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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 number of secretory cells in the mammary gland is often cited as a major determinant of milk production. However, literature data for proxy measures of secretory cell number do not fully support such a claim. In particular, measurements of total mammary DNA in livestock explain only <25% of variation in milk yield, probably because of tissue heterogeneity for DNA concentration. Relative to BW, measurements of udder size in dairy cattle, as total DNA or udder weight, are approximately double those seen in most other livestock classes. Therefore, selection for dairy production, not surprisingly, has resulted in cows with greater secretory capacity. There is limited evidence that genetic selection is still increasing udder size in some cattle populations, but more recent data are needed. It is contended that the most important period of mammary growth for determination of milk yield is that occurring in pregnancy and early lactation. Mammary development is largely complete, at term, in sheep, goats, and cattle, but in pigs, the udder continues to grow during the first 3 wk of lactation, depending, in part, on litter size. Increased litter size in sheep and goats will enhance the extent of mammary development at the end of gestation (and milk yield) by 20 to 25%. However, twinning in dairy cattle does not affect milk production and, by inference only, is not likely to affect numbers of secretory cells at term. Milking frequency and suckling intensity in very early lactation will increase milk yield in cows and increase mammogenesis and milk yield in sheep, indicating that even at term, the ruminant gland retains some capacity for further development, if demand requires it. There is limited understanding of the hormonal signals in pregnancy that regulate mammary development relative to the number of young carried. Furthermore, the genetic differences between dairy and beef cattle that lead to substantially greater udder size in the dairy breeds have not been identified. During lactation, the drivers for secretory cell loss in relation to milking frequency and nutritional status are still not known. Measurement of mammary development and using this phenotype in genomewide association studies to identify key genetic variants for mammogenesis will provide knowledge that is fundamental to understanding the quantitative regulation of milk production.

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 7d (mid lactation) or 2 or 4d (late lactation) after which cows were milked out and twice-daily milking resumed for 4d. 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 2d 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., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 mid- and 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 (R(2)=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 (R(2)=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., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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, genotypes 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 posttrial 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., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 N(1)-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 alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-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.

New molecular and quantitative genetic technologies are the latest in a long list of technologies that have been introduced to dairy industries over many decades to improve the performance of cows. The catalysts for future advances will be sequencing of the bovine genome and development of high-throughput technologies to identify and exploit relevant variation in DNA sequences. The new technologies will allow the selection of animals based on specific genotypes that suit specific applications such as once-daily (OAD) milking or extended lactation. These technologies will also allow exploitation of between-cow variation in milk composition, which is currently hidden by bulking of milk on and between farms. Thus, there are opportunities to produce milk from herds of cows selected for specific milk composition that might be, for example, more suitable for cheese-making or have milkfat with specific properties to enhance human health. Identification of genes and gene polymorphisms associated with improved resistance to specific diseases in cows and other farm species also represents a real opportunity to improve animal health over the coming decade. New levels of genetic regulation have been identified, e.g. microRNA and epigenetics, the impacts of which on the performance of cows and humans are only just beginning to be understood. There is a potential role for veterinarians to provide or be the interface for provision of genetic advice to farmers in much the same way that nutritional advice is currently given.

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.

A mathematical model of biological mechanisms regulating lactation is constructed. In particular, the model allows prediction of the effect of milking frequency on milk yield and mammary regression, and the interaction of nutrition and milking frequency in determining yield. Possible interactions of nutrition with milking frequency on alveolar dynamics are highlighted. The model is based upon the association of prolonged engorgement (as a consequence of milk accumulation) of active secretory alveoli with changes in gene expression that result in impairment and, ultimately, cessation of milk secretion. The emptying of alveoli at milking, following alveolar contraction induced by oxytocin, prevents this process and also allows quiescent alveoli to reactivate. Prolonged engorgement results in apoptosis of the secretory cells and, hence, regression of the mammary gland. Milk yield is linked to alveolar populations, with secretion rates being modulated by nutrition and udder fill effects. The model was used to investigate different management scenarios, and is in agreement with experimental results. The model shows that while milking frequency drives alveolar population, and therefore potential milk production, actual production varies considerably with nutrition. A significant portion of the loss associated with once-daily milking was due to udder fill rather than loss of secretory tissue. The model showed qualitative agreement with experimental data, on the acute and chronic effects of temporary once-daily milking.

The responses of the mammary microvasculature in lactating goats (n = 8) during feed withdrawal (18-20 h) and mammary engorgement (26-28 h of milk accumulation) were compared using an indicator-dilution technique with FITC-albumin and [(14)C]sucrose as the intravascular and diffusible indicators, respectively. Feed withdrawal and mammary engorgement caused a 50-60% decrease in mammary arterial flow and in the permeability-surface area product (PS) values for sucrose. Only feed withdrawal increased the mean transit time [from 17.3 to 30.0 s, SE of the difference (SED) = 2.16, P < 0.01] of FITC-albumin, whereas only mammary engorgement reduced sucrose extraction (0.63 to 0.51, SED = 0.04, P < 0.05). Mammary engorgement also caused a substantial reduction in the sucrose-accessible extravascular space from 92 to 44 ml (SED = 15.2, P < 0.01). In a separate experiment using five goats, milking after mammary engorgement did not immediately restore arterial flow or sucrose extraction, indicating that the effect of milk accumulation was not mediated simply via increased intramammary pressure. In conclusion, feed withdrawal resulted in slower flow in the capillary bed but apparently no change in capillary recruitment, whereas mammary engorgement caused capillary derecruitment.

The objective of this study was to investigate the role of amylin (a pancreatic hormone) in regulating metabolism in support of lactation. Rat amylin was infused (320 pmol.kg LW(-1).h(-1)) for 6 h via an external pudic (mammary) artery into six lactating goats. This dose of amylin led to a sixfold increase in plasma concentrations of amylin relative to baseline. Amylin infusion increased plasma concentrations (jugular) of glucose and NEFA up to 16 and 168%, respectively, relative to saline infusion. In contrast, plasma concentrations of Ca and PO4 during amylin infusion were reduced by 18 and 30%, respectively, relative to saline infusion. Plasma concentrations of IGF-I, insulin, and Mg were not different between the two treatments, although IGF-I concentrations in the amylin-infused group, 1 and 6 h postinfusion, were significantly higher than those in the saline-infused group. Similarly, amylin infusion failed to affect milk yield and major constituents of milk except protein; milk protein content decreased progressively until the end of amylin infusion and remained low thereafter. Amylin also had no effect on minerals in milk (Ca, PO4, Mg, Fe, Sr, S, K, or Na) except Zn, which was significantly decreased from 56.8+/-5.8 micromol/L at 0 h to 44.5+/-2.4 micromol/L at 6 h postinfusion. Mammary blood flow (measured with a transit-time blood flow probe) increased up to 26% during amylin infusion, although this effect lasted only for the first 3 h. In conclusion, amylin increased plasma concentrations of glucose and NEFA, and mammary blood flow, while decreasing plasma concentrations of Ca and PO4. Despite these metabolic changes, amylin infusion did not increase milk yield of lactating goats.

Experiments were undertaken to validate a method (using adrenaline injection) for determination of the size of cisternal and alveolar compartments in the udder, to use this method to determine the pattern of milk accumulation in the udder over time and to determine the relationship between the size of the alveolar and cisternal compartments and tolerance of once daily milking. Cows received intrajugular injections of adrenaline (3 mg) immediately before milking, to block milk ejection and allow harvesting of the cisternal milk fraction. This was followed by removal of the alveolar fraction 30 min later after intrajugular oxytocin (5 i.u.) injection. Results obtained were similar to those obtained by catheter drainage. The alveolar compartment was 90% full at 16 h post milking while the cisternal compartment filled more slowly and was only 70% full at 24 h post milking. At full capacity (measured at 40 h), the volumes of milk contained in the cisternal and alveolar compartments were similar. In a further experiment involving identical twin cows, it was shown that the greater the degree of filling of the cisternal compartment at 24 h, the lower was the production loss on once daily milking. This suggests that the freedom of the alveoli to drain was an important factor in the production loss on once daily milking. Although there were significant correlations within twin sets for milk yield and the size of udder compartments, the relationship within twin sets for yield loss on once daily milking was not statistically significant.

Eight cows in early lactation were used to study the effect of milk accumulation on the state of mammary tight junctions and to examine alpha-lactalbumin as an indicator of tight junction permeability in vivo. During three successive periods, the cows were milked twice (4 days), once (6 days), and twice daily (4 days). Plasma lactose, alpha-lactalbumin, and milk sodium concentrations were used as indicators of tight junction permeability. Furthermore, four cows were used to study the clearance of lactose and alpha-lactalbumin from the blood. Milk yield during once-daily milking decreased by 15.4% (P < 0.001). All indicators of mammary tight junction patency increased (P < 0.05) transiently during once-daily milking and indicated that tight junctions opened after approximately 18 h. Plasma alpha-lactalbumin and lactose were highly correlated (r = 0.82, P < 0.001), indicating the suitability of plasma alpha-lactalbumin as an indicator of tight junction status in vivo. Clearance of alpha-lactalbumin and lactose from the blood was best described by a biexponential model. Elimination half-lives for lactose and alpha-lactalbumin were 44 and 40 min, respectively. This study showed that milk stasis during early established lactation induces tight junctions to switch to a leaky state after approximately 18 h and to revert to the closed state shortly after milking.

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.

The suitability of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to induce disruption of mammary tight junctions (TJ) and its effect on milk secretion were investigated in six goats. EGTA was administered via the teat of one gland as an isosmotic (300 mosmol/l) K-EGTA solution (68 mM EGTA), whereas the control gland received an isosmotic sucrose solution. Lactose, Na, K, and Cl in milk, blood lactose, and the presence of Evans blue (EB) in mammary lymph were used as indicators of TJ disruption. EGTA caused transient (approximately 60 h) changes (P < 0.05) in the concentration of lactose, K, Na, and Cl in milk, consistent with loss of TJ integrity. This was confirmed by a rapid (< 1 h) increase (P < 0.05) in blood lactose levels. Moreover, EB appeared in lymph < 1 h after EGTA+EB treatment. Milk secretion declined unilaterally by 15% (P < 0.05) after EGTA and did not return to baseline until approximately 60 h after EGTA. EGTA caused a unilateral, temporary (first 7 h) increase in mammary blood flow. This study shows that a rapid temporary disruption of mammary TJ can be successfully induced in vivo and that such disruption compromises milk secretion.

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation. With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lactating sheep. alpha- and beta-casein protein synthesis, beta-lactoglobulin synthesis, and alpha-casein, beta-casein, and beta-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of alpha-S1-casein, beta-lactoglobulin, and alpha-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled alpha-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.

The aim of this study was to compare the plasma concentration profile, mammary blood flow response and transfer into milk of intact IGF-I with that of its truncated analogue, des(1-3)IGF-I (des-IGF-I). Each peptide was infused for 24 h into the pudic artery supplying one mammary gland of lactating goats (n = 5). Concentrations of IGF-I in plasma (from the jugular vein) rose rapidly during infusion of IGF-I or des-IGF-I to reach 510 +/- 62 and 640 +/- 32 ng/ml (mean +/- S.E.M.) respectively, compared with 262 +/- 35 ng/ml after a similar infusion of saline. Ligand blotting analysis indicated a significant increase in the intensity of [125I]IGF-I binding to the 40-43 kDa doublet (binding protein-3 (BP-3), P < 0.01) and the band at 31 kDa (P < 0.05) during infusion of either IGF-I or des-IGF-I, as compared with saline. Furthermore des-IGF-I induced a significant increase in intensity of binding to the 35 and 24 kDa bands, but IGF-I did not. Whereas [125I]IGF-I was distributed between BP-3 and the other binding proteins, [125I]des-IGF-I bound exclusively to BP-3. Mammary blood flow (MBF) increased 48 +/- 6% after 12 h of infusion of des-IGF-I, compared with an increase of 22 +/- 6% during IGF-I. The difference in response was significant at P < 0.05. In addition, more IGF-I was secreted into the milk of the infused than the non-infused gland during either infusion of IGF-I or des-IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Human synthetic parathyroid hormone-related protein (PTHrP) increased mammary blood flow (MBF) following close-arterial infusion via the external pudic artery in goats during mid-lactation. MBF increased 74 +/- 8% within 30 min of the start of continuous infusion of PTHrP compared with 10 +/- 3% in controls. MBF decreased by 90 min, however, and was not different from control values for the remainder of the infusion. The increase in plasma concentrations of calcium and decrease in phosphate during PTHrP suggests that this was not due to altered activity of PTHrP, but may relate to downregulation of response or production of counter-regulatory vasoconstrictive agents within the gland. This problem was alleviated when PTHrP was infused in a pulsatile fashion. An average 14-40% increase in MBF was achieved over 6 h, but this did not alter the rate of milk secretion, suggesting that mammary hyperaemia is not sufficient by itself to increase milk yield in the normally lactating goat. MBF increased in a dose-dependent fashion, although the lowest dose used to give a detectable response was approximately 40-fold higher than the concentration normally present in the mammary venous circulation. Thus, endogenous PTHrP may not be an important regulator of MBF during lactation in the goat.

Five lactating goats were infused, via an external pudic arterial catheter, directly into the mammary gland with 0.9% (w/v) NaCl (20 ml/h), recombinant human insulin-like growth factor-I (IGF-I; 80 nmol/h), recombinant human IGF-II (133 nmol/h) or IGF-I and IGF-II combined. The infusion was for 6 h and milk yield was determined every 2 h. The ratio of milk yield in the infused relative to the non-infused gland was changed only slightly by saline (2%), but increased to 9% (P < 0.05) in response to IGF-I and 8% (P < 0.05) in response to IGF-II. When combined, both peptides increased this ratio by 6%. These effects were elicited within 2-4 h of the beginning of infusion. Mammary blood flow increased 50-80% (P < 0.05) during all IGF infusions, but only 28% during saline treatment. Plasma insulin decreased 50% (P < 0.01) during the infusion of IGF-I alone or in combination with IGF-II and 25% in response to IGF-II alone. Whereas plasma glucose increased by approximately 10% during infusion of IGF-I alone or with IGF-II, it was not altered by infusion of IGF-II only. The rapidity and unilateral nature of the milk-yield response to IGF-I and IGF-II is consistent with their acting directly on mammary tissue itself. Thus, the present results demonstrate similar local and systemic actions induced by intramammary infusion of IGF-II and IGF-I, although the magnitude of the response to IGF-II tends to be less than that to IGF-I.

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.

Plasma and mammary efferent lymph concentrations of insulin-like growth factor I (IGF-I) were determined in lactating ewes before and after treatment with GH (10 mg/day) for 3 days. The lymph:plasma ratio of IGF-I increased from 0.34 to 0.47 after GH treatment when the IGF-I content of plasma increased by 19.4 nmol/l (from 32.1 nmol/l) and lymph by 13.7 nmol/l (from 10.7 nmol/l). This increase in the relative content of IGF-I in lymph was associated with increased lymph content of IGF-I in a lower molecular mass pool (nominally 50 kDa) derived by size exclusion chromatography. GH treatment increased the total binding capacity for IGF-I in both high (150 kDa) and low (50 kDa) molecular mass pools of plasma and the 150 kDa pool in lymph but there was a proportionally greater increase in 50 kDa total binding in lymph relative to plasma. Further, GH treatment increased the 'saturation' of the 50 kDa binding proteins but decreased the 'saturation' of the 150 kDa fraction, in both plasma and lymph. Ligand blot analysis of IGF-binding proteins (IGFBPs) in plasma and lymph showed that GH treatment of lactating sheep increased IGFBP-3 and decreased IGFBP-2 in plasma and lymph. Radioimmunoassay of IGFBP-2 showed that while GH treatment reduced the plasma content of IGFBP-2 by about half, the lymph:plasma ratio was increased from 0.68 to 0.87. GH treatment of lactating ewes not only increased the IGF-I content of plasma but increased the apparent efficiency of transfer of IGF-I across capillary endothelium to mammary efferent lymph.

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.

Five goats were injected with GH (15 mg/day), three goats received systemic infusions of insulin-like growth factor (IGF)-I (43 nmol/h) and four goats received systemic infusions of physiological saline (20 ml/h) on days 4-6 of a 10-day experimental period during mid-lactation. Milk yield increased by an average of 24% in GH-treated goats by the time of the third injection. In contrast, milk yield of IGF-I-infused goats did not differ from saline-infused animals although two of three goats did show a small increase (12%) after 36 h of IGF-I infusion. With GH and IGF-I treatments plasma IGF-I concentrations increased similarly, reaching maxima of 100-130 nmol/l within 24 h. Plasma IGF-I concentration was relatively constant in saline-infused goats at about 50 nmol/l throughout the experiment. Total IGF-I bound to 50 kDa and 150 kDa binding proteins in plasma was increased by GH and IGF-I treatments but, in contrast to IGF-I, GH increased the proportion of IGF-I bound to 150 kDa binding protein. In a second experiment, four goats received systemic infusion of IGF-I (43 nmol/h) and four goats received systemic infusion of physiological saline (20 ml/h). There was no evidence that milk yield was changed during IGF-I infusion. However, when those goats which had previously received IGF-I infusions were injected with GH, milk yield increased by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Four cows received thyroxine injections (T4; 20 mg/d) and three cows received growth hormone injections (GH; 44 mg/d) for 4 d during successive 16-d experimental periods. Measurement was made of milk yield, protein yield, mammary tyrosine and phenylalanine uptake, blood plasma hormone concentrations, mammary blood flow and cardiac output. Milk yield increased by 25% with T4 and 21% with GH treatment. Milk protein content tended to decline during T4 treatment and increase following GH treatment. Cardiac output increased by 8.9 liter/min (20%) and 4.6 liter/min (10%) with T4 and GH injection. Mammary blood flow (half-udder) increased from 3.6 to 4.9 liter/min (35%) and from 3.3 to 4.4 liter/min (33%) with T4 and GH treatment, respectively. These increases calculated on a whole-udder basis, accounted for 28% (T4) and 48% (GH) of the increases in cardiac output. The proportion of cardiac output perfusing the (whole) udder increased to 19.1% (T4) and 18.7% (GH), increases of 17 and 30%, respectively. Heart rate increased with T4 (but not GH treatment) from 80 to 115/min. Ratio of blood flow to milk yield was not changed by either treatment. The proportion of cardiac output perfusing the udder likely plays a major role in facilitating the partitioning of nutrients for milk synthesis.

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.

Examination of data pertaining to milk yield and litter growth rate of guinea-pigs at peak lactation showed that milk yield increased with increasing number of pups carried by the dam during pregnancy and also with increasing maternal weight. There was a significant linear correlation (P less than 0.05) of mammary gland weight (expressed per unit of maternal body weight) with litter size. Increasing litter size, within 18 h of parturition, from 1 pup to 5 pups did not elicit a response in milk yield. Decreasing litter size from 5 pups to 1 pup reduced milk yield to the level expected of a monotocous dam. Data are presented showing the ability of the guinea-pig pup to survive from birth without access to milk and the inadequacy of milk supply to meet litter requirements when the litter size is more than 2 pups. The mechanisms controlling lactation in the guinea-pig are discussed in relation to data obtained in other species.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.

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.

(1) The arterio-venous difference technique, previously used to measure mammary substrate uptake with cows and goats, was used to measure amino acid uptake by the mammary gland of the lactating Merino ewe. Possession of a single large superficial epigastric vein by the Merino ewe makes the Merino breed the most suitable for this type of study. (2) A method was developed enabling hourly measurement of milk yield without causing undue stress. Milk yield was essentially constant over a 7-8 h period. (3) Mammary extraction of most non-essential amino acids was low relative to output in milk protein and showed a greater variability with time than that found for the essential amino acids. There was a significant mammary extraction of ornithine and citrulline, neither amino acid being found in ovine milk protein. (4) Of the essential amino acids valine, isoleucine, leucine and arginine were taken up in excess of their requirement for milk protein synthesis. (5) On the basis of the extent of mammary extraction, methionine, lysine and leucine were first-, second- and third-limiting to the rate of milk protein synthesis. (6) Despite fluctuations in arterial amino acid concentrations the arterio-venous differences of the essential amino acids were relatively constant over a 7-8 h period. (7) The pattern of mammary amino acid uptake in the ewe is contrasted with that found in similar studies carried out with the lactating cow and goat.

Three cows received injections of thyroxine (T4; 20 mg/day), four cows GH (40 mg/day) and three cows saline (control; 10 ml/day) on days 5-8 of a 16-day experimental period during peak lactation. Milk yield increased 13% in cows given GH (from 14.6 to 16.5 kg/day) and 15% in cows given T4 (from 15.8 to 18.2 kg/day) but did not change in control cows. Injection of T4 increased milkfat and lactose content but reduced milk protein content. Injection of GH was without effect on milk composition during the injection period but milk protein rose after injections ceased. Injection of T4 increased plasma concentrations of T4 and tri-iodothyronine six- to sevenfold, with maxima occurring on day 9. Injection of GH increased the plasma concentration of GH five- to tenfold 5 h after injection. The plasma concentration of insulin-like growth factor I (IGF-I) was increased in cows given GH in both morning (08.30 h) and afternoon (14.30 h) blood samples, the difference being greatest in afternoon samples in which plasma IGF-I content increased from 3.3 to 6.8 nmol/l. Injection of T4 reduced the plasma concentration of IGF-I in morning samples but the concentration in afternoon samples remained relatively constant throughout the 16-day experimental period. The plasma concentration of IGF-II rose in morning samples in all treatment groups to reach a maximum of 200-250 nmol/l by day 9. The galactopoietic response to injection of GH but not T4 was associated with an increase in plasma concentration of IGF-I. Changes in plasma concentration of IGF-II were not associated with changes in milk yield.

(1) The validity of using the arterio-venous concentration difference of methionine to calculate mammary blood flow in the ewe, on the basis of the Fick principle, is discussed. (2) Calculation of mammary blood flow in the lactating Merino ewe indicated that blood flow per unit weight of tissue and the ratio of blood flow : milk yield were approximately twice that found in the lactating cow and goat. (3) Calculated mammary blood flow in Merino ewes was used in conjunction with glucose arterio-venous difference to determine mammary glucose uptake. Glucose uptake per unit weight of tissue in the ewe was almost double that found in the cow and goat. The ratio of mammary glucose uptake to lactose output was also higher in the ewe than that found in the cow and goat. The utilization of glucose by the mammary gland of the ewe is discussed in relation to the possible greater requirement for NADPH and glycerol for milk fat synthesis in this species.

The change in epithelial cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) causing loss of secretory activity and apoptosis. Down-regulation of β1-integrin and TJ protein expression reflect a loss of cell-extracellular matrix communication and TJ integrity, respectively, during mammary apoptosis. This study examined the effect of acute physical distension of rat mammary glands on apoptosis and the protein expression of β1-integrin and TJ components (occludin, claudin-1 and zonula occludens-1 (ZO-1)). Sprague-Dawley rats at peak lactation each had two teats sealed to induce mammary engorgement. Another gland was acutely distended by infusing 1 ml of isosmotic sucrose solution (6h of milk secretion) up-the-teat prior to sealing. The remaining unsealed teats served as suckled controls. Mammary tissue was collected post-mortem at 0, 1, 3, and 6h after teat sealing (n=6 per time point). A dramatic increase (P<0.001) in the number of apoptotic nucleioccurred at 1, 3 and 6h in infused glands, and at 6h in engorged glands, compared with controls. By 3h and at 6h, apoptosis in infused glands was also significantly greater than engorged glands. This was accompanied by a reduction in the expression of β1-integrin in infused glands compared with control (P<0.05) and engorged (P<0.1) glands by 6h. Occludin protein expression was significantly up-regulated in infused glands immediately following physical distension at 0h compared with control and engorged glands, but then declined to be down-regulated within 6h of teat-sealing. Claudin-1 and ZO-1 protein expression were significantly decreased in engorged glands, but not infused glands, compared with controls by 6h. These results indicate that acute physical distension of rat mammary glands accelerates the induction of apoptosis and loss of β1-integrin and occludin protein expression compared with milk accumulation alone. In conclusion, we suggest a role for mechanotransduction during apoptosis and involution of mammary glands. [ABSTRACT FROM AUTHOR]

Extended periods of milk accumulation result in reduced milk secretion, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased TJ permeability between mammary epithelial cells and down-regulated expression of the major transmembrane TJ proteins, occludin and claudin-1. This study investigated the temporal expression of the cytoplasmic TJ component ZO-1 relative to the onset of apoptosis during mammary engorgement. Sprague-Dawley rats at peak lactation had three abdominal inguinal glands on one side sealed to induce mammary engorgement; the remaining glands were not sealed and acted as suckled controls. Mammary tissue was collected post-mortem at 0, 6, 12, 18, 24 and 36h after teat sealing (n = 6 per time point). A dramatic increase (P<0.001) in the number of apoptotic nuclei located within alveolar epithelia or lumina was observed by 18, 24 and 36h in the engorged glands compared with suckled controls. The number of apoptotic nuclei was generally greater (P<0.001) in the epithelial layer than within alveolar lumens for both control and engorged glands, except at 36h when a similar number was detected in both locations for engorged glands. Apoptotic nuclei present in the alveolar lumen of engorged glands at 18, 24 and 36h were associated with the presence of leukocytes. These changes were accompanied by a significant reduction in ZO-1 protein expression in engorged glands compared with control glands within 12h following teat-sealing. Furthermore, quantitative real-time RT-PCR analysis showed that mRNA expression of ZO-1 was decreased 1.8-fold by 12h (P<0.05). Responses to mammary engorgement were locally regulated as no changes were detected in suckled controls. In conclusion, the down-regulation of TJ proteins is consistent with a loss of epithelial cell-cell integrity and communication during mammary apoptosis and involution. [ABSTRACT FROM AUTHOR]

Context: In New Zealand, cows are usually milked twice a day (TAD), but in ~8% of herds, cows are milked once a day (OAD) for the entire lactation. If a genetic correlation (r g) of the same trait expressed in two environments (such as TAD and OAD) is substantially <1.0, then the genetic merit assessed from TAD herds may not be reliable for predicting genetic merit in OAD herds. Initial evaluation of sires has been undertaken from progeny test herds with TAD milking, and the best sires have then been made widely available for use in TAD or OAD herds. Aim: The study was designed to test formally whether sire re-rankings occur in widely used sires at different milking frequencies. Methods: Regression coefficients and rank correlations (r S) for estimated breeding values (EBVs) of 242 widely used sires (86 Holstein-Friesian, 96 Jersey and 60 crossbred) were calculated for yields of milk, fat and protein, and for somatic cell score (SCS). The r S values were contrasted with expected rank correlations (r E) between TAD and OAD EBVs assuming r g = 1.0 between true BVs expressed at the two milking frequencies and accounting for the fact that sires were highly selected. Key results: Within and across breeds, regression coefficients of OAD on TAD EBVs for the same traits were <1.0, but r S values between TAD and OAD EBVs of the same sires were strong (>0.75) for milk yield, moderate–strong for fat and protein yield (0.55–0.77), and moderate for SCS (0.41–0.65). Estimates of r S were higher than their critical r E values, indicating no significant sire re-ranking across milking frequencies. Conclusions: On the basis of the results, a separate selection program to generate sires for use in OAD herds is not justified. Implications: Farmers operating under OAD systems can rely on genetic evaluation of sires evaluated in TAD systems and used in the OAD population. However, producers should recognise that the realised productive and economic advantage of daughters of elite sires born in OAD herds is diminished relative to that expected in TAD herds. In New Zealand, cows are milked once or twice daily. However, bulls are evaluated only in the twice-daily system. Testing for changes in the genetic evaluation across milking environments could reveal whether once-daily populations need a separate selection scheme, and was done by comparing estimated breeding values of the same bulls with a simulated expected correlation. Correlations of estimated breeding values were greater than their expected correlations, indicating that farmers can rely on the evaluation made in twice-daily systems. [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]

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]