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Lactoferrin is a multifunctional glycoprotein with a range of antimicrobial and immune-related properties that is found at >10-fold higher concentration in human milk (~1.7 g/L) relative to bovine milk (~0.15 g/L). Consumer demand is increasing for bovine lactoferrin through a wide range of nutritional and cosmetic consumer products. Increasing lactoferrin yield and concentration in bovine milk could assist in satisfying this increasing demand and may also help in increasing resistance to bovine mammary infection. Two experiments with cows in mid and late lactation were carried out to examine milking strategies to increase milk lactoferrin concentration and yield. Milking was suspended in cows normally milked twice daily, for periods of 2, 4, or 7 d (mid lactation) or 2 or 4 d (late lactation) after which cows were milked out and twice-daily milking resumed for 4 d. In all groups, lactoferrin concentration was significantly increased during the remilking period, approaching concentrations similar to those found in human milk (~1 g/L). Lactoferrin yields were significantly higher in all treatment groups, although increasing the nonmilking period beyond 2 d offered no advantage. Milk yield was lower initially after resumption of milking but recovered to preexperimental values by the fourth day of remilking in all groups, except the 4-d nonmilking group in late lactation. Milk somatic cell count was significantly elevated in all groups at the start of remilking but had substantially reduced by d 4 and reached a preexperimental level in the 2-d nonmilking group of mid-lactation cows. In summary, extended milking intervals can be used as a tool to produce a short-term increase in the concentration and yield of lactoferrin from bovine milk during established lactation, without any apparent long-term effects on milk yield and quality. [ABSTRACT FROM AUTHOR]

The main objectives of this study were to establish the relative value of milk yields under twice-daily milking (TDM) as a predictor of yield and yield loss under once-daily milking (ODM), and to understand the role of residual milk and udder storage capacity-related traits in regulating yield and yield loss during ODM. A Holstein-Friesian × Jersey crossbred herd was established over 2 seasons (years), as 2 individual cohorts on the same farm, managed on a pasture-based system over 4 lactations. Short-term (1-wk) ODM studies, with a starting total of 690 cows, were undertaken in midand late-lactation in lactation 2 and in mid-lactation in lactation 3 for each cohort. A 10-wk study of ODM performance began in mid-lactation in lactation 3, whereas lactation 4 was a full-lactation assessment of ODM. In the short-term studies, milk yield under ODM was well predicted (R2 = 0.7 to 0.8 in 5 of 6 studies) by the daily yield under TDM in the week before ODM. Yield loss (kg/d) increased with increasing milk yield and with increasing somatic cell count (SCC), although predictions were relatively poor (R2 = 0.09 to 0.30). Yield loss (%) decreased with increasing TDM yield in 3 of the 6 studies and was positively correlated with SCC during ODM. Nevertheless, ODM yield loss, in absolute or percentage terms, was a poorly repeatable trait in grazing cows. Part of the variation in yield loss percentage (30%) was positively associated with residual milk (%), measured pretrial, during measurement of functional udder capacity in lactation 3. Total production (kg of milk) over the full-lactation ODM study in lactation 4 was correlated with total production in the 10-wk trial in lactation 3 (r = 0.72 and 0.63 for cohorts 1 and 2, respectively). Identifying the highest- and lowest-producing 10% of animals during the full lactation of ODM indicated that poor production was associated with high yields of residual milk (measured in lactation 3) and, conversely, high production was associated with low yields of residual milk, relative to the other 80% of animals. These same "high" and "low" production groups from lactation 4 had similar differences in performance in the earlier short-term studies and a larger or smaller percentage yield loss associated with the residual milk measurement. Breeding strategies for ODM may benefit, therefore, from greater emphasis on selecting for a low residual milk fraction to optimize milking performance. Nevertheless, the level of milk production under TDM is a strong phenotypic predictor of milk production under ODM. [ABSTRACT FROM AUTHOR]

A genomic prediction for residual feed intake (RFI) developed in growing dairy heifers (RFIgro) was used to predict and test breeding values for RFI in lactating cows (RFIlac) from an independent, industry population. A selection of 3,359 cows, in their third or fourth lactation during the study, of above average genetic merit for milk production, and identified as at least 15/16ths Holstein-Friesian breed, were selected for genotyping from commercial dairy herds. Genotyping was carried out using the bovine SNP50 BeadChip (Illumina Inc., San Diego, CA) on DNA extracted from ear-punch tissue. After quality control criteria were applied, geno-types were imputed to the 624,930 single nucleotide polymorphisms used in the growth study. Using these data, genomically estimated breeding values (GEBV) for RFIgro were calculated in the selected cow population based on a genomic prediction for RFIgro estimated in an independent group of growing heifers. Cows were ranked by GEBV and the top and bottom 310 identified for possible purchase. Purchased cows (n = 214) were relocated to research facilities and intake and body weight (BW) measurements were undertaken in 99 "high" and 98 "low" RFIgro animals in 4 consecutive groups [(beginning at d 61 ± 1.0 standard error (SE), 91 ± 0.5 SE, 145 ± 1.3 SE, and 191 ± 1.5 SE d in milk, respectively)] to measure RFI during lactation (RFIlac). Each group of ~50 cows (~25 high and ~25 low RFIgro) was in a feed intake facility for 35 d, fed pasture-alfalfa cubes ad libitum, milked twice daily, and weighed every 2 to 3 d. Milk composition was determined 3 times weekly. Body weight change and BW at trial mid-point were estimated by regression of pre- and post-trial BW measurements. Residual feed intake in lactating cows was estimated from a linear model including BW, BW change, and milk component yield (as MJ/d); RFIlac differed consistently between the high and low selection classes, with the overall means for RFIlac being +0.32 and -0.31 kg of dry matter (DM) per day for the high and low classes, respectively. Further, we found evidence of sire differences for RFIlac, with one sire, in particular, being highly represented in the low RFIgro class, having a mean RFIlac of -0.83 kg of DM per day in 47 daughters. In conclusion, genomic prediction of RFIgro based on RFI measured during growth will discriminate for RFIlac in an independent group of lactating cows. [ABSTRACT FROM AUTHOR]

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution, Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N¹-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes αs1-β-, and κ-casein, and a-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas β-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in a-lactalbumin and β-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors β-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution. [ABSTRACT FROM AUTHOR]

The impact of nutrition on lactation can be separated into acute effects, affecting day-to-day yield, and chronic effects, which govern the persistency of lactation and rate of decline of the lactation curve. A mathematical model of the mammary gland was constructed to investigate both acute and chronic effects. Mammary growth is expressed in terms of the dynamics of populations of active (secreting) and quiescent (engorged) alveoli. The secretion rate of active alveoli is expressed in terms of the energy status of the dam. The model was fitted to data from a 2 x 2 factorial trial in which lactation curves were measured for heifers of two different genotypes (North American and New Zealand Holstein-Friesians) fed two different diets [grass and total mixed rations (TMR)]. Total formation of alveoli during pregnancy and lactation was statistically the same across all groups despite differences between diets, in the rate of formation of alveoli at parturition. The senescence rate of alveoli was significantly higher for heifers fed grass compared with heifers fed TMR, which corresponds to better persistency for heifers fed TMR. Heifers fed TMR had a higher rate of reactivation of quiescent alveoli than heifers fed grass, which also contributes to increased persistence for heifers fed TMR. There was a genotype x diet interaction in the rate of quiescence of active alveoli: the North American-Grass group had a higher rate of quiescence than the other three groups, perhaps reflecting differences in selection pressures between the New Zealand and North American genotypes. [ABSTRACT FROM AUTHOR]

Presents a study which combined the alveolar model and animal model to produce a lactation model that is sensitive to both intake and milking frequency. Schema of lactation model; Rate of generation of alveoli; Reactivation of quiescent alveoli; Role of alveoli and the secretion rate act in milk production.

Most dairy cows throughout the world are milked twice daily. In intensive dairying systems, however, it is not uncommon to increase milking frequency to between 3 and 6 times daily to increase milk production. Reducing milking frequency is much less common; however, once-daily milking of dairy cows, practiced either strategically during certain parts of the lactation or for the entire lactation, is not uncommon in key dairying countries where less emphasis is placed on milk production per cow. The practice fits well with more extensive dairy production systems, particularly those based on grazed pasture. A feature of once-daily milking is that it reduces milk yield by approximately 22%, depending on stage of lactation, breed, and parity, and it may adversely affect lactation length and persistency. However, it can offer several positive farm management options, especially related to labor requirements and farm working expenses. In addition, it may provide a tool to better manage the metabolism and energy balance of cows during early lactation or during periods of pasture deficit, and it may help to improve reproductive performance and animal health and welfare. Once-daily milking, representing one extreme of the mammary function spectrum, has attracted considerable research interest over the years. Consequently, substantial scientific information is available on its effects on mammary function, at both the physiological and molecular levels. This review focuses instead on the management of the cow milked once daily, covering the production response in relation to breed, stage of lactation, and parity, and its effect on energy status, reproduction, health and welfare, as well as on milk composition and processability. [ABSTRACT FROM AUTHOR]

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 μmol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate heterogeneity in tissue morphology, milk protein and immune-related gene expression, and apoptosis of epithelial cells in the lactating and involuting mammary glands of the dairy cow. Mammary tissue from different regions of the gland (alveolar, cisternal, and peripheral) was collected postmortem from nonpregnant, pasture-fed, Holstein-Friesian primiparous cows in mid-lactation that were killed at different time points postmilking: 0, 6, 12, 18, 24, 36, and 72 h (n = 6 per time point). The CSN1NS1 and LALBA mRNA was decreased in alveolar, cisternal, and peripheral tissue by 12 to 36 h postmilking. In contrast, lactoferrin (LF) and mammary serum amyloid A3 (M-SAA3) mRNA was increased in these regions by 36 to 72 h. During lactation, more variability was present in gene expression in alveolar tissue between cows and between quarters within a cow, than within quarters. Histological analysis indicated the alveolar tissue from lactating cows was mostly uniform in structure; however, in situ hybridization indicated that although most of the alveolar tissue expressed milk proteins, the level of expression varied within and between alveoli. This heterogeneity became more pronounced with involution and with increasing regions of alveoli expressing lactoferrin, indicating that alveoli enter involution asynchronously. The peripheral and cisternal tissue had more variability in gene expression between cows compared with the alveolar tissue. The M-SAA3 signal was more intense in the cisternal tissue and less intense than the peripheral compartment compared with LF particularly in the earlier time points. In addition, between cows within the later time points, differences were observed in tissue morphology, the levels of milk protein and immune-related gene expression, and phosphorylated signal transducer and activator of transcription (STAT) 5-P and STAT3-P proteins, and degree of apoptosis, indicating that involution of the mammary gland occurs at different rates between cows. Understanding the mechanisms initiating the process of involution of the mammary gland provides an opportunity for enhancing milk production of the dairy cow. [ABSTRACT FROM AUTHOR]

X chromosome inactivation (XCI) is a process by which 1 of the 2 copies of the X chromosomes present in female mammals is inactivated. The transcriptional silencing of one X chromosome achieves dosage compensation between XX females and XY males and ensures equal expression of X-linked genes in both sexes. Although all mammals use this form of dosage compensation, the complex mechanisms that regulate XCI vary between species, tissues, and development. These mechanisms include not only varying levels of inactivation, but also the nature of inactivation, which can range from being random in nature to driven by parent of origin. To date, no data describing XCI in calves or adult cattle have been reported and we are reliant on data from mice to infer potential mechanisms and timings for this process. In the context of dairy cattle breeding and genomic prediction, the implications of X chromosome inheritance and XCI in the mammary gland are particularly important where a relatively small number of bulls pass their single X chromosome on to all of their daughters. We describe here the use of RNA-seq, whole genome sequencing and Illumina BovineHD BeadChip (Illumina, San Diego, CA) genotypes to assess XCI in lactating mammary glands of dairy cattle. At a population level, maternally and paternally inherited copies of the X chromosome are expressed equally in the lactating mammary gland consistent with random inactivation of the X chromosome. However, average expression of the paternal chromosome ranged from 10 to 90% depending on the individual animal. These results suggest that either the mammary gland arises from 1 or 2 stem cells, or a nongenetic mechanism that skews XCI exists. Although a considerable amount of future work is required to fully understand XCI in cattle, the data reported here represent an initial step in ensuring that X chromosome variation is captured and used in an appropriate manner for future genomic selection. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms have been the DNA variant of choice for genomic prediction, largely because of the ease of single nucleotide polymorphism genotype collection. In contrast, structural variants (SV), which include copy number variants (CNV), translocations, insertions, and inversions, have eluded easy detection and characterization, particularly in nonhuman species. However, evidence increasingly shows that SV not only contribute a substantial proportion of genetic variation but also have significant influence on phenotypes. Here we present the discovery of CNV in a prominent New Zealand dairy bull using long-read PacBio (Pacific Biosciences, Menlo Park, CA) sequencing technology and the Sniffles SV discovery tool (version 0.0.1; https://github.com/fritzsedlazeck/Sniffles). The CNV identified from long reads were compared with CNV discovered in the same bull from Illumina sequencing using CNVnator (read depth-based tool; Illumina Inc., San Diego, CA) as a means of validation. Subsequently, further validation was undertaken using whole-genome Illumina sequencing of 556 cattle representing the wider New Zealand dairy cattle population. Very limited overlap was observed in CNV discovered from the 2 sequencing platforms, in part because of the differences in size of CNV detected. Only a few CNV were therefore able to be validated using this approach. However, the ability to use CNVnator to genotype the 557 cattle for copy number across all regions identified as putative CNV allowed a genome-wide assessment of transmission level of copy number based on pedigree. The more highly transmissible a putative CNV region was observed to be, the more likely the distribution of copy number was multimodal across the 557 sequenced animals. Furthermore, visual assessment of highly transmissible CNV regions provided evidence supporting the presence of CNV across the sequenced animals. This transmission-based approach was able to confirm a subset of CNV that segregates in the New Zealand dairy cattle population. Genome-wide identification and validation of CNV is an important step toward their inclusion in genomic selection strategies. [ABSTRACT FROM AUTHOR]

Residual feed intake (RFI), as a measure of feed conversion during growth, was estimated for around 2,000 growing Holstein-Friesian heifer calves aged 6 to 9 mo in New Zealand and Australia, and individuals from the most and least efficient deciles (low and high RFI phenotypes) were retained. These animals (78 New Zealand cows, 105 Australian cows) were reevaluated during their first lactation to determine if divergence for RFI observed during growth was maintained during lactation. Mean daily body weight (BW) gain during assessment as calves had been 0.86 and 1.15 kg for the respective countries, and the divergence in RFI between most and least efficient deciles for growth was 21% (1.39 and 1.42 kg of dry matter, for New Zealand and Australia, respectively). At the commencement of evaluation during lactation, the cows were aged 26 to 29 mo. All were fed alfalfa and grass cubes; it was the sole diet in New Zealand, whereas 6 kg of crushed wheat/d was also fed in Australia. Measurements of RFI during lactation occurred for 34 to 37 d with measurements of milk production (daily), milk composition (2 to 3 times per week), BW and BW change (1 to 3 times per week), as well as body condition score (BCS). Daily milk production averaged 13.8 kg for New Zealand cows and 20.0 kg in Australia. No statistically significant differences were observed between calf RFI decile groups for dry matter intake, milk production, BW change, or BCS; however a significant difference was noted between groups for lactating RFI. Residual feed intake was about 3% lower for lactating cows identified as most efficient as growing calves, and no negative effects on production were observed. These results support the hypothesis that calves divergent for RFI during growth are also divergent for RFI when lactating. The causes for this reduced divergence need to be investigated to ensure that genetic selection programs based on low RFI (better efficiency) are robust. [ABSTRACT FROM AUTHOR]

Feed makes up a large proportion of variable costs in dairying. For this reason, selection for traits associated with feed conversion efficiency should lead to greater profitability of dairying. Residual feed intake (RFI) is the difference between actual and predicted feed intakes and is a useful selection criterion for greater feed efficiency. However, measuring individual feed intakes on a large scale is prohibitively expensive. A panel of DNA markers explaining genetic variation in this trait would enable cost-effective genomic selection for this trait. With the aim of enabling genomic selection for RFI, we used data from almost 2,000 heifers measured for growth rate and feed intake in Australia (AU) and New Zealand (NZ) genotyped for 625,000 single nucleotide polymorphism (SNP) markers. Substantial variation in RFI and 250-d body weight (BW250) was demonstrated. Heritabilities of RFI and BW250 estimated using genomic relationships among the heifers were 0.22 and 0.28 in AU heifers and 0.38 and 0.44 in NZ heifers, respectively. Genomic breeding values for RFI and BW250 were derived using genomic BLUP and 2 Bayesian methods (BayesA, BayesMulti). The accuracies of genomic breeding values for RFI were evaluated using cross-validation. When 624,930 SNP were used to derive the prediction equation, the accuracies averaged 0.37 and 0.31 for RFI in AU and NZ validation data sets, respectively, and 0.40 and 0.25 for BW250 in AU and NZ, respectively. The greatest advantage of using the full 624,930 SNP over a reduced panel of 36,673 SNP (the widely used BovineSNP50 array) was when the reference population included only animals from either the AU or the NZ experiment. Finally, the Bayesian methods were also used for quantitative trait loci detection. On chromosome 14 at around 25 Mb, several SNP closest to PLAG1 (a gene believed to affect stature in humans and cattle) had an effect on BW250 in both AU and NZ populations. In addition, 8 SNP with large effects on RFI were located on chromosome 14 at around 35.7 Mb. These SNP may be associated with the gene NCOA2, which has a role in controlling energy metabolism. [ABSTRACT FROM AUTHOR]

Selection for divergence between individuals for efficiency of feed utilization (residual feed intake, RFI) has widespread application in the beef industry and is usually undertaken when animals are fed diets based on silages with grain. The objective of this research was to develop a feeding system (using Gallagher, Hamilton, New Zealand, electronics) to measure RFI for growth in Holstein-Friesian heifers (aged 5--9 mo), and identify divergent individuals to be tested for RFI during lactation. A dry forage diet (alfalfa cubes) was fed because intakes could be measured accurately, and the New Zealand dairy industry (4.4 million milking cows in lactation) relies heavily on forage feeding. The evaluation was undertaken over 3 yr with 1,052 animals fed in a facility for 7 wk, and weighed 3 times weekly. The mean age at the start of measurements was 215 d, body weight (BW) 189 kg, and mean daily dry matter intakes averaged 6.7 kg. Body weight gain (all animals) averaged 0.88 kg/d. The RFI was determined as the residuals from the regression of mean intake on mean BW0.75 and daily BW gain of individuals. Actual and fitted intakes were strongly related (R² = 0.82). In terms of gross efficiency (feed intake/BW gain), RFI + year explained 43% of the variation, BW gain + year explained 66%, and RFI + BW gain + year explained 79% of the variation (all P < 0.001). Daily BW gains (kg) of the most and least efficient 10% averaged (± standard deviation) 0.88 ± 0.15 and 0.88 ± 0.12 (P = 0.568), respectively, and the divergence between mean intakes was 1.46 kg of dry matter/d. The most and least efficient animals will be tested for RFI during lactation and genetic markers will be identified for the trait. [ABSTRACT FROM AUTHOR]

Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. A bovine cDNA microarray comprising approximately 22,000 expressed sequence tags was used to evaluate the transcriptional changes that occur in the mammary gland after the onset of clinical Strep. uberis mastitis. Five lactating Friesian heifers were intramammary infused in an uninfected quarter with approximately 1,000 to 1,500 cfu of a wild-type strain of Strep. uberis. Microarray results showed that Strep. uberis mastitis led to the differential expression of more than 2,200 genes by greater than 1.5-fold compared with noninfected control quarters. The most highly upregulated genes were associated with the immune response, programmed cell death, and oxidative stress. Quantitative real-time reverse transcription PCR analysis confirmed the increase in mRNA expression of immune-related genes complement component 3, clusterin, IL-8, calgranulin C, IFN-γ IL-10, IL-1β, IL-6, toll-like receptor-2, tumor necrosis factor-α, serum amyloid A3, lactoferrin, LPS-bonding protein, and oxidative stress-related genes metallothionein 1A and superoxide dimutase 2. In contrast, a decrease of mRNA levels was observed for the major milk protein genes. Bovine mammary epithelial cells in culture challenged with the same Strep. uberis strain used to induce clinical mastitis in the in vivo animal experiment did not cause a change in the mRNA levels of the immune-related genes. This suggests that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not Strep. uberis. However, challenging epithelial cells with different Strep. uberis strains and Staphylococcus aureus resulted in an increase in the mRNA expression of a subset of the immune-related genes measured. In comparison, an Escherichia coli challenge caused an increase in the majority of immune-related genes measured. Results demonstrate the complexity of the bovine mammary gland immune response to an infecting pathogen and indicate that a coordinated response exists between the resident, recruited, and inducible immune factors. [ABSTRACT FROM AUTHOR]

The membrane spanning enzyme γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) catalyses the breakdown of the tripeptide glutathione and uses free amino acids (AA) to form γ-glutamyl transpeptidase (GT) AA that become transported into cells and converted back into free AA. γ-Glutamyl transpeptidase activity has been shown to be important for mammary AA uptake in rodent systems, and while γ-GT activity is high in lactating bovine mammary tissue, the role of this enzyme in milk protein synthesis of the ruminant has not been defined. The present study shows that γ-GT activity in the ovine mammary gland, like that of rodents, increases during pregnancy and peaks early in lactation. Acivicin, a well-known inhibitor of γ-GT, decreased γGT activity in acini isolated from the ovine mammary gland and did not have secondary toxicity effects on cell viability or the uptake of radiolabeled amino-isobutyric acid. Isolated ovine acini were incubated in the presence of radiolabeled leucine, and incorporation of label into secreted protein increased during incubation. Incubation of acini with acivicin decreased milk protein secretion by 75%, indicating that γ-GT plays an important role in milk protein production in the ruminant. Acivicin did not inhibit secretion of specific caseins but caused a global decrease in individual proteins suggesting that γ-GT may be responsible for providing a complement of AA for milk protein synthesis. [ABSTRACT FROM AUTHOR]

Accurate and timely pregnancy diagnosis is an important component of effective herd management in dairy cattle. Predicting pregnancy from Fourier-transform mid-infrared (FT-MIR) spectroscopy data is of particular interest because the data are often already available from routine milk testing. The purpose of this study was to evaluate how well pregnancy status could be predicted in a large data set of 1,161,436 FT-MIR milk spectra records from 863,982 mixed-breed pasture-based New Zealand dairy cattle managed within seasonal calving systems. Three strategies were assessed for defining the nonpregnant cows when partitioning the records according to pregnancy status in the training population. Two of these used records for cows with a subsequent calving only, whereas the third also included records for cows without a subsequent calving. For each partitioning strategy, partial least squares discriminant analysis models were developed, whereby spectra from all the cows in 80% of herds were used to train the models, and predictions on cows in the remaining herds were used for validation. A separate data set was also used as a secondary validation, whereby pregnancy diagnosis had been assigned according to the presence of pregnancy-associated glycoproteins (PAG) in the milk samples. We examined different ways of accounting for stage of lactation in the prediction models, either by including it as an effect in the prediction model, or by pre-adjusting spectra before fitting the model. For a subset of strategies, we also assessed prediction accuracies from deep learning approaches, utilizing either the raw spectra or images of spectra. Across all strategies, prediction accuracies were highest for models using the unadjusted spectra as model predictors. Strategies for cows with a subsequent calving performed well in herd-independent validation with sensitivities above 0.79, specificities above 0.91 and area under the receiver operating characteristic curve (AUC) values over 0.91. However, for these strategies, the specificity to predict nonpregnant cows in the external PAG data set was poor (0.002-0.04). The best performing models were those that included records for cows without a subsequent calving, and used unadjusted spectra and days in milk as predictors, with consistent results observed across the training, herd-independent validation and PAG data sets. For the partial least squares discriminant analysis model, sensitivity was 0.71, specificity was 0.54 and AUC values were 0.68 in the PAG data set; and for an image-based deep learning model, the sensitivity was 0.74, specificity was 0.52 and the AUC value was 0.69. Our results demonstrate that in pasture-based seasonal calving herds, confounding between pregnancy status and spectral changes associated with stage of lactation can inflate prediction accuracies. When the effect of this confounding was reduced, prediction accuracies were not sufficiently high enough to use as a sole indicator of pregnancy status., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to estimate genetic correlations among milk fatty acid (FA) concentrations in New Zealand dairy cattle. Concentrations of each of the most common FA, expressed as a percentage of the total FA, were determined by gas chromatography on a specific cohort of animals. Using this data set, prediction equations were derived using mid-infrared (MIR) spectroscopy data collected from the same samples. These prediction equations were applied to a large data set of MIR measurements in 34,141 milk samples from 3,445 Holstein-Friesian, 2,935 Jersey, and 3,609 crossbred Holstein-Friesian × Jersey cows, sampled an average of 3.42 times during the 2007-2008 season. Data were analyzed using univariate and bivariate repeatability animal models. Heritability of predicted FA concentration in milk fat ranged from 0.21 to 0.42, indicating that genetic selection could be used to change the FA composition of milk. The de novo synthesized FA (C6:0, C8:0, C10:0, C12:0, and C14:0) showed strong positive genetic correlations with each other, ranging from 0.24 to 0.99. Saturated FA were negatively correlated with unsaturated (-0.93) and polyunsaturated (-0.84) FA. The saturated FA were positively correlated with milk fat yield and fat percentage, whereas the unsaturated FA were negatively associated with fat yield and fat percentage. Our results indicate that bovine milk FA composition can be changed through genetic selection using MIR as a phenotypic proxy., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The use of Fourier-transform mid-infrared (FTIR) spectroscopy is of interest to the dairy industry worldwide for predicting milk composition and other novel traits that are difficult or expensive to measure directly. Although there are many valuable applications for FTIR spectra, noise from differences in spectral responses between instruments is problematic because it reduces prediction accuracy if ignored. The purpose of this study was to develop strategies to reduce the impact of noise and to compare methods for standardizing FTIR spectra in order to reduce between-instrument variability in multiple-instrument networks. Noise levels in bands of the infrared spectrum caused by the water content of milk were characterized, and a method for identifying and removing outliers was developed. Two standardization methods were assessed and compared: piecewise direct standardization (PDS), which related spectra on a primary instrument to spectra on 5 other (secondary) instruments using identical milk-based reference samples (n = 918) analyzed across the 6 instruments; and retroactive percentile standardization (RPS), whereby percentiles of observed spectra from routine milk test samples (n = 2,044,094) were used to map and exploit primary- and secondary-instrument relationships. Different applications of each method were studied to determine the optimal way to implement each method across time. Industry-standard predictions of milk components from 2,044,094 spectra records were regressed against predictions from spectra before and after standardization using PDS or RPS. The PDS approach resulted in an overall decrease in root mean square error between industry-standard predictions and predictions from spectra from 0.190 to 0.071 g/100 mL for fat, from 0.129 to 0.055 g/100 mL for protein, and from 0.143 to 0.088 g/100 mL for lactose. Reductions in prediction error for RPS were similar but less consistent than those for PDS across time, but similar reductions were achieved when PDS coefficients were updated monthly and separate primary instruments were assigned for the North and South Islands of New Zealand. We demonstrated that the PDS approach is the most consistent method to reduce prediction errors across time. We also showed that the RPS approach is sensitive to shifts in milk composition but can be used to reduce prediction errors, provided that secondary-instrument spectra are standardized to a primary instrument with samples of broadly equivalent milk composition. Appropriate implementation of either of these approaches will improve the quality of predictions based on FTIR spectra for various downstream applications., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

In cattle, the X chromosome accounts for approximately 3 and 6% of the genome in bulls and cows, respectively. In spite of the large size of this chromosome, very few studies report analysis of the X chromosome in genome-wide association studies and genomic selection. This lack of genetic interrogation is likely due to the complexities of undertaking these studies given the hemizygous state of some, but not all, of the X chromosome in males. The first step in facilitating analysis of this gene-rich chromosome is to accurately identify coordinates for the pseudoautosomal boundary (PAB) to split the chromosome into a region that may be treated as autosomal sequence (pseudoautosomal region) and a region that requires more complex statistical models. With the recent release of ARS-UCD1.2, a more complete and accurate assembly of the cattle genome than was previously available, it is timely to fine map the PAB for the first time. Here we report the use of SNP chip genotypes, short-read sequences, and long-read sequences to fine map the PAB (X chromosome:133,300,518) and simultaneously determine the neighboring regions of reduced homology and true pseudoautosomal region. These results greatly facilitate the inclusion of the X chromosome in genome-wide association studies, genomic selection, and other genetic analysis undertaken on this reference genome., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

The genetic merit of a herd is a key determinant in productivity for dairy farmers. However, making breeding decisions to maximize the rate of genetic gain can be complex because there is no certainty about which cows will become pregnant with a heifer calf. In this study, breeding worth (BrW) was used as a measure of genetic merit, and several mating strategies were evaluated. These strategies included randomly mating whole herds to the entire bull team, excluding low-ranked cows from producing replacement heifers, and nominating high-ranked cows to the most highly ranked bulls. Simulations were undertaken using 4 bull teams generated from bulls currently marketed in New Zealand and a selection of New Zealand dairy herds. Average replacement heifer BrW was calculated for 1,000 iterations of each combination of mating strategy, herd, and bull team (scenario). Variation in resulting average replacement heifer BrW within scenarios was due to random sampling of which cows became pregnant with a heifer calf. Relative to mating the whole herd to an entire bull team, excluding the lowest ranked cows from producing replacements resulted in the greatest increase in average replacement heifer BrW across all herds and bull teams, with a gain of approximately 0.4 BrW point for each 1% of cows excluded. Nominating top-ranking cows to the highest ranking bulls in the team had little effect (0.06-0.13 BrW increase for each 1% of top cows nominated) in improving BrW of replacement heifers. The number of top bulls nominated had a variable effect depending on the BrW spread of the entire bull team. Although excluding cows with the lowest BrW from producing replacement heifers is most effective for improving BrW, it is important to ensure that the number of heifers born is sufficient to replace cows leaving the herd. It is likely that optimal strategies for improving BrW will vary from farm to farm depending not only on the BrW structure of the herd, the bull team available, and the reproduction success on farm but also on farm management practices. This simulation study provides expected outcomes from a variety of mating strategies to allow informed decision making on farm., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).)

Our objective was to test the hypothesis that local production of the vasorelaxant nitric oxide could regulate mammary blood flow. In four lactating Saanen goats, the response of mammary blood flow to intraarterial infusion of the nitric oxide donor diethylamine NONOate and the inhibitor of nitric oxide synthesis N omega-nitro-arginine was measured. Diethylamine NONOate induced a rapid and sustained increase of mammary blood flow in the infused gland only, suggesting a direct effect on vasculature of the mammary gland. In contrast, infusion of N omega-nitro-arginine decreased mammary blood flow by up to 35%, and the coinfusion of arginine, the nitric oxide precursor, with N omega-nitro-arginine markedly reduced its ability to decrease mammary blood flow. The distribution of nitric oxide synthase was investigated in cryosections of caprine and bovine mammary tissue by histochemical staining for NADPH-diaphorase activity and by immunocytochemistry using specific antibodies against two nitric oxide synthase isoforms. Both techniques revealed nitric oxide synthase in the vascular endothelium and secretory epithelium of the two species. Only antibodies against nitric oxide synthase-III showed specific staining. These results suggest that the mammary gland produces and responds to nitric oxide and, further, raise the possibility that the epithelium may control its own blood supply by secreting nitric oxide.

Accumulation of lactoferrin mRNA in mammary tissue from virgin, pregnant, lactating, and involuting ewes and cows was localized using 35S-labeled cRNA probes. Expression of lactoferrin was low in the glands of virgin animals. In the glands of animals in early pregnancy, very high expression occurred in the ducts and immature alveoli, but expression tended to decrease as the alveoli matured. In the lactating and involuting gland, expression was generally low or absent in actively secreting alveoli and high in alveoli that had an accumulation of vesicles in the lumen and secretory epithelium, which was indicative of stasis. Occasionally, expression of lactoferrin was seen in cells that appeared to be secretory, particularly in involuting glands. Lactoferrin mRNA was expressed not only at different sites from other milk protein genes, such as alpha-lactalbumin and alpha s1-casein, but also during different stages of mammary development, supporting the view that the expression of lactoferrin is regulated differently from that of other milk proteins. For all ewes and cows, lactoferrin mRNA was detected in the epithelial ducts of the mammary parenchyma and the teat in a gradient that increased in ducts nearer the teats. The expression of lactoferrin in the ductal epithelium close to the teat was consistent with the antibacterial role of lactoferrin.

Although milk yield of cows and goats is known to be closely related to the total flow of blood through the udder, a number of studies suggest that milk yield can vary independently. No studies have attempted to measure the proportion of total flow that is nutritive. Within the mammary gland, capillary networks form a basket-like architecture surrounding each alveolus. Notably, flow in individual capillaries is not constant and varies among capillaries. Capillary flow (measured by intravital microscopy) was decreased by oxytocin, which generally increased total flow in the mammary artery, suggesting that the proportion of total flow that is nutritive can vary. In addition to classic metabolic regulators (e.g., carbon dioxide and oxygen) of tissue blood flow, the mammary gland produces a number of vasodilatory compounds, including parathyroid hormone-related protein, insulin-like growth factor-I, prostacyclin, nitric oxide, and endothelin. All of these compounds have been shown to alter mammary blood flow. Mammary tissue also contains kallikrein and angiotensin-converting enzyme, which convert circulating kinins and angiotensin, respectively, into potent vasoactive compounds. A number of these compounds are produced by epithelial cells themselves, providing a mechanism for the functioning epithelium to control its own blood supply and, hence, nutrient flow for milk synthesis. In this review, we examine the nature of the mammary microcirculation, its behavior under different conditions, and some of the regulatory features of the mammary microvasculature.

Eight primiparous cows in midlactation were used to determine a method for the mammary biopsy of standing cows in full lactation. Cows were mildly sedated; therefore, preoperative feed withdrawal was not necessary. A core of secretory tissue (0.75 to 1 g) was extracted using a rotating stainless steel cannula with a retractable blade at the cutting edge. Postoperative recovery was rapid, taking only 15 min per cow, and the method was reliable and efficient. The presence of secretory tissue was verified by histology and in situ hybridization with alpha s1-casein and alpha-lactalbumin probes. The capsular end of the core contained more connective tissue, and the parenchyma showed heterogeneous expression of alpha s1-casein and alpha-lactalbumin. Despite some postoperative bleeding, milk yield and composition in the biopsied gland were affected only transiently. Yield recovered by 3.5 d after biopsy, and composition recovered by 6.5 d after biopsy. Yield and composition of milk from the control glands were not affected by the procedure. Biopsy sites healed rapidly and without infection. No clinical mastitis was observed in any of the biopsied cows throughout the remainder of the lactation.

Six pairs of identical twin cows during late lactation (213 d) were used to study the effect of milking frequency (twice vs. once daily) and bST during once daily milking on the activity of plasminogen activator, plasminogen, and plasmin in milk. Less frequent milking increased the activity of plasminogen, plasmin, and plasminogen activator in milk. The ratio of plasminogen to plasmin, a measure that is independent of milk volume, decreased during less frequent milking, suggesting that at least part of the increase in activity of plasmin was due to the accelerated conversion of plasminogen to plasmin. Changes in the activity of plasminogen and plasmin in milk were positively correlated with increases in the concentrations of milk BSA and plasma lactose, both of which are indicators of disruption of tight junctions between mammary epithelial cells, indicating that paracellular leakage may have contributed to increased protease activity in milk during less frequent milking. No correlation existed between changes in plasminogen activator and indicators of tight junction disruption, suggesting that increased activity of plasminogen activator in milk was not due to leakage across the mammary epithelium, but rather to increased local production in the mammary gland. Administration of bST during once daily milking did not significantly affect milk protease activity.

Six pairs of monozygous Friesian twin cows during late lactation were used to assess the effect of once daily milking and bST treatment on yields and tight junction permeability of mammary epithelial cells. During the first 7 d, all cows were milked twice daily; on d 8 through d 21, all cows were milked once daily, but one cow of each twin pair was treated daily with 20 mg of bST on d 13 through 21; and, finally, during d 22 through 28, all cows were again milked twice daily. Once daily milking, a common management practice in New Zealand, resulted in a small (7%) but significant decrease in milk yield. Treatment with bST increased milk yield by 19%, thereby exceeding the milk yield loss from once daily milking. The integrity of mammary tight junctions was assessed indirectly by measuring concentrations of plasma lactose and milk BSA. Once daily milking temporarily disrupted tight junction integrity, based on a 4- to 5-fold increase in plasma lactose and a 42 to 55% increase in the concentrations of milk BSA. In the present study, bST did not affect the permeability of mammary tight junctions.

The timing and relation of changes in mammary epithelial cell tight junction integrity and mammary blood flow during a 36-h milking interval were studied in six lactating Saanen goats. An increase in lactose concentration in plasma, a decrease in transepithelial potential difference, and changes in ionic milk composition were used to indicate tight junction patency. After 36 h of milk accumulation, mammary tight junctions had become disrupted. Further analyses indicated that this disruption began after 21 h of milk accumulation and that mammary blood flow also started to decline after 21 h. The time when both events occurred was not significantly different from the time when milk secretion began to decline (19 h). Moreover, positive but nonsignificant correlations existed between these events. Mammary tight junctions became disrupted when milk secretion declined, suggesting that impairment of mammary tight junction integrity is associated with decreased milk secretion during an extended milking interval. The decline in mammary blood flow may be the result of a negative feedback response to a reduced demand for metabolites, which is due to a reduced rate of milk secretion.

The physiological responses to somatotropin supplementation of ruminants are reviewed with particular reference to the dairy industry. Exogenous somatotropin improves lactational performance by altering the partitioning of absorbed nutrients. Lipid accretion is reduced, lipid mobilization is increased, and glucose uptake by peripheral tissues and whole body oxidation of glucose and amino acids are reduced. The net effect of these changes is that the limited supply of glucose and amino acids is spared for synthesis of milk components, and lipid reserves are preferentially used as an energy source. The increase of cardiac output and mammary blood flow are prominent physiological responses to somatotropin by the dairy cow. These responses permit a greater share of nutrient input to the mammary gland. The lack of evidence for functional membrane receptors for somatotropin in mammary tissue demands the involvement of endocrine mediators to explain the increase of mammary gland metabolism with somatotropin treatment. An increase of high affinity somatotropic receptors in the liver and increased plasma insulin-like growth factor-I and -II appear to be one of the most prominent endocrine responses to somatotropin treatment. However, the relative role of the insulin-like growth factors and the multiple forms of their plasma-binding proteins and receptors remain to be resolved.

In ruminants, mammary supply of substrate varies with rate of mammary blood flow and concentrations of blood substrates. Blood concentrations of most mammary substrates, except acetate and tryptophan, do not vary greatly with feed intake, short term. Fasting reduces mammary blood flow, whereas milking and injection of growth hormone or thyroxine increase flow. It is proposed that the fraction of cardiac output that perfuses the udder of lactating ruminants plays a role in regulation of nutrient partitioning between milk and body tissues. In fed animals this fraction is 15 to 16% of cardiac output, which declines on fasting to 8 to 9% and increases slightly following growth hormone treatment to 17.6%. Following realimentation of fasted cows or goats, mammary blood flow takes several hours to return to normal. Investigation of the mechanism of this response, in terms of the ability of the animal to recognize its nutritional status and partition nutrients accordingly, should prove fruitful to understanding causes of variations of milk production in response to feed quantity and quality. Several substrates show increased mammary arteriovenous difference with increasing blood concentrations. This may reflect differing ratios of blood flow:milk yield. The steep gradient of concentration of substrates across the mammary epithelial cell membrane suggests that a major impediment to substrate supply for milk synthesis is the rate of substrate transport across the membrane.

An intravaginal sponge impregnated with 500 mg estradiol-17 beta and 1000 mg progesterone to induce lactation in nonpregnant dairy cattle was investigated. Sponge retention (greater than 95%) was achieved by its attachment to a nylon collar. Treatment with the sponge for 10 days resulted in lactogenesis in 25% of treated cows compared with an 89% success rate when an adjunct injection of dexamethasone esters (20 mg intramuscular) was given on day 6 or a 96% success rate when reserpine injections (2.5 mg intramuscular) were given on days 6, 8, and 10 10 after sponge insertion. Milking was begun 12 days after sponge insertion (2 days after removal). Peak milk yield was unaffected by adjunct treatments but was greater in spring (mean 11.5 kg/day) than in autumn treated cows (mean 3 and 6 kg/day) in 2 yr of trials. Concentration of estradiol-17 beta in plasma rose rapidly after sponge insertion to a peak similar to that in cows in late pregnancy. Measurement of estradiol-17 beta concentration in milk indicated less, even at first milking than in commercially distributed pasteurized milk. Milk fat and protein content were higher in milk from induced lactations compared with that from normally calving cows on the same day.

The physiology of growth hormone and the insulin-like growth factors are reviewed with particular reference to the dairy industry. Growth hormone secretion in the ruminant is pulsatile in nature and nutritional factors have a major impact on its secretion. Isolation of growth hormone-releasing factor has allowed further progress in understanding the mechanisms underlying growth hormone release. The receptors appear to be under active endocrine and metabolic control, and nutritional influences on the somatotropic axis are in large part mediated through changes in somatotropic receptors. The mode of action of growth hormone to induce acute metabolic affects and lipolysis remains to be resolved, but there is increasing evidence that its anabolic actions are mediated by the insulin-like growth factors. Recent studies of measurement of insulin-like growth factor-1 and -2 in the ruminant and the role of growth hormone, nutrition, insulin, and sex steroids in their regulation are reviewed. The relative role of the two factors and the multiple forms of their receptors remain to be resolved. It is well-documented that growth hormone is galactopoietic. The evidence that this effect is largely due to enhanced nutrient supply to the mammary gland is not convincing. Effects of growth hormone are indirect and may be mediated by the insulin-like growth factors. The potential is considerable for manipulating the growth hormone insulin-like growth factor axis to enhance lactation.