The imprinted insulin-like growth factor 2 gene (IGF2) encodes a fetal mitogenic hormone protein (IGF-II) and has previously been shown to be associated with performance in dairy cattle. In this study we assessed genotype-phenotype associations between four single nucleotide polymorphisms (SNPs) located within the bovine IGF2 locus on chromosome 29 and a range of performance traits related to milk production, animal growth and body size, fertility and progeny survival in 848 progeny-tested Irish Holstein-Friesian sires. Two of the four SNPs (rs42196909 and IGF2.g-3815A>G), which were in strong linkage disequilibrium (r2=0·995), were associated with milk yield (P⩽0·01) and milk protein yield (P⩽0·05); the rs42196901 SNP was also associated (P⩽0·05) with milk fat yield. Associations (P⩽0·05) with milk fat percentage and milk protein percentage were observed at the rs42196901 and IGF2.g-3815A>G SNPs, respectively. The rs42196909 and IGF2.g-3815A>G SNPs were also associated with progeny carcass conformation (P⩽0·05), while an association (P⩽0·01) with progeny carcass weight was observed at the rs42194733 SNP locus. None of the four SNPs were associated with body size, fertility and progeny survival. These findings support previous work which suggests that the IGF2 locus is an important biological regulator of milk production in dairy cattle and add to an accumulating body of research showing that imprinted genes influence many complex performance traits in cattle.

Although Corynebacterium bovis and coagulase-negative staphylococci are frequently the most commonly isolated bacteria from milk samples submitted for identification of pathogens causing intramammary infection, the individual quarter somatic cell count (SCC) from those samples is most often low. The present study aimed at evaluating the difference in bacteriology results from milk sampled by the standard technique (as recommended by the National Mastitis Council) and by the use of a teat cannula surpassing the teat canal, since C. bovis is often only found in the teat canal. Single quarter milk samples were collected in duplicate from 132 dairy cows on a commercial dairy farm using the standard milk sampling technique and also using a cannula introduced into the teat. Two groups of quarters were sampled: a group that was selected randomly at cow and quarter level and a group that was selected based on having SCC >200 000 cells/ml at the previous milk recording at cow level and on California mastitis test result at quarter level. Bacteriological culture performed on the samples yielded 29 Corynebacterium spp. isolates from the samples collected with the standard technique and 6 isolates from the samples collected with a cannula. Bacteriological culture yielded 73 and 100 culture negative samples respectively with the standard and the alternative sampling technique. A significant difference between the two sampling techniques was observed for recovery of Corynebacterium spp. and for no-growth samples. There was no significant difference in the isolation of Corynebacterium spp. or other bacterial species when using the standard technique before or after sampling with the cannula; thus the observed difference in bacteriology results could not be attributed to a particular sampling order. No significant change was observed overall in individual quarter SCC measured on the sampling day and 7 d later. Our results agree with several studies showing that Corynebacterium bovis often colonizes the teat canal, without causing true intramammary infection.

The effect of freeze-drying on viability and probiotic properties of a microbial mixture containing selected bacterial and yeast strains isolated from kefir grains (Lactobacillus kefir, Lactobacillus plantarum, Lactococcus lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) was studied. The microorganisms were selected according to their potentially probiotic properties in vitro already reported. Two types of formulations were performed, a microbial mixture (MM) suspended in milk and a milk product fermented with MM (FMM). To test the effect of storage on viability of microorganisms, MM and FMM were freeze-dried and maintained at 4°C for six months. After 180 days of storage at 4°C, freeze-dried MM showed better survival rates for each strain than freeze-dried FMM. The addition of sugars (trehalose or sucrose) did not improve the survival rates of any of the microorganisms after freeze-drying. Freeze-drying did not affect the capacity of MM to inhibit growth of Shigella sonnei in vitro, since the co-incubation of this pathogen with freeze-dried MM produced a decrease of 2 log in Shigella viability. The safety of freeze-dried MM was tested in mice and non-translocation of microorganisms to liver or spleen was observed in BALB/c mice feed ad libitum during 7 or 20 days. To our knowledge, this is the first report about the effect of freeze-drying on viability, in vitro probiotic properties and microbial translocation of a mixture containing different strains of both bacteria and yeasts isolated from kefir.

An investigation of the shape of the lactation curve and the mastitis incidence was conducted to identify whether management interventions of the lactation curve constitute a potential for reducing incidence of mastitis at herd level. Lactation curves were estimated to describe the variation of daily milk yield during the 305-d lactation period in Norwegian Red cows. Associations between mastitis incidence at herd level and lactation curve characteristics such as production level at onset of lactation, magnitude and time of peak milk yield, and increase and decrease of milk yield rates were studied. Data from 250 303 lactations occurring during 2005 and 2006 from 14 766 herds were obtained from the Norwegian Dairy Herd Recording System. Besides veterinary treatments, the records included information on monthly test-day milk yields. The shapes of the lactation curves at herd level were parameterized using a modified Wilmink model in two separate mixed model analyses. In the first analysis a subset of lactations with no records of veterinary treatments was used. Lactation curves from herds with high (>0·31 cases/305-d lactation) and low (<0·07 cases/305-d lactation) herd mastitis incidence rate were parameterized and compared for three separate strata of parity. The result showed that high herd mastitis incidence rate was associated with a low intercept (P<0·05), a steep slope before peak milk yield (P<0·01) and a rapid decline after peak milk yield (P<0·01). In the second analysis a subset of high-yielding lactations with veterinary treatments of mastitis only and lactations with no records of veterinary treatment were compared. This was done to investigate whether the findings at herd level were also reflected at cow level. These results showed that lactation curves from lactations with mastitis cases were associated with a steep slope before peak milk yield (P<0·05) in second and later parities and a rapid decline after peak milk yield (P<0·01) in all three parity groups.

The effects of the caprine αs1-casein (CSN1S1) polymorphisms on milk quality and cheese yield have been widely studied in French and Italian goat breeds. Much less is known about the consequences of κ-casein (CSN3) genotype on the technological and coagulation properties of goat milk. In the current study, we have performed an association analysis between polymorphisms at the goat CSN1S1 and CSN3 genes and milk coagulation (rennet coagulation time, curdling rate and curd firmness) and technological (time to cutting of curd and cheese yield) properties. In this analysis, we have included 193 records from 74 Murciano-Granadina goats (with genotypes constituted by different combinations of alleles B, E and F of the gene CSN1S1 and alleles A and B of the gene CSN3) distributed in three herds, which were collected bimonthly during a whole lactation. Data analysis, using a linear mixed model for repeated observations, revealed significant associations between CSN1S1 genotypes and the rate of the curdling process. In this way, milk from EE goats had a significantly higher curdling rate than milk from BB individuals (P<0·05). Contrary to previous experiments performed in French breeds, cheese yield was not significantly different in BB, EE and EF goats. Moreover, we have shown that CSN3 genotype has a significant effect on the rennet coagulation time (BB>AB, P<0·05) but not on cheese yield. No interaction between the CSN1S1 and CSN3 genotypes was observed.

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex® test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically ‘coagulase-negative variant of Staph. aureus’. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis ‘coagulase-negative staphylococci (CNS)’ in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.

This study was undertaken to compare flow cytometry (FC) and direct microscopic leucocyte count (MDLC) for the differentiation of macrophages, lymphocytes and polymorphonuclear leucocyte (PMN) and to evaluate leucocyte distribution in ewe milk with low and high somatic cell count (SCC). Milk samples were grouped for somatic cell count in low SCC (LSCC) when the content was lower than 5·00×105/ml and high SCC (HSCC) when the content was higher than 1·00×106/ml. No differences were found between the two methods tested suggesting that FC could be used as a routine test for rapid discrimination of leucocytes. Percentages of lymphocytes in ewe milk were higher in LSCC (50%) than in HSCC (39%) and count ranged from 273·91±56·62×103cells/ml (LSCC) to 308·90±46·15×103cells/ml (HSCC). PMN number was lower in LSCC than in HSCC (248·83±46·87×103cells/ml v. 444·38±58·62×103cells/ml); accordingly the percentage was lower in LSCC (40%) than in HSCC (57%). No differences were found for macrophages which were 36·36±5·51×103cells/ml and 39·32±6·83×103cells/ml in LSCC and HSCC, respectively. Lymphocytes in ewe milk did not vary with increased number of somatic cells and were the predominant cell type in LSCC. PMN represented the main population detected in HSCC and the correlation with SCC evidenced that this leucocyte class could be useful in differentiating ewe milk cell count, being strictly responsible for the SCC increase.

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

Flaxseed hull, a co-product obtained from flax processing, is a rich source of n-3 fatty acids (FA) but there is little information on its value for dairy production. Monensin supplementation is known to modify biohydrogenation of FA by rumen microbes. Therefore, the main objective of the experiment was to determine the effect of feeding a combination of monensin and flaxseed hulls on ruminal fermentation characteristics and FA profile of ruminal fluid and milk. Four ruminally fistulated multiparous Holstein cows averaging 665±21 kg body weight and 190±5 d in milk were assigned to a 4×4 Latin square design (28-d experimental periods) with a 2×2 factorial arrangement of treatments. Treatments were: 1) control, neither flaxseed hulls nor monensin; 2) diet containing (dry matter basis) 19·8% flaxseed hulls; 3) diet with monensin (16 mg/kg dry matter); 4) diet containing 19·8% (dry matter basis) flaxseed hulls and 16 mg monensin/kg. Flaxseed hull supplementation decreased the acetate to propionate ratio in ruminal fluid and monensin had no effect. Concentrations of trans-18:1 isomers (trans9,trans11,trans13/14+6/8) and cis9,12,15-18:3 in ruminal fluid and milk fat were higher and those of cis9,12-18:2 in milk fat tended (P=0·07) to be higher for cows supplemented with flaxseed hulls than for cows fed no flaxseed hulls. Monensin had little effect on milk fatty acid profile. A combination of flaxseed hulls and monensin did not result in better milk fatty acid profile than when feeding only flaxseed hulls.

This study was designed to evaluate the effects of a single intramammary infusion of Panax ginseng extract on cell proliferation and death mechanism in bovine mammary gland during early involution. Eight mammary quarters from six non-pregnant cows in late lactation were infused with 10 ml of ginseng solution (3 mg/ml), six quarters were treated with 10 ml of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Animals included in the three groups were slaughtered 7 d after inoculation and samples for histological analysis were taken. Morphometric analysis showed a significant increase in percentages of mammary tissue area occupied by stroma in ginseng-treated quarters compared with controls. A significant increase of immunostained area for bax protein and active caspase-3 was observed in ginseng-treated quarters compared with controls, whereas no differences were observed for bcl-2 immunostaining. Expression of bax mRNA was significantly higher in ginseng-treated quarters than in controls. The bax/bcl-2 ratio indicated a significant predominance of bax over bcl-2 mRNA expression in ginseng-treated quarters compared with controls. The rise of epithelial and stromal cell apoptosis in situ by TUNEL was more marked in quarters treated with ginseng than in controls. Ginseng inoculation had no effect on the number of epithelial and stromal proliferating cells labelled with Ki-67 antibody. Ratio of apoptotic to proliferating cells was higher in quarters treated with ginseng compared with controls, indicating a net loss of cells in parenchymal components. Also, the intramammary inoculation of ginseng extract at drying off increased the rate of mammary cell apoptosis without inhibiting cell proliferation. Taken together, these changes are indicative of mammary regression enhancement during early involution.

The aim of this study was to evaluate the antioxidant, antihypertensive and immunomodulatory characteristics of skim milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340. Supernatants obtained from the ferments after centrifugation were subjected to ultrafiltration and yielded four peptidic fractions of 10–5 kDa, 5–3 kDa, 3–1 kDa, and <1·0 kDa. Peptides in 5–3 kDa range exhibited a good antioxidant activity. The peptides (<1·0 k) was applied to Superdex-30 G column fractionation and produced six fractions (F1–6). Fraction F2 presented the highest angiotensin I-converting enzyme inhibition activity with IC50 of 67·71±7·62 mg/ml. Moreover, fraction F6, which displayed a good immunomodulatory activity, had a positive effect on murine spleen lymphocyte proliferation with Stimulation Index of 0·729±0·123. The present data showed the potential of the milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340 as a functional food, however, further research is needed to evaluate the biofunctional activity of this fermentation product in vivo using model animal.

The effect of freezing on the properties of a raw ewes’-milk semi-soft cheese (Serpa cheese) was studied using small amplitude oscillatory (SAOS) and texture measurements, colour and chemical parameters. The freezing was introduced at three different stages of the ripening process (28, 35 and 42 days), and the cheeses were maintained frozen for 12 months. Cheeses were submitted to a slow or fast freezing method, and to different storage temperatures: −10 and −20°C (three replicates for each set conditions). Chemical data showed that only the proteolysis indicators exhibited differences between frozen and non-frozen samples; frozen samples showed higher values of NPN than the non-frozen samples, indicating that the freezing process did not prevent the secondary proteolysis of cheese. Frozen samples showed a significantly (P<0·05) stronger structure than the non-frozen, as indicated by hardness. However, the differences between the frozen and non-frozen samples were not significantly for storage modulus (G′1Hz) and loss tangent (tan δ1Hz) (P>0·05). Freezing affected mainly colour parameters: frozen samples were more luminous, and more yellow-green. The results allowed us to conclude that the damages caused by freezing to cheese properties could be minimized if this type of storage is introduced at the end of ripening (42 d) using a freezing temperature of −20°C.

Dairy herds are expanding and, with increasing numbers of animals in each herd, there is a need for automatic recording of indicators in milk in order to detect mastitis, inflammation of the udder. A number of biomarkers for mastitis have been suggested over the years. Mastitis usually occurs in one of the four udder quarters and since it is now possible to milk each udder quarter separately in automated milking systems, it is important to evaluate the normal variation in the biomarkers at udder quarter level. This study evaluated the normal variations between milkings for some biomarkers in clinically healthy cows, determined by repeated somatic cell count and bacteriological analysis. The biomarkers studied were serum amyloid A (SAA), haptoglobin (Hp), lactate dehydrogenase (LDH), N-acetyl-β-d-glucosaminidase (NAGase) and alkaline phosphatase (AP), parameters that have been suggested as markers for mastitis. Ten cows were monitored on 42 consecutive milking occasions through collection of udder quarter milk samples and representative cow composite milk samples, giving a total of 2100 individual milk samples. Each cow had its individual profile for the concentrations and variations in the parameters analysed. Although there was relatively large variation between cows for the biomarkers analysed, the variation between milkings in clinically healthy quarters within cows was often below 10%. The biomarker with the lowest variation in this study was LDH. The results suggest that comparing quarters within an individual cow can identify deviations from the natural variations between milkings. This could be a valuable tool instead of, or in combination with, a cut-off value for each parameter in order to detect changes in the milk indicating mastitis.

Experiments were designed to investigate the suitability of a combination of a short manual teat stimulation with a short latency period before teat cup attachment to induce and maintain oxytocin release and milk ejection without interruption. In Experiment 1, seven dairy cows in mid lactation were manually pre-stimulated for 15, 30 or 45 s, followed by either 30 s or 45 s of latency period. It was shown that all treatments induced a similar release of oxytocin without interruption until the end of milking. In particular, the latency period of up to 45 s did not cause a transient decrease of oxytocin concentration. In Experiment 2, milking characteristics were recorded in seven cows each in early, mid, and late lactation, respectively. Because the course of milk ejection depends mainly on the degree of udder filling, individual milkings were classified based on the actual degree of udder filling which differs between lactational stages but also between morning and evening milkings. All animals underwent twelve different udder preparation treatments, i.e. 15, 30, or 45 s of pre-stimulation followed by latency periods of 0, 30, 45, or 60 s. Milking characteristics were recorded. Total milk yield, main milking time and average milk flow rate did not differ between treatments if the degree of udder filling at the start of milking was >40% of the maximum storage capacity. However, if the udder filling was <40%, main milking time was decreased with the duration of a latency period up to 45 s, independent of duration of pre-stimulation. Average milk flow at an udder filling of <40% was highest after a pre-stimulation of 45 s followed by a latency period of another 45 s. In contrast, average milk flow reached its lowest values at a pre-stimulation of 15 s without additional latency period. However, average milk flow after a 15-s pre-stimulation increased with increasing latency period. In conclusion, a very short pre-stimulation when followed by a latency period up to 45 s before teat cup attachment remains a suitable alternative for continuous stimulation to induce milk ejection.

A method for evaluating cleaning effect based on Bacillus cereus spores was developed and tested in a model system designed to resemble actual farm conditions. A test rig with four removable sampling plates was mounted in a milk line. The plates were attached at the end of T-junctions protruding either 1·5 or 3-times the milk pipe diameter from the main loop to reflect different levels of cleaning difficulty. In each cleaning test, B. cereus spores were applied to the four sampling plates to simulate soil. A series of cleaning tests was conducted at 35, 45, 55 and 65°C with six commercial chlorine-free, alkaline detergents; three liquid and three powder-based products. A commercial alkaline detergent with chlorine, a sodium hydroxide solution, a sodium hydroxider/hypochlorite solution and pure water were also tested. Triplicate tests were performed with each cleaning solution, giving a total of 120 cleaning tests. The cleaning effect was evaluated by comparing the number of spores before and after cleaning. At all temperatures, the two chlorine-based cleaning solutions gave significantly greater reductions in B. cereus spores than the chlorine-free products. All six commercial chlorine-free, alkaline detergents generally gave similar cleaning effects, with no differences in the performance of powder-based and liquid forms. The mechanical spore reduction effect with water alone was greater (1·5–1·8 log-units) than the additional chemical effect of sodium hydroxide or chlorine-free detergents (0·5–1·2 log-units). The chlorine-based solutions had a considerably more powerful chemical effect (2–4 log-units depending on temperature). In general, an increase in cleaning solution temperature up to 55°C gave a greater reduction in spores. A further increase to 65°C did not improve cleaning effectiveness.

Nutritional value is a priority in new product development. Using vegetable or marine oils, rich in polyunsaturated fatty acids, in dairy beverage formulations is an option to provide the consumers with healthier products. However, these formulations are prone to oxidation, which is responsible for rapid flavour degradation and the development of potentially toxic reaction products during storage. Flaxseed lignans, secoisolariciresinol diglucoside (SDG), and its mammalian metabolites have antioxidant activity and could be used in beverage formulations to prevent oxidation. Commercially available SDG extract was added to the formulation of dairy beverages enriched with flaxseed oil. As an alternative approach, dairy beverages were produced from milk naturally rich in SDG metabolites obtained through the alteration of cow diet. Resistance to oxidation was determined from the kinetics of hexanal and propanal production during heat and light exposure treatments. Increasing SDG concentration in dairy beverage slightly reduced redox potential but had no effect on oxygen consumption during oxidation treatments. The presence of SDG in dairy beverage significantly improved resistance to heat- and light-induced oxidation. However, purified enterolactone, a mammalian metabolite from SDG, prevented oxidation at much lower concentrations. The use of milk from dairy cow fed flaxseed meal did not improve resistance to oxidation in dairy beverage. Enterolactone concentration in milk was increased by the experimental diet but it remained too low to observe any significant effect on dairy beverage oxidation.

Subclinical mastitis (SM) is a major concern in the dairy industry because it causes economic losses and can lead to clinical mastitis. The mechanisms of the onset and progression of SM are not fully understood, and a new procedure for the detection and appropriate prediction of SM leading to clinical mastitis is necessary for dairy cow management. Inflammatory cytokines such as interleukin (IL)-6 are known to be present in inflamed mammary glands at the onset of mastitis, and IL-6 concentrations correlate with the levels of inflammation. In this study, the detection of IL-6 was examined for the evaluation for the future prediction of SM in 77 quarter milk samples from 20 cows. IL-6 concentrations in quarter milk were measured by sandwich ELISA, and the data were compared with milk somatic cell count (SCC) levels to diagnose SM. Average IL-6 concentration was significantly higher in SM quarter milk (207·0±441·6 pg/ml) than in healthy control quarter milk (12·6±33·4 pg/ml, P<0·01). Results of the cross-tabulation table show that SM prediction accuracy based on IL-6 concentration was almost equal or superior to SM prediction accuracy based on SCC. The detection of IL-6 in milk indicated SM earlier than did the detection of elevated SCC. Thus, the detection of IL-6 in milk could be a future prediction marker for SM.

Massese is an Italian sheep breed, with black or grey coat colour, mainly reared in the Tuscany and Emilia Romagna regions. Recently, the emerging interests in this breed have resulted in the production of Pecorino cheese obtained with only Massese milk. In order to be profitable, this marketing link between Massese breed and its products should be defended against fraudsters who could include milk of other sheep breeds or cow milk in Massese labelled productions. To identify the genetic factors affecting coat colour in sheep, we have recently analysed the melanocortin 1 receptor (MC1R) gene and identified several single nucleotide polymorphisms (SNPs). In this work, as a first step to set up a DNA based protocol for authentication of Massese dairy products, we further investigated the presence and distribution of one of these SNPs (c.-31G>A) in 143 Massese sheep and in another 13 sheep breeds (for a total of 351 animals). The Massese breed was fixed for allele c.-31A, whereas in all other breeds allele c.-31 G was the most frequent or with frequency of 0·50. At the same nucleotide position the cattle MC1R gene carries the G nucleotide. Using these data we developed a method to detect adulterating milk (from other sheep breeds or from cow) in Massese dairy products based on the analysis of the c.-31G>A SNP. We first tested the sensitivity of the protocol and then applied it to analyse DNA extracted from ricotta and Pecorino cheese obtained with only Massese milk or obtained with unrestricted sheep and cattle milk. To our knowledge, this system represents the first one that can be used for breed authentication of a sheep production and that, at the same time, can reveal frauds derived from the admixture of milk of an unreported species.