air 350 ml/min; make-up gas (helium) 45 ml/min.
FA peaks were identified by comparing with a standard mixture of fatty acid methyl esters (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö, Sweden). CLA isomers were identified by comparing samples chromatograms with those of single purified isomers (CLA cis-9, trans-11; CLA trans-10, cis-12; CLA cis-9, trans-11; CLA trans −9, trans-11) (Larodan Fine Chemicals, AB, Limhamnsgårdens Malmö, Sweden).
4
Goat Milk Composition and Fatty Acid Profile
On days 10 and 30 after delivery, milk samples were collected from each goat twice a day (at 7:00 am and 6:00 pm) and pooled together within each group. Milk was stored at 4 °C and immediately transported to the laboratory for the analysis of fat, protein, lactose, and total solids using an infrared milk analyzer (Milkoscan 133-B, Foss Electric, Hillerød, Denmark) previously standardized for goat’s milk. Ash content was detected after burning a sample in a muffle furnace at 550 °C for 5 h.
Total lipids were extracted according to the chloroform/methanol method described by Folch et al. [25 (link)]. FAs were methylated using a BF3-methanol solution (12% v/v) [26 ]. The FA profile was assessed by using a Chrompack CP 9000 gas chromatograph, with a silicate glass capillary column (70% cyanopropyl polysilphenylene-siloxane BPX 70 of SGE Analytical Science, length 50 m, internal diameter 0.22 mm, film thickness 0.25 μm). The temperature program was 135 °C for 7 min, followed by increases of 4 °C per minute up to 210 °C. Fatty acid peaks were identified using a comparative analysis with standard reference mixtures. The fatty acid content was expressed as the percentage of total fatty acid methyl esters (FAME).
Total lipids were extracted according to the chloroform/methanol method described by Folch et al. [25 (link)]. FAs were methylated using a BF3-methanol solution (12% v/v) [26 ]. The FA profile was assessed by using a Chrompack CP 9000 gas chromatograph, with a silicate glass capillary column (70% cyanopropyl polysilphenylene-siloxane BPX 70 of SGE Analytical Science, length 50 m, internal diameter 0.22 mm, film thickness 0.25 μm). The temperature program was 135 °C for 7 min, followed by increases of 4 °C per minute up to 210 °C. Fatty acid peaks were identified using a comparative analysis with standard reference mixtures. The fatty acid content was expressed as the percentage of total fatty acid methyl esters (FAME).
Corresponding organizations : University of Bari Aldo Moro, University of Basilicata
5
Comprehensive Dairy Cow Lactation Assessment
During lactation, cows were milked twice a day at the stand (0600 and 1600 h), and milk yield was weighed after each milking. Starting from 120 DRC, milk yield was recorded monthly on milk test days. Milk samples were collected twice weekly for the first 120 DRC (every Monday and Thursday) at the morning milking, kept in a water bath at 28°C, and analyzed within 2 h for fat, protein, and lactose contents using a mid-infrared analyzer (Milko Scan 133B, Foss Electric), SCC using an automated counter (Fossomatic 180, Foss Electric), pH with a combined electrode, and titratable acidity measuring the milliliters of NaOH (0.25 N) necessary to maintain the pH of 50 mL of milk at 8.65 using an automatic titrator (Micro TT 2050; Crison). Fat and protein output were calculated by multiplying the relative content by the milk yield. The SCC was log 10 transformed and reported as geometric mean. Urea was determined on skim milk using a spectrometric assay (IL Test Urea Nitrogen kit; Werfen) using the ILAB-600 clinical auto-analyzer (Werfen).
Ten milliliters of milk was used to measure coagulation properties (McMahon and Brown, 1982) (link) after adding 0.2 mL of rennet [Chr. Hansen A/S, 1:15,000 in a 1.5% (vol/vol) solution in distilled water] and measuring the thromboelastogram at 35°C for a total time of 30 min using the Formagraph (Foss Electric). The lag time (min) before the beginning of coagulation was defined as the rennet clotting time (RCT), the time (min) necessary to reach 20 mm of width of the thrombus was defined as curd firming rate (k 20 ), and the width of the thrombus after 30 min (mm) was defined as curd firmness (a 30 ). 7, 21, 35, 49, 63, 90 , and 120 DRC immediately before the delivery of morning concentrate (0700 h), blood samples were harvested from the jugular vein. For the metabolic profile assessment, samples were collected into 10-mL heparinized vacuum tubes (Vacutainer, Becton Dickinson) and placed on ice until centrifugation. Within 1 h of collection, a small amount of blood was used for the determination of packed cell volume (Centrifugette 4203, ALC International Srl), and the remainder was centrifuged at 3,500 × g for 16 min at 4°C. Aliquots of the plasma obtained were frozen at -20°C until further analysis. Blood metabolites were analyzed at 37°C using a clinical auto-analyzer (ILAB 600; Werfen). Commercial kits were used to determine plasma concentrations of glucose, urea, triglycerides, Mg, Ca, P, total protein, albumin, cholesterol, bilirubin, aspartate aminotransferase-glutamate oxaloacetate transaminase (GOT), γ-glutamyltransferase (GGT), and creatinine (Werfen), nonesterified fatty acids (NEFA), Zn (Wako Chemicals GmbH), and BHB (Randox Laboratories Ltd.); Na, K, and Cl were measured using a potentiometer method (ion selective electrode). Ceruloplasmin was determined following the method proposed by Sunderman and Nomoto (1970) (link) and haptoglobin as proposed by Skinner et al. (1991) (link). Globulin was calculated as the difference between total protein and albumin and albumin-to-globulin ratio as the ratio between albumin and globulin. Methods and kit catalog numbers for each biomarker are reported in Supplemental Table S2 (https: / / doi .org/ 10 .6084/ m9 .figshare .21679712 .v2; Cattaneo et al., 2023) (link).
Ten milliliters of milk was used to measure coagulation properties (McMahon and Brown, 1982) (link) after adding 0.2 mL of rennet [Chr. Hansen A/S, 1:15,000 in a 1.5% (vol/vol) solution in distilled water] and measuring the thromboelastogram at 35°C for a total time of 30 min using the Formagraph (Foss Electric). The lag time (min) before the beginning of coagulation was defined as the rennet clotting time (RCT), the time (min) necessary to reach 20 mm of width of the thrombus was defined as curd firming rate (k 20 ), and the width of the thrombus after 30 min (mm) was defined as curd firmness (a 30 ). 7, 21, 35, 49, 63, 90 , and 120 DRC immediately before the delivery of morning concentrate (0700 h), blood samples were harvested from the jugular vein. For the metabolic profile assessment, samples were collected into 10-mL heparinized vacuum tubes (Vacutainer, Becton Dickinson) and placed on ice until centrifugation. Within 1 h of collection, a small amount of blood was used for the determination of packed cell volume (Centrifugette 4203, ALC International Srl), and the remainder was centrifuged at 3,500 × g for 16 min at 4°C. Aliquots of the plasma obtained were frozen at -20°C until further analysis. Blood metabolites were analyzed at 37°C using a clinical auto-analyzer (ILAB 600; Werfen). Commercial kits were used to determine plasma concentrations of glucose, urea, triglycerides, Mg, Ca, P, total protein, albumin, cholesterol, bilirubin, aspartate aminotransferase-glutamate oxaloacetate transaminase (GOT), γ-glutamyltransferase (GGT), and creatinine (Werfen), nonesterified fatty acids (NEFA), Zn (Wako Chemicals GmbH), and BHB (Randox Laboratories Ltd.); Na, K, and Cl were measured using a potentiometer method (ion selective electrode). Ceruloplasmin was determined following the method proposed by Sunderman and Nomoto (1970) (link) and haptoglobin as proposed by Skinner et al. (1991) (link). Globulin was calculated as the difference between total protein and albumin and albumin-to-globulin ratio as the ratio between albumin and globulin. Methods and kit catalog numbers for each biomarker are reported in Supplemental Table S2 (https: / / doi .org/ 10 .6084/ m9 .figshare .21679712 .v2; Cattaneo et al., 2023) (link).
Corresponding organizations : Università Cattolica del Sacro Cuore
Top 5 most cited protocols using «milkoscan 133b»
1
Milk Composition and Stearoyl-CoA Desaturase Analysis
Milk was completely suckled by kids up to day 60; therefore, yield was recorded every day and representative samples (weighted average of the two daily milking), were analyzed monthly, from April to August, for protein and fat concentration by Milkoscan 133B (Foss Matic, Hillerod, Denmark). Total fat was obtained by a mixture of hexane/isopropane (3/2 v/v) [23 (link)].
The FA profile was determined using the same procedures for feeds with additional standards for CLA isomers (Larodan Fine Chemicals, AB, Limhamnsgardens Malmo, Sweden).
According to Lock and Garnsworthy [24 (link)] the C14:1/C14:0 ratio was used to represent the SCD activity index.
Milk samples were centrifuged in order to obtain the milk somatic cell pellet. The total RNA extraction and the SCD gene expression were determined as described by Tudisco et al. [25 (link)]. Complementary DNA (cDNA) samples obtained by Quantiscript Reverse transcriptase (Qiagen) were amplified using RT-PCR (ABI Prism 7300 System, Applied Biosystems, Foster City, California, USA). The threshold cycle numbers (Ct) of the SCD mRNA were normalized using the mean Ct of housekeeping genes following the formula: 2−(CTSCD− CThousekeeping).
The expression of miRNA 103 was evaluated in whole milk, briefly, a 200 µL/sample of milk was purified using mirVana miRNA isolation kit (Ambion, USA) following the manufacturer’s instructions. The quantity and quality of purified total RNA were determined as described by Tudisco et al. [25 (link)] for SCD expression. Total RNAs were stored at −80 °C for further use. For amplification of miRNA, DNase I-digested total RNA was polyadenylated and reverse transcribed using a Mir-X™ miRNA First Strand Synthesis Kit (Clontech Laboratories, Inc., CA, USA) to prepare cDNAs, and subjected to quantitative real-time PCR using a SYBR Advantage qPCR Premix (Clontech Laboratories, Inc., CA, USA) with the provided miRNA reference gene (U6). The qRT-PCR analysis was performed in triplicate on ABI Prism 7300 System (AppliedBiosystems, Foster City, California, USA). The 25 μL PCR contained 2.0 μL of the RT product (template), 12.5 μL 2× SYBR Advantage Premix, 9 μL ddH2O, 0.5 μL 50× ROX Dye, 0.5 μL miRNA-specific primer (10 μM) and 0.5 μL mRQ 3′primer. The default thermal profile used for PCR amplification consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 60 s. The same conditions were performed on an equal amount of RNase-free water as a negative control. The miR-103-specific primer (3’-AGCAGCATTGTACAGGGCTA) was synthesized by Eurofins Genomics (Eurofins Genomics S.r.l., Milano, Italy). Ct values determined for each sample were normalized against the values for U6. The relative fold change in expression to U6 was calculated using 2−(Ct103miRNA− CtU6).
The FA profile was determined using the same procedures for feeds with additional standards for CLA isomers (Larodan Fine Chemicals, AB, Limhamnsgardens Malmo, Sweden).
According to Lock and Garnsworthy [24 (link)] the C14:1/C14:0 ratio was used to represent the SCD activity index.
Milk samples were centrifuged in order to obtain the milk somatic cell pellet. The total RNA extraction and the SCD gene expression were determined as described by Tudisco et al. [25 (link)]. Complementary DNA (cDNA) samples obtained by Quantiscript Reverse transcriptase (Qiagen) were amplified using RT-PCR (ABI Prism 7300 System, Applied Biosystems, Foster City, California, USA). The threshold cycle numbers (Ct) of the SCD mRNA were normalized using the mean Ct of housekeeping genes following the formula: 2−(CTSCD− CThousekeeping).
The expression of miRNA 103 was evaluated in whole milk, briefly, a 200 µL/sample of milk was purified using mirVana miRNA isolation kit (Ambion, USA) following the manufacturer’s instructions. The quantity and quality of purified total RNA were determined as described by Tudisco et al. [25 (link)] for SCD expression. Total RNAs were stored at −80 °C for further use. For amplification of miRNA, DNase I-digested total RNA was polyadenylated and reverse transcribed using a Mir-X™ miRNA First Strand Synthesis Kit (Clontech Laboratories, Inc., CA, USA) to prepare cDNAs, and subjected to quantitative real-time PCR using a SYBR Advantage qPCR Premix (Clontech Laboratories, Inc., CA, USA) with the provided miRNA reference gene (U6). The qRT-PCR analysis was performed in triplicate on ABI Prism 7300 System (AppliedBiosystems, Foster City, California, USA). The 25 μL PCR contained 2.0 μL of the RT product (template), 12.5 μL 2× SYBR Advantage Premix, 9 μL ddH2O, 0.5 μL 50× ROX Dye, 0.5 μL miRNA-specific primer (10 μM) and 0.5 μL mRQ 3′primer. The default thermal profile used for PCR amplification consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 60 s. The same conditions were performed on an equal amount of RNase-free water as a negative control. The miR-103-specific primer (3’-AGCAGCATTGTACAGGGCTA) was synthesized by Eurofins Genomics (Eurofins Genomics S.r.l., Milano, Italy). Ct values determined for each sample were normalized against the values for U6. The relative fold change in expression to U6 was calculated using 2−(Ct103miRNA− CtU6).
Corresponding organizations : University of Naples Federico II, Magna Graecia University, University of Sassari
2
Dairy Cow Nutrient Utilization Assessment
Feed refusals and feces were collected daily at 10:30 h before feeding for 5 days (from day 9 to day 13 of each experimental period) and were pooled for analysis. Samples of feed, refusals, and feces were dried at 60°C in a forced air oven and ground through a 1-mm screen in a mill before analysis. The DM, CP, CF, ether extract, and ash of feed, refusals, and feces were determined using a standard procedure (AOAC, 1990) . The NDF and ADF of feed were analyzed according to the procedure of Van Soest et al. (1991) . The cows were milked twice daily at 08:30 and 18:00 h and the milk yield was recorded. Sample collected at each milking were analyzed for fat, protein, lactose and solids-not-fat (SNF) contents using a Foss Milko-Scan 133B (N. Foss Electric A/S, Hillerød, Denmark). Urine was collected daily at the 10:30 h for 5 days (from day 9 to day 13) and was pooled for analysis. The gross energy of all samples was measured using an adiabatic bomb calorimeter (CA-4P; Shimadzu Corp., Kyoto, Japan). Milk and urine samples were placed in polyethylene bags, freeze-dried, and then burnt using calorimeter (Itoh and Tano, 1977) . Body weight was measured at 10:30 h on days 0 and 13. Rectal temperature was measured daily at 8:30 hr.
At the end of the each experimental period, blood samples were taken from the jugular vein using a heparinized tube. Plasma was separated by centrifugation at 1,500 g for 15 min and stored at -20°C until analysis. The TP, albumin, and urea nitrogen in plasma were measured using an automatic analyzer (7080 Clinical Analyzer; Hitachi Ltd., Tokyo, Japan). The 3MH in plasma was measured using HPLC method after converting the fluorescamine derivative in the acidic condition at 80°C for 1 h (Wassner et al., 1980) . The HPLC system (Hitachi Ltd, Tokyo, Japan) was consisted of a pump (model L-7100), syringe-loading sample injector containing a 5-µl loop, a column oven (model L-7300) and a fluorescence detector (model L-7480). The analytical reversed-phase column was a LiChrospher 100 RP-18 (e) (particle size 5 µm, 250×4 mm i.d.) protected by guard column (10×4 mm i.d.) containing with the same material (both obtained from Kanto Chemical, Co., Inc., Tokyo, Japan). The column temperature was maintained at 40°C. The isocratic mobile phase was composed of 24% acetonitrile/76% 10 mM sodium phosphate buffer (pH 6.5, vol/vol). The flow rate was 1.2 ml/min. Detection was performed at 360 nm (excitation wavelength) and 485 nm (emission wavelength).
At the end of the each experimental period, blood samples were taken from the jugular vein using a heparinized tube. Plasma was separated by centrifugation at 1,500 g for 15 min and stored at -20°C until analysis. The TP, albumin, and urea nitrogen in plasma were measured using an automatic analyzer (7080 Clinical Analyzer; Hitachi Ltd., Tokyo, Japan). The 3MH in plasma was measured using HPLC method after converting the fluorescamine derivative in the acidic condition at 80°C for 1 h (Wassner et al., 1980) . The HPLC system (Hitachi Ltd, Tokyo, Japan) was consisted of a pump (model L-7100), syringe-loading sample injector containing a 5-µl loop, a column oven (model L-7300) and a fluorescence detector (model L-7480). The analytical reversed-phase column was a LiChrospher 100 RP-18 (e) (particle size 5 µm, 250×4 mm i.d.) protected by guard column (10×4 mm i.d.) containing with the same material (both obtained from Kanto Chemical, Co., Inc., Tokyo, Japan). The column temperature was maintained at 40°C. The isocratic mobile phase was composed of 24% acetonitrile/76% 10 mM sodium phosphate buffer (pH 6.5, vol/vol). The flow rate was 1.2 ml/min. Detection was performed at 360 nm (excitation wavelength) and 485 nm (emission wavelength).
Corresponding organizations : Kyushu Okinawa Agricultural Research Center, Institute of Livestock and Grassland Science
3
Impact of Negative Energy Balance on Milk Fatty Acids
A total of 25 Holstein cows (9 primiparous, 9 in the second, and 7 in the third and subsequent parity) calved within one month were included in the study. Therefore, breed, year as well as seasonal effects were excluded. The average daily milk yield ranged from 11.34 l to 47.2 l of milk with an average of 28.60 l. All cows selected for observations were without reproduction and health disorders in previous lactations. Body condition was evaluated monthly in relation to the habit practiced by breeders. A body condition index (BCS; a 5-point scale with 0.25 point increments) was used for body condition evaluation (Ferguson et al. 1994) . BCS ranged from 1.5 to 3.75 points with an average of 2.69. The cows were loose housed in a straw-bedded cubicle barn. All the animals were fed a total mixed ration (TMR) consisting of maize silage, lucerne silage, straw, grass hay, lucerne hay, concentrates, brewery draff, bakery waste, and mineral supplements. The ingredient composition of the diet corresponded to the current daily milk yield of individual cows, and feed rations were completely balanced for energy, protein and fat as well as mineral and vitamin content. Feed rations consisted of the same ingredients throughout the entire experimental period.
Sample collection and analyses. Sampling of each dairy cow's milk started on the 7 th day after calving. Milk samples (n = 425) were collected at 7-day periods during the first 17 weeks of lactation. Two aliquot milk samples were collected from each cow in accordance with the milk recording system on every sampling day. The first sample with a preservative was heated to 39 ± 1°C and applied for fat (F) and protein (P) percentage content determination using Milkoscan 133B (N. Foss Electric, Hillerød, Denmark). Subsequently the fat to protein ratio (FPR) was computed. The second sample, without a preservative, was used for the fat extraction and fatty acid (FA) content determination in accordance with the methodology described by Ducháček et al. (2012c) . The content (mg/100 g) of individual FA (28) and six FA groups (SFA, its parts HCFA and VFA; UFA, its parts MUFA and PUFA) was investigated. Subsequently, the proportions (%) of the six FA groups observed in milk fat were determined and evaluated. The HCFA group included lauric, myristic and palmitic FA in accordance with Kontkanen et al. (2011) . The VFA group was represented by the content of butyric, caproic, caprylic, and capric FA in accordance with the study by Pešek et al. (2006) .
Statistical analysis. The data were evaluated by the SAS 9.3 statistical software (SAS/STAT ® 9.3, 2011) using UNIVARIATE, CORR, and MIXED procedures. The best model for evaluation was selected in accordance with the values of the Akaike information criterion (AIC). A model including the effects of parity, negative energy balance, and regression on lactation week corresponding with milk yield recorded as well as on milk FPR both specifying the energy status of dairy cow (Moallem et al. 2007 ) was designed for evaluation of FA group (SFA, HCFA, VFA, UFA, MUFA, PUFA) proportions in the total milk fat content. The main effect of NEB was represented by two levels (YES -within the NEB period, NO -NEB period overcome) expressed according to the individual BCS changes and additionally by the course of current milk yield as well as milk FPR values during weeks of observation used as a regression. Cows with BCS decline were considered within the NEB period, while those with balanced or increased BCS were labelled as NEB overcome. The length of average NEB was 10.12 weeks, when average BCS continually declined from 3.13 points in calving to 2.52 points in the 12 th week. Subsequently, BCS slowly, however continuously increased to 2.55 points in the 16 th week post partum. The NEB course was specified on the level of individual dairy cows using their current milk yield and corresponding FPR during three weeks before balancing/increasing BCS. In accordance with Duffield et al. (1997) and Vacek et al. (2011) , the threshold of FPR on the level 1.3 was taken as a criterion for within NEB (> 1.3) and/ or NEB overcome (< 1.3). Regressions on lactation week and milk FPR applied within the model clarified determination of the NEB breakpoint because of the monthly period of BCS evaluation. The Tukey-Kramer method was used for evaluation of differences in the least squares means. The model equation used for the evaluation was as follows:
where: Y ijk -dependent variable (SFA, HCFA, VFA, UFA, MUFA, PUFA in %); µ -mean value of dependent variable; PAR i -fixed effect of i th number of lactation (i = 1 st lactation, n = 153; 2 nd lactation, n = 153; 3 rd and subsequent lactations, n = 119); NEB j -fixed effect of j th NEB period occurrence (j = YES -within NEB period, n = 242; NO -NEB period overcome, n = 183); b 1 *(WEEK) -regression on the lactation week order; b 2 *(FPR) = regression on the fat to protein ratio in milk sample; e ijk -random error Significance levels P < 0.05, P < 0.01, and P < 0.001 were used to evaluate the differences between groups.
Sample collection and analyses. Sampling of each dairy cow's milk started on the 7 th day after calving. Milk samples (n = 425) were collected at 7-day periods during the first 17 weeks of lactation. Two aliquot milk samples were collected from each cow in accordance with the milk recording system on every sampling day. The first sample with a preservative was heated to 39 ± 1°C and applied for fat (F) and protein (P) percentage content determination using Milkoscan 133B (N. Foss Electric, Hillerød, Denmark). Subsequently the fat to protein ratio (FPR) was computed. The second sample, without a preservative, was used for the fat extraction and fatty acid (FA) content determination in accordance with the methodology described by Ducháček et al. (2012c) . The content (mg/100 g) of individual FA (28) and six FA groups (SFA, its parts HCFA and VFA; UFA, its parts MUFA and PUFA) was investigated. Subsequently, the proportions (%) of the six FA groups observed in milk fat were determined and evaluated. The HCFA group included lauric, myristic and palmitic FA in accordance with Kontkanen et al. (2011) . The VFA group was represented by the content of butyric, caproic, caprylic, and capric FA in accordance with the study by Pešek et al. (2006) .
Statistical analysis. The data were evaluated by the SAS 9.3 statistical software (SAS/STAT ® 9.3, 2011) using UNIVARIATE, CORR, and MIXED procedures. The best model for evaluation was selected in accordance with the values of the Akaike information criterion (AIC). A model including the effects of parity, negative energy balance, and regression on lactation week corresponding with milk yield recorded as well as on milk FPR both specifying the energy status of dairy cow (Moallem et al. 2007 ) was designed for evaluation of FA group (SFA, HCFA, VFA, UFA, MUFA, PUFA) proportions in the total milk fat content. The main effect of NEB was represented by two levels (YES -within the NEB period, NO -NEB period overcome) expressed according to the individual BCS changes and additionally by the course of current milk yield as well as milk FPR values during weeks of observation used as a regression. Cows with BCS decline were considered within the NEB period, while those with balanced or increased BCS were labelled as NEB overcome. The length of average NEB was 10.12 weeks, when average BCS continually declined from 3.13 points in calving to 2.52 points in the 12 th week. Subsequently, BCS slowly, however continuously increased to 2.55 points in the 16 th week post partum. The NEB course was specified on the level of individual dairy cows using their current milk yield and corresponding FPR during three weeks before balancing/increasing BCS. In accordance with Duffield et al. (1997) and Vacek et al. (2011) , the threshold of FPR on the level 1.3 was taken as a criterion for within NEB (> 1.3) and/ or NEB overcome (< 1.3). Regressions on lactation week and milk FPR applied within the model clarified determination of the NEB breakpoint because of the monthly period of BCS evaluation. The Tukey-Kramer method was used for evaluation of differences in the least squares means. The model equation used for the evaluation was as follows:
where: Y ijk -dependent variable (SFA, HCFA, VFA, UFA, MUFA, PUFA in %); µ -mean value of dependent variable; PAR i -fixed effect of i th number of lactation (i = 1 st lactation, n = 153; 2 nd lactation, n = 153; 3 rd and subsequent lactations, n = 119); NEB j -fixed effect of j th NEB period occurrence (j = YES -within NEB period, n = 242; NO -NEB period overcome, n = 183); b 1 *(WEEK) -regression on the lactation week order; b 2 *(FPR) = regression on the fat to protein ratio in milk sample; e ijk -random error Significance levels P < 0.05, P < 0.01, and P < 0.001 were used to evaluate the differences between groups.
Corresponding organizations : Czech University of Life Sciences Prague
4
Milk Composition and Quality Analysis
Milk samples were analysed for pH and total protein, fat, lactose and urea content using an I.R. spectrophotometer (Milko-Scan 133B Foss Electric®, DK-3400 Hillerød, Denmark) according to the International Dairy Federation (IDF) standard (IDF 141C:2000) ; somatic cell count (SCC) using an automatic cell counter (Fossomatic 90, Foss Electric®) according to IDF 148A:1995; total mesophilic count (TMC) using a Bacto-Scan, Foss Electric® (IDF 358:2000) ; freezing point (FP) in Hortvet degrees (H°) by a thermistore cryoscope (IDF 108:2002) .
Corresponding organizations : University of Sassari
5
Comprehensive Milk Quality Assessment Protocol
The analytical procedures were described in the previous studies of Hanuš et al. (2007) (link) or Janů et al. (2007) (link). Briefly, composition and physical-chemical and technological properties of milk were carried out according to the following methods:
-Fat, lactose and solids-not-fat were measured using MilkoScan 133B (Foss Electric, Denmark).
-Crude protein (CP), true protein (TP), and casein (Cas) were determined by reference Kjeldahl's method using the instrument line Tecator with a Kjeltec 2200 autodistillation unit (Foss-Tecator AB, Sweden) according to ČSN 57 0530 (1973) .
-Somatic cell count (SCC) was determined using Fossomatic 90 instrument (Foss Electric, Denmark) according to ČSN EN ISO 13366-3 (1998) .
-Urea was determined using an Ureakvant apparatus (Agroslužby Olomouc, Czech Republic) based on specific enzymatic and conductometric method.
-Acetone (Ac) was determined by spectrophotometry at 485 nm wavelength using a Spekol 11 instrument (Carl Zeiss Jena, Germany).
-Electric conductivity was measured using an OK 102/1 (Radelkis, Hungary) conductometer at 20 • C (in mS cm -1 ).
-Active acidity (pH) was measured using a CyberScan 510 pH meter (Eutech Instruments) at 20 • C.
-Titration acidity was measured according to the standard ČSN 57 0530 (1973) .
-Milk alcohol stability was determined with the help of milk titration (5 mL) by 96 % ethanol for the creation of the first visible milk protein precipitated flakes.
-Rennet coagulation time -time for enzymatic coagulation from the addition of rennilase (microbial enzyme) to milk to the beginning of coagulation (s).
-Curd quality -subjective estimation of curd cake quality determined by inspection and touch from first (excellent) to fourth (poor) class.
-Cheese curd firmness -depth of penetration of the corpuscle falling into curd cake in a standard way (mm; the higher the firmness, the fewer millimetres).
-Whey volume -obtained during the process of enzymatic cheese making from curd cake for 60 min (mL). The following milk indicators were calculated: whey protein, non-protein nitrogen (NPN), fat to crude protein ratio (fat / CP), urea N in NPN ratio, casein number on crude protein basis (Cas-CP), and casein number on true protein basis (Cas-TP).
The feeding rations consisted of roughage (see Table 1) and supplements of concentrate mixture according to milk yield and standard demands. Samples of feedstuffs were collected at the same time as the IMSs. Commercially available quantitative enzyme-linked immunosorbent assay (ELISA) kits (Veratox, Neogen Corp., Lansing, MI, USA) were used for measuring the presence of the mycotoxins deoxynivalenol (DON), fumonisins (FUM), zearalenone (ZEA), aflatoxin (AFL), and T-2 toxin according to the manufacturer's instructions. Based on the obtained results for each analysed feedstuff, a class of mycotoxin load was calculated as a mean from classes assigned to individual mycotoxins (see Table 2). Four classes were created under the correlation of mycotoxin amount with recommendation limits. Class 1 was represented by samples with no mycotoxins, in class 2 there were samples under the concentration limit, and in class 4 there were samples with more than 3 times the concentration limit. Finally, the load of herds was classified as negligible (Load 1: mean class 1), low (Load 2: mean class 2), and medium (Load 3: mean class 3).
-Fat, lactose and solids-not-fat were measured using MilkoScan 133B (Foss Electric, Denmark).
-Crude protein (CP), true protein (TP), and casein (Cas) were determined by reference Kjeldahl's method using the instrument line Tecator with a Kjeltec 2200 autodistillation unit (Foss-Tecator AB, Sweden) according to ČSN 57 0530 (1973) .
-Somatic cell count (SCC) was determined using Fossomatic 90 instrument (Foss Electric, Denmark) according to ČSN EN ISO 13366-3 (1998) .
-Urea was determined using an Ureakvant apparatus (Agroslužby Olomouc, Czech Republic) based on specific enzymatic and conductometric method.
-Acetone (Ac) was determined by spectrophotometry at 485 nm wavelength using a Spekol 11 instrument (Carl Zeiss Jena, Germany).
-Electric conductivity was measured using an OK 102/1 (Radelkis, Hungary) conductometer at 20 • C (in mS cm -1 ).
-Active acidity (pH) was measured using a CyberScan 510 pH meter (Eutech Instruments) at 20 • C.
-Titration acidity was measured according to the standard ČSN 57 0530 (1973) .
-Milk alcohol stability was determined with the help of milk titration (5 mL) by 96 % ethanol for the creation of the first visible milk protein precipitated flakes.
-Rennet coagulation time -time for enzymatic coagulation from the addition of rennilase (microbial enzyme) to milk to the beginning of coagulation (s).
-Curd quality -subjective estimation of curd cake quality determined by inspection and touch from first (excellent) to fourth (poor) class.
-Cheese curd firmness -depth of penetration of the corpuscle falling into curd cake in a standard way (mm; the higher the firmness, the fewer millimetres).
-Whey volume -obtained during the process of enzymatic cheese making from curd cake for 60 min (mL). The following milk indicators were calculated: whey protein, non-protein nitrogen (NPN), fat to crude protein ratio (fat / CP), urea N in NPN ratio, casein number on crude protein basis (Cas-CP), and casein number on true protein basis (Cas-TP).
The feeding rations consisted of roughage (see Table 1) and supplements of concentrate mixture according to milk yield and standard demands. Samples of feedstuffs were collected at the same time as the IMSs. Commercially available quantitative enzyme-linked immunosorbent assay (ELISA) kits (Veratox, Neogen Corp., Lansing, MI, USA) were used for measuring the presence of the mycotoxins deoxynivalenol (DON), fumonisins (FUM), zearalenone (ZEA), aflatoxin (AFL), and T-2 toxin according to the manufacturer's instructions. Based on the obtained results for each analysed feedstuff, a class of mycotoxin load was calculated as a mean from classes assigned to individual mycotoxins (see Table 2). Four classes were created under the correlation of mycotoxin amount with recommendation limits. Class 1 was represented by samples with no mycotoxins, in class 2 there were samples under the concentration limit, and in class 4 there were samples with more than 3 times the concentration limit. Finally, the load of herds was classified as negligible (Load 1: mean class 1), low (Load 2: mean class 2), and medium (Load 3: mean class 3).
Corresponding organizations : University of Veterinary Sciences Brno, Mendel University in Brno
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