Local zebu cattle breeds in Indian villages are crossbred with exotic breeds at an unprecedented rate without utilising the full potential of genomic technologies. In addition to agriculture produce, livestock, particularly cattle, constitute a vital source of livelihood for farmers in India. Age-old agricultural practices, errant monsoon, and frequent crop failures have resulted in Maharashtra having the highest number of farmer suicides in the country. Local cattle breeds are considered low-yield breeds and thus are primarily used as beasts of burden. Information on functional gene variants in Indian cattle breeds is scant and limited to PCR-RFLP study in few breeds. In this study, 32 samples from 8 cattle breeds were obtained from remote villages of Maharashtra state. By using the re-sequencing technology, we sequenced 403 bp of the exon IV CSN3 allele and inferred its haplotypes. From 32 samples, 14 genotypes (G1–G14) defined by 7 single-nucleotide polymorphisms (SNP's) were identified. From these 14 genotypes, we reconstructed 3 haplotypes (H01, H02, and H03) and estimated their frequencies. Of the 3 haplotypes, two (H01 and H03) corresponded to CSN3*B4 and CSN3*B and CSN3*H and CSN3*G alleles, respectively. The third haplotype H02 was identified as a new allele and differed from CSN3*B4 and CSN3*B and CSN3*H and CSN3*G alleles by one nonsynonymous mutation at the position C5306T-Ile100Thr. The neighbour-joining tree reconstruction revealed haplotype sharing or hybridisation between B. t. indicus and B. t. taurus because the 3 haplotypes originated in this study clustered with A1, B2, B4, B, G, and H alleles, which were reported previously in both the subspecies of B. taurus. The occurrence of one new allele in a small sample size highlights the urgency to screen local zebu cattle breeds by using genomic tools for circumventing genetic erosion that is widespread in Indian villages in India.

Bovine spongiform encephalopathy (BSE) involves insertion/deletion (in/del) polymorphisms in the prion protein gene (PRNP) promoter region that are associated with vulnerability to disease progression. Recently, a second member of the prion gene family, prion-like protein gene (PRND), has been reported to show the PRND R132Q polymorphism, which is associated with the susceptibility to BSE in German Fleckvieh breeds. The objective of this study was to examine the genotype, allele, and haplotype frequencies of PRND gene in Korean cattle and evaluate their susceptibility to BSE. We did this in 277 Korean native cattle (Hanwoo) and 124 Korean dairy cattle (Holstein) by direct sequencing and compared the R132Q genotype frequency between BSE-affected German cattle and Korean cattle. The results indicated a total of 5 single nucleotide polymorphisms (SNPs) including PRND c.149G > A (p.50Arg > His; R50H), PRND c.285C > T (C4819T), PRND c.395G > A (p.132Arg > Gln; R132Q) and PRND c.528T > A (T5063A) in the open reading frame (ORF) and c.602C > G in the 3′ untranslated region (UTR) of exon 2 in Korean Holstein and Hanwoo cattle. Except for c.149G > A, the remaining 4 SNPs showed significantly different genotype and allele frequencies between the Korean Holstein and Hanwoo (P < 0·01). There were no significant differences in genotype distribution of c.395G > A SNP between BSE-affected German and Korean Holstein cattle (P = 0·6778), but a significant difference was detected between BSE-affected German cattle and Hanwoo cattle (P = 0·0028). The results suggest that Hanwoo cattle may possess a relatively more BSE-resistant genotype than Korean Holstein cattle.

The aims of the study reported in the Research Communication were to evaluate changes of macrominerals and major calcitropic hormone concentrations in 51 healthy dairy cows over the peripartum period, and to compare those cows with 51 cows subject to subclinical hypocalcaemia (SCH) on calving day. The results showed that serum calcium (Ca) and potassium (K) concentrations were lowest in the healthy periparturient dairy cows at parturition, while sodium (Na) and parathyroid hormone (PTH) concentrations were highest. Serum magnesium (Mg), chloride (Cl) and phosphorus (P) concentrations were highest 1 wk prepartum, however, the concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was highest 1 wk postpartum. The dairy cows with subclinical hypocalcaemia (SCH) had significantly lower serum levels of Ca, K, Na, Mg, Cl, 1,25(OH)2D3 and glucose and had significantly higher levels of serum P, non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) than their healthy counterparts on calving day. However, the serum calcitonin and PTH concentrations were not significantly different between the two groups. These data demonstrated that the concentrations of macrominerals and major calcium regulating hormones of cows change dramatically over the peripartum period. Furthermore, SCH was associated with a risk of the development of ketosis and fatty liver.

This research paper addresses the hypothesis that dietary pioglitazone (PGT), as synthetic and specific ligand for PPAR-γ or walnut meal (WM) as a natural ligand for PPAR-γ, affect plasma metabolites and reduce the oxidative status in high body condition score (BCS) dairy cows (≥4 BCS). Total of 36 multiparous Holstein cows were randomly assigned to one of the dietary treatments: 1- Control (basal diet; CTR), 2- Walnut meal (9·45% walnut meal of DMI; WM), and 3- Pioglitazone (6 mg/kg BW; PGT). The experimental diets were fed from parturition time to 21 d postpartum. Results showed that the PGT supplementation increased dry matter intake (DMI) (22·95 kg/d) compared to the CTR (21·45 kg/d) and WM (21·78 kg/d) groups. Results showed that milk yield and milk composition were not affected by the experimental diets. Body condition score losses tended to be higher in the CTR group compared to the PGT and WM cows. The PGT group had higher plasma insulin compared to the CTR group (11·84 vs. 10·68 mIU/l), and WM cows had intermediate plasma insulin. The PGT cows had lower plasma non esterified fatty acid (NEFA) and tended to have lower β-hydroxy butyric acid (BHBA) than the CTR group. Feeding pioglitazone decreased plasma malondialdehyde (MDA) and increased plasma total antioxidant capacity (TAC) and superoxide dismutase (SOD) compared to the CTR and WM groups. It was concluded that dietary pioglitazone had positive effects on DMI, BCS change, blood metabolites and oxidative status in fresh dairy cows with high pre-calving BCS. The anti-oxidant effects of walnut meal were not supported by the present data.

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

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

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

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

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

In this Research Communication we report the results of a controlled study conducted under field conditions in which we analysed milk ejection curves in cows with chronic mastitis, and assessed the influence of antinflammatory treatment with ketoprofen. Total milking time was reduced in chronic mastitis cows, irrespective of ketoprofen treatment, and the proportion of bimodal flow curves was increased. This latter effect was partially reversed by ketoprofen. To our knowledge, this is the first study showing that chronic mastitis has a significant effect on the milk ejection curve. Antinflammatory treatment with ketoprofen was shown to be efficacious in reducing these negative effects, re-establishing a pattern close to the one observed in healthy cows.

Heat stress (HS) negatively affects milk production and has been associated with decreased immune function, and increased rate of intramammary infections (IMI). Research has shown that HS affects gene expression, cell cycle, and cell metabolism in bovine mammary epithelial cells (BMEC). Since BMEC are an initial target of mastitis pathogens, we studied adherence to and internalisation of S. uberis into HS-BMEC, as well as the effect that this interaction has on host cells by measuring HS-BMEC viability and membrane integrity. Results reported in this Research Communication showed that HS reduced cell viability and induced membrane damage. However, these pathological changes, as well as the rate of adherence and internalisation of S. uberis into BMEC, were augmented when S. uberis was cocultured with HS-BMEC. These results may help to understand the pathogenesis of S. uberis IMI as well as the increased susceptibility of mammary glands to IMI in cows subjected to HS.

The study reported in this Research Communication was conducted to characterise Staphylococcus aureus isolates recovered from mastitic bovine milk from dairy herds in Argentina. A total of 829 mastitic milk samples, both clinical and subclinical, were collected from 21 farms by veterinarians and submitted to the laboratory for testing from which 229 S. aureus isolates were recovered, an isolation rate of 28·1%. These isolates were tested for susceptibility to the antibiotics penicillin, erythromycin and clindamycin. Of the 229 isolates, 53 (23·1%) were resistant to penicillin, 31 (13·5%) to erythromycin and 28 (12·2%) to clindamycin. All isolates were negative for the mecA, mecC and pvl genes by PCR. Southernblot hybridisation revealed that the ermC gene was located on plasmid bands. Eighty isolates were randomly selected from the 229 for further characterisation. Restriction analysis of chromosomal DNA with Cf9I followed by PFGE of the 80 isolates revealed 23 distinct pulsotypes at 80% similarity. Seven major types (A, B, N, P, S, T, U and V) accounted for 68·7% of these isolates and 12 pulsotypes (A, B, F, G, J, K, M, N, P, S, T and U) occurred on more than one farm indicating genetic diversity within the farms. MLST of a representative isolate from dominant types identified the STs 97 705, 746, 2102 and 2187 with ST97 being the most predominant. Antibiotic susceptibility testing showed that 53·7% of the 80 randomly selected isolates were resistant to at least one of the three antibiotics tested. To our knowledge, this study represents the first large scale molecular studies on S. aureus isolates from dairy farms in Argentina.

This Research Paper addresses the hypothesis that using teatcups with automatic valves, without cutting off the vacuum prior to cluster removal, could increases the risk of mastitis and affect other milking variables on goats. A first trial used 46 intramammary infection (IMI)-free goats that had been milked with normal teatcups (without automatic valves) during a pre-experimental period of 8 ± 2 d postpartum. These animals were divided into two groups (n = 23), randomly assigning each group to teatcups with automatic valves (teatcups A) or without automatic valves (teatcups B) for a 20-week experimental period. During this period, several strategies were applied to increase teat exposure to pathogens in both experimental groups. In the first eight weeks of the experimental period, the new IMI rate per gland was significantly higher (P < 0·05) in the group of animals milked with teatcups A (6 of 46; 13%) than in the group milked with teatcups B (1 of 46; 2%). However, throughout the rest of the experimental period the same number of glands appeared with new IMI (n = 7) in both animal groups. SCC was higher in goats milked with teatcups A, but no significant differences were found in the remaining variables (milk production and composition, frequency of liner slips + teatcup fall-off). In a second experiment, in a crossover design (54 goats in fourth month of lactation, 2 treatments – teatcups A and B – in 2 experimental periods each lasting 1 week), no differences were observed in total milk, average milk flow, total milking time or teat thickness changes after milking between both teatcups. However, teatcups A worsened slightly the maximum milk flow. We concluded that the use of teatcups with automatic valves, without cutting off the vacuum prior to cluster removal, increases the risk of mastitis on goat livestock farms.

Oxytocin release, milking characteristics, and teat condition were investigated with reduced claw vacuum and pulsation settings compared to milking at regular settings with or without pre-stimulation. The reduced vacuum and pulsation settings during low milk flow are expected to protect the teat tissue before the occurrence of milk ejection at the start of milking, and at the end of milking during a potential overmilking period, i.e. at a milk flow <200 g/min. Seven cows were machine-milked either after a 60 s manual pre-stimulation, or without pre-stimulation and reduced vacuum and pulsation settings, or at full vacuum and normal pulsation during the start of milking. Plasma oxytocin (OT) concentration increased similarly in response to manual pre-stimulation and to both milking with reduced, or with full vacuum and pulsation settings, however delayed by 1 min if the cluster was attached without pre-stimulation. In all treatments OT concentrations remained elevated throughout milking. Milk flow curves were mostly non-bimodal at milkings after manual pre-stimulation and bimodal at milkings without pre-stimulation. The main milking time was shorter and average milk flow was higher during milking after pre-stimulation, but did not differ between treatments without pre-stimulation. Milk yields and peak flow rates were not affected by treatments. Either reduced or full vacuum settings were again applied during an intended overmilking from 200 to 100 g/min of milk flow towards the end of milk harvest. Pre-milking teat ultrasound cross sections were recorded one day before the experiment started. Post-milking ultrasound cross sections were performed at 15 min after each experimental milking. Teat wall thickness was increased after milking as compared to pre-milking but did not differ among treatments. In conclusion, OT release and milking performance are similar if milking is performed with pre-stimulation, or without pre-stimulation but reduced claw vacuum and b-phase during low milk flow.

This Research Communication addresses the hypothesis that fat, protein, lactose and total solids content can be predicted using daily milk yield (DMY), pH, electrical conductivity (MEC) and refractive index (RI) of milk as predictors. It also addresses the possibility of these measurements being used for on-farm benchmarking activities towards selecting the highest yielding animals and flocks regarding milk quality traits (MQT). A total of 308 purebred Frizarta ewes were used for the study. From each individual ewe, a composite milk sample was collected. pH, MEC and RI of milk were measured and the samples were assayed for fat, protein, lactose and total solids content, using an automatic infrared milk analyser. The predictive value of DMY, pH, MEC and RI of milk on its MQT was assessed using multiple linear regression analysis. Significant regression equations were produced for all of the studied traits. RI and MEC were significant and reliable predictors for all studied MQT, whereas DMY was a significant predictor for most MQT with the exception of protein content. pH was a marginally significant predictor for some of the MQTs at the initial development of the equations but proved unreliable after bootstraping. Using these equations a number of ewes varying from 75 (for fat) to 97 (for protein) out of the 100 highest MQT yielders were correctly predicted, whereas, none of the ewes out of the 100 lowest MQT yielders was mispredicted as a high yielder for protein-, lactose- and total solids- content. Three out of 100 lowest fat-yielders were mispredicted as high fat-yielders. Similar equations can be used for benchmarking activities towards selecting the highest protein-, fat-, lactose- and total solids- yielding animals and flocks in cases where laboratories for MQT analyses are not readily available or the cost of chemical analyses is high. The method can be regarded as a handy tool for the dairy industry to readily assess milk quality at the farm level.

This Research Communication investigated the potential of mid-infrared spectroscopy to predict detailed mineral composition of bovine milk. A total of 153 bulk milk samples were analysed for contents of Ca, Cl, Cu, Fe, K, Mg, Na, P and Zn. Also, soluble and colloidal fractions of Ca, Mg and P were quantified. For each milk sample the mid-infrared spectrum was captured and stored. Prediction models were developed using partial least squares regression and the accuracy of prediction was evaluated using both cross- and external validation. The proportion of variance explained by the prediction models in cross-validation ranged from 34% (Na) to 77% (total P), and it ranged from 13% (soluble Mg) to 54% (Cl−) in external validation. The ratio of the standard deviation of each trait to the standard error of prediction in external validation, which is an indicator of the practical utility of the prediction model, was low and never greater than 2. Results from the current study supported the limited usefulness of mid-infrared spectroscopy to predict minerals present in low concentration in bulk milk. For major mineral components, results from the present research did not match previous findings demonstrating the need for further studies using larger reference datasets.

We examined the latent structure of 26 cheese related phenotypes in dairy cattle. Traits related to milk yield and quality (8 traits), milk protein fractions (8 traits), coagulation and curd firmness indicators (CF, 5 traits) and cheese-making phenotypes (cheese yields (%CY) and nutrient recoveries in the curd (REC), 5 traits) were analysed through multivariate factor analysis (MFA) using a varimax rotation. All phenotypes were measured in 1264 Brown Swiss cows. Ten mutual orthogonal, latent variables (factors; Fs) were obtained explaining 74% of the original variability. These Fs captured basic concepts of the cheese-making process. More precisely, the first 4 Fs, sorted by variance explained, were able to capture the underlying structure of the CY percentage (F1: %CY), the CF process with time (F2: CFt), the milk and solids yield (F3: Yield) and the presence of nitrogen (N) in the cheese (F4: Cheese N). Moreover, 4 Fs (F5: as1-β-CN, F7: κ-β-CN, F8: as2-CN and F9: as1-CN-Ph) were related to the basic milk caseins and 1 factor was associated with the α-LA whey protein (F10: α-LA). A factor describing udder health status (F6: Udder health), mainly loaded on lactose, other nitrogen compounds in the milk and SCS, was also obtained. Further, we inferred the effects of some potential sources of variation (e.g. stage of lactation and parity) including feeding and management systems. Stage of lactation had a significant effect for 7 of the 10 Fs, followed by parity of the cow (3 Fs), dairy system and feeding (3 Fs). Our work demonstrates the usefulness of MFA in reducing a large number of variables to a few latent factors with biological meaning and representing groups of traits that describe a complex process like cheese-making. Such an approach would be a valuable tool for studying the influence of different production environments and individual animal factors on protein composition and cheese-making related traits.

Cronobacter sakazakii is a life-threatening foodborne pathogen found in powdered infant formula and dairy products. Kefir is a dairy probiotic product and its antimicrobial activity against C. sakazakii was reported in our previous study. To identify key microorganisms that mediate growth suppression, we tested the antimicrobial activity of culture supernatants derived from lactic acid bacteria found in kefir. Lactobacillus kefiri DH5, L. kefiranofaciens DH101, and Bifidobacterium longum 720 (a commercial probiotic strain that served as a positive control) all significantly inhibited the growth of C. sakazakii ATCC 29544, delaying the initiation of exponential growth from 3 to 9 h in the nutrient broth. Among them, L. kefiri DH5 exerted the strongest antimicrobial effects against C. sakazakii, showing bactericidal effect at the addition of 300 µl of supernatant in 1 ml of nutrient broth. Interestingly, the supernatant of L. kefiri DH5 has higher pH and lower titrable acidity than that of L. kefiranofaciens DH101, suggesting metabolites produced by heterofermentation of L. kefiri acted more effectively to antagonise the growth of C. sakazakii. In addition, the supernatant of L. kefiri DH5 induced the leakage of cytoplasmic materials including nucleic acid and proteins, suggesting L. kefiri DH5 disrupted the cellular membrane integrity of C. sakazakii. Considering that pH neutralisation reduced the L. kefiri-dependent growth suppression, it is inferred that this activity is mainly due to organic acids produced during the fermentation process.

In this study, the relationship between the dissociation of κ-casein from casein micelles due to heat-induced denaturation and the strength of acid milk gel was investigated. The κ-casein-dissociated micelles were fractionated by gel filtration chromatography and two-dimensional polyacrylamide gel electrophoresis, and their zeta potential and surface hydrophobicity were measured. The negative charge of the κ-casein-dissociated micelles was lower than that of native micelles, and micellar surface hydrophobicity was higher. For confirmation, the isoelectric point of the casein micelles was measured. The κ-casein-dissociated micelles were found to cohere at an earlier stage of acidification than the native micelles. These results demonstrated that the heat-induced increase in the strength of acid milk gel was partly due to the decrease in micellar surface charge and partly to the increase in surface hydrophobicity caused by the dissociation of κ-casein.