Once-daily Milking Research Articles

Reducing milking frequency from twice to once daily as an adjunct treatment for ketosis in lactating dairy cows—A randomized controlled trial

This randomized controlled trial investigated the effects of temporarily reducing milking frequency (MF) on the resolution of ketosis and milk production in dairy cows in early lactation. To detect ketosis [blood β-hydroxybutyrate (BHB) ≥1.2 mmol/L], Holstein cows were screened daily from 3 to 16 d in milk using a cow-side meter. Cows diagnosed with ketosis (n = 104) were randomly assigned to twice-daily milking (TDM) or reduced to once-daily milking (ODM) for 2 wk, then returned to twice-daily milking. Both treatment groups received a 5-d treatment of an oral propylene glycol drench (PG; 300 g) beginning on the afternoon of the diagnosis; cows received additional 5-d PG treatments if they had a ketotic test result (blood BHB ≥1.2 mmol/L) at least 4 d after finishing the first PG treatment. Blood BHB tests were conducted for the first 3 d after ketosis diagnosis, and then once every 3 d for 21 d of trial (DOT). Milk and milk component data were collected weekly for 15 wk following trial enrollment. The ODM group showed rapidly and markedly decreased blood BHB concentrations (primiparous cows: 1 DOT, 0.92 ODM vs. 1.22 TDM, 15 DOT, 0.55 vs. 0.81 mmol/L; multiparous cows: 1 DOT, 1.01 vs. 1.40, 15 DOT, 0.78 vs. 1.65 mmol/L). In addition, a logistic regression model indicated that ODM cows were less likely to have blood BHB concentrations ≥1.2 mmol/L [primiparous cows: 3 DOT: ODM 1% (95% confidence interval: 0-10%) vs. TDM 43% (30-58%), 15 DOT ODM 0% (0-0.2%) vs. TDM 22% (13-36%); multiparous cows: 3 DOT: ODM 33% (24-44%) vs. TDM 59% (48-69%), 15 DOT ODM 20.9% (13-31%) vs. TDM 64% (53-74%)]. The proportion of ODM cows that required additional treatments of PG were substantially lower than the TDM group (ODM: 39%; TDM: 64%) than the TDM cows during the initial 21-d period. However, during the 2-wk treatment period, cows in the ODM group produced 26% less milk and 25% less energy-corrected milk than the TDM cows. During wk 3 to 15, when all cows were milked twice daily, ODM cows produced less milk (-14%) and energy-corrected milk (-12%) compared with the TDM group. Milk protein percentage was greater, and milk fat percentage and linear score tended to be greater in the ODM group over 15 wk. In conclusion, a 2-wk reduction of MF in ketotic cows from twice to once daily with treatment with PG resolved ketosis and decreased blood BHB concentrations more effectively than treating TDM cows with PG alone. However, the 2-wk MF reduction had immediate and long-term (up to 13 wk after cessation of MF reduction) negative effects on milk production.

Bovine Teat Cistern Microbiota Composition and Richness Are Associated With the Immune and Microbial Responses During Transition to Once-Daily Milking

The relationship between microbiota and health has been widely reported in humans and animals. We established a link between teat cistern microbiota composition and bovine mastitis, an inflammatory disease often due to bacterial infections. To further decipher the relationships between teat cistern microbiota and immune and microbial responses, a switch from twice- to once-daily milking (ODM) in 31 initially healthy quarters of dairy cows was used to trigger an udder perturbation. In this study, a temporal relationship was reported between initial teat cistern microbiota composition and richness, the immune response to ODM, and mastitis development. Quarters with a low initial microbiota richness and taxonomic markers such as Bacteroidetes and Proteobacteria were associated with a higher rate of mastitis during ODM. Quarters with a higher richness and taxonomic markers such as Firmicutes, including the Lachnospiraceae family, and genera such as Bifidobacterium and Corynebacterium displayed early inflammation following transition to ODM but without developing mastitis (no infection). Short-term compositional shifts of microbiota indicates that microbiotas with a higher initial richness were more strongly altered by transition to ODM, with notably the disappearance of rare OTUs. Microbiota modifications were associated with an early innate immune system stimulation, which, in turn, may have contributed to the prevention of mastitis development.

A redefinition of the modeled responses of mammary glands to once-daily milking

Milking cows once daily is a management tool that has been implemented to improve physical and financial results of seasonal pasture-based dairy farms. The Molly cow model integrates physiology and metabolism of dairy cattle; however, milk production during short-term changes in milking frequency (e.g., 1× milking) is not well represented. The model includes a representation of variable rates of cell quiescence and death. However, the rate constants governing cell death and the return of quiescent to active cells are not affected by milking frequency. An empirical assessment of the problem was conducted, and it was hypothesized that changing the current representation of the rate of cell death in response to short-term 1× milking would more accurately represent active and quiescent cells and improve predictions of milk production. An extra senescent cell flux was added to account for cell loss during periods of 1× milking. Additional changes included a gradual decline in the rate of 1× stimulated senescence during 1× milking, and a structural change in cell cycling between active and quiescent cells during and after short-term 1× milking. Data used for parameter estimation were obtained from 5 studies where 1× milking or different feeding strategies were tested. Parameter estimates of cell loss indicated that 1× milking would affect a small proportion of quiescent cells to cause extra cell death. This added cell senescence was influenced by the length of 1× milking such that cell senescence peaked on d 1 of 1× milking and decayed from that point. The new structure in the model includes a variable rate of cell death in response to 1× milking and a gradual rate of return of quiescent cells back to the active pool in response to switching to 2× milking after short-term 1× milking. Root mean square errors, mean bias, and slope bias declined by at least 50% for predictions of energy-corrected milk yield and fat percent. The model showed quantitative agreement with production data from short-term 1× milking. The accuracy of predictions was improved and the error was reduced by implementing modifications in the model in response to changes in milking frequency.

Effect of milking frequency and α-tocopherol plus selenium supplementation on sheep milk lipid composition and oxidative stability

The aim of this research was to study the effect of milking frequency [once-daily milking (ODM) vs. twice-daily milking (TDM)] and antioxidant (AOX) supplementation on fatty acid (FA) profile and oxidative stability in sheep milk. Sixteen Assaf ewes were used; 8 did not receive any vitamin-mineral supplement (control), and the other 8 received an oral dose of 1,000 IU of α-tocopherol and 0.4 mg of Se daily. The experiment consisted of 2 consecutive periods; the first was 3 wk with TDM of both mammary glands. The second period was 8 wk and consisted of ODM of one mammary gland and TDM of the other gland. All ewes were fed ad libitum the same total mixed ration from lambing and throughout the experiment. There were no differences in plasma or milk Se concentrations between control and AOX ewes. However, plasma and milk α-tocopherol concentrations and AOX capacity were increased in ewes receiving the AOX supplement. Milk FA profile was practically unaffected after 21 d of AOX supplementation. However, after 77 d, AOX supplementation increased the relative percentage of C16:0 and cis-9 C18:1 and reduced the proportions of some saturated FA with less than 16 carbons and cis-9 C12:1. Antioxidant supplementation had no effect on the proportions of conjugated linoleic acid or total polyunsaturated FA (PUFA) but decreased the proportion of trans-7,cis-9 C18:2 and increased that of n-6 C20:3. Once-daily milking did not affect α-tocopherol, Se, or fat resistance to oxidation in milk. Total monounsaturated FA, cis-9 C16:1, and several cis and trans isomers of C18:1 were increased and total saturated FA were decreased in milk from ODM glands. Compared with TDM, ODM increased the proportions of cis-9,cis-12 C18:2 and several isomers of C18:2 and reduced those of cis-9,cis-12,cis-15 C18:3 and some PUFA of 20 and 22 carbons, but total proportion of PUFA was unaffected. Once-daily milking and AOX supplementation modified milk FA profile, but the effects of ODM could be considered of little biological relevance for consumer health. Supplementing ewes with α-tocopherol plus Se could be considered an effective strategy to improve plasma AOX status and reduce milk fat oxidation without substantial changes in the milk FA profile.

Response profiles of dairy cows to a single 24-h milking interval in relation with milk proteolysis, udder expansion and immune traits

An extended milking interval of 24 h (24-h milking interval (24h-MI)) constitutes the acute phase of cow adaptation to once-daily milking (ODM). A recent trial including 724 24h-MI challenges demonstrated that milk yield responses to this acute phase of ODM are highly variable (from+22% to −52% of milk yield when switching to the 24h-MI, mean=−25.3%) and that factors such as stage of lactation parity and milk yield level influenced cows’ responses but did not account for all individual variability. Additional traits related to physiological, immune and behavioural adaptation were measured on a subset (96 observations) of this data set. This study aimed to determine (1) the relationship of these traits with cows’ milk yield responses, (2) their ability – combined with previously identified traits – to help predict milk yield responses to 24h-MI (adaptive profiles). The 24h-MI challenge consisted of three successive periods: one control week of twice-daily milking (cTDM), one single day of 24h-MI and then 13 days of TDM (pTDM). Milk yield responses to the 24h-MI (corrected for effects of stage of lactation, parity, milk yield level and milk yield) were related to physiological traits measured during cTDM (milk flow rate, presence or absence of interleukin-8) and to their changes during the 24h-MI (absolute increase in milk flow rate and relative udder distension). Analysis of associations between milk yield responses, stage of lactation, parity, milk yield level, proteolysis, udder expansion and immune traits found three adaptive cow profile clusters. Cows in cluster 1 had a less compliant udder than cows in cluster 2, and they lost more milk during the 24h-MI than cluster-2 and cluster-3 cows. After resuming twice daily-milking (TDM), cluster-2 cows fully recovered the milk they had lost during the 24h-MI. On the opposite, cluster-3 cows did not recover the milk they lost, likely due to udder inflammation during cTDM, as suggested by elevated concentrations of interleukin-8 in their milk. These results combining new traits with stage of lactation, parity and milk yield level constitute a first step towards predicting individual cow responses to a 24h-MI.

Genetic parameters of milk production traits in response to a short once-daily milking period in crossbred Holstein × Normande dairy cows

Despite its potential utility for predicting cows' milk yield responses to once-daily milking (ODM), the genetic basis of cow milk trait responses to ODM has been scarcely if ever described in the literature, especially for short ODM periods. This study set out to (1) estimate the genetic determinism of milk yield and composition during a 3-wk ODM period, (2) estimate the genetic determinism of milk yield responses (i.e., milk yield loss upon switching cows to ODM and milk yield recovery upon switching them back to twice-daily milking; TDM), and (3) seek predictors of milk yield responses to ODM, in particular using the first day of ODM. Our trial used 430 crossbred Holstein × Normande cows and comprised 3 successive periods: 1 wk of TDM (control), 3 wk of ODM, and 2 wk of TDM. Implementing ODM for 3 wk reduced milk yield by 27.5% on average, and after resuming TDM cows recovered on average 57% of the milk lost. Heritability estimates in the TDM control period and 3-wk ODM period were, respectively, 0.41 and 0.35 for milk yield, 0.66 and 0.61 for milk fat content, 0.60 and 0.80 for milk protein content, 0.66 and 0.36 for milk lactose content, and 0.20 and 0.15 for milk somatic cell score content. Milk yield and composition during 3-wk ODM and TDM periods were genetically close (within-trait genetic correlations between experimental periods all exceeding 0.80) but were genetically closer within the same milking frequency. Heritabilities of milk yield loss observed upon switching cows to ODM (0.39 and 0.34 for milk yield loss in kg/d and %, respectively) were moderate and similar to milk yield heritabilities. Milk yield recovery (kg/d) upon resuming TDM was a trait of high heritability (0.63). Because they are easy to measure, TDM milk yield and composition and milk yield responses on the first day of ODM were investigated as predictors of milk yield responses to a 3-wk ODM to easily detect animals that are well adapted to ODM. Twice-daily milking milk yield and composition were found to be partly genetically correlated with milk yield responses but not closely enough for practical application. With genetic correlations of 0.98 and 0.96 with 3-wk ODM milk yield losses (in kg/d and %, respectively), milk yield losses on the first day of ODM proved to be more accurate in predicting milk yield responses on longer term ODM than TDM milk yield.

343 Effect of once daily milking on mammary transcriptome and cell turnover in dairy goat

Once-daily milking is known to modify cell number and activity in the bovine mammary gland. However, in goats, the effect of once-daily milking on mammary cell death is controversial. To assess the effect of once-daily milking on mammary transcriptome and cell turnover and the effect of the duration of once-daily milking, 10 goats producing 2.5 kg of milk per day at 100 DIM were divided into 2 groups. All goats were twice daily milked during a pre-experimental period of 2 wk. Then, the goats were once daily milked for the 3 following weeks. Mammary biopsy samples were collected at d −1 and 7 after the start of once-daily milking for one group of 5 goats and at d −1 and 21 for a second group of 5 goats. Cell apoptosis and proliferation rates were analyzed in mammary tissue by immunohistological analyses after TUNEL and PCNA staining, respectively. Ribonucleic acid was extracted from mammary tissues. A transcriptomic analysis using the Agilent Bovine 4x44k microarrays has been performed to compare the effect of once-daily milking on mammary transcript profiles. Data were normalized and statistical significant raw P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. A similar reduction in milk yield (−19%) was observed in both groups of goats during once-daily milking compared with twice-daily milking. Cell apoptosis was higher during once-daily milking than during twice-daily milking for both groups of goats (P < 0.05) whereas cell proliferation did not vary (P = 0.27). The transcriptomic analysis showed a differential gene expression of 4,039 transcripts, 2,238 and 1,801 transcripts up- and downregulated, respectively, by once-daily milking compared with twice-daily milking. More than 1,000 transcripts were commonly regulated between the 2 groups of goats. IPA analysis showed that these transcripts were part of networks associated with DNA replication, cellular growth and proliferation, and cell-to-cell signaling and interaction in both groups of goats. RT-qPCR analysis of 11 genes confirmed the differential gene expression with a downregulation of genes involved in milk synthesis and an upregulation of genes involved in cellular cycle and apoptosis. These results showed that once-daily milking induce cell turnover in goat mammary tissue, with a small impact of time.

Intramammary infections and milk leakage following gradual or abrupt cessation of milking

The objective of this study was to evaluate the effect of milking cessation method (abrupt or gradual) and daily milk yield before dry-off on milk leakage following dry-off and intramammary infections (IMI) at calving. Data from 1,086 quarters of 285 cows from 5 Ohio dairy herds were analyzed. All cows that were due to be dried off within a week were assigned to the same study group to facilitate management. Abrupt-cessation cows kept the farm’s regular milking schedule through dry-off, and gradual-cessation cows were milked once daily for the final week of lactation. Aseptic technique was used to collect quarter foremilk samples at the time of enrollment (7 to 14 d before expected dry-off), the final milking before dry-off (D-O), and within 7 d of calving. Cows in the gradual-cessation group were observed for milk leakage during the period of once-daily milking. In the only herd that did not use internal teat sealants at dry-off, milk leakage after dry-off was recorded in both abrupt and gradual groups. Gradual cessation decreased milk production by 33.4% during the final week of lactation, causing milk yield at D-O to be lower for these cows compared with abrupt-cessation cows (13.2 vs. 19.8kg/d, respectively). Logistic regression models were used to model the probability of a quarter being infected at calving with any pathogen, accounting for clustering of quarters within cows and cows within herds. The final model investigating the probability of IMI at calving was stratified by parity of cows at the time of dry-off (primiparous and multiparous). Among quarters of cows that ended their first lactation, abrupt cessation of milking before dry-off and milk leakage after dry-off were associated with an increased risk of IMI at calving. Among quarters of multiparous cows, on the other hand, gradual cessation of milking before dry-off, presence of IMI at D-O, and thrice-daily milking during lactation increased the odds of IMI at calving. These results indicate that implementation of differing management practices near dry-off for different parity groups may improve mammary health within a herd.

Once-daily milking during late lactation in pasture-fed dairy cows has minor effects on feed intake, body condition score gain, and hepatic gene expression

Milking cows once daily (1×) is a management practice occasionally used during mid/late lactation in pasture-based systems. It has been postulated that 1× milking will reduce dry matter intake (DMI) and increase body condition score (BCS) gain; however, this has not been quantified. Lactating, pregnant Holstein-Friesian dairy cows (n=52) were allocated to either 1× or twice-daily (2×) milking in mid-January (summer, 175d in milk). To obtain accurate DMI measurements, cows underwent 4 periods in a Calan gate indoor feeding facility, interspersed with grazing outdoors. Milk production, body weight (BW), and BCS were recorded 2 wk before treatment start (−2 wk) and weekly thereafter. Blood variables were recorded at −2 wk and weekly when indoors. Liver was biopsied at −2, 2, and 10 wk, and hepatic gene expression measured using quantitative PCR. Milking cows 1× tended to lower DMI (17.8 vs. 18.2kg of dry matter), but increased BCS gain (0.36 vs. 0.13 BCS units) and BW (546 vs. 533kg) at wk 12 relative to 2×. The greater BCS and BW of cows milked 1× compared with 2× were reflected in lower plasma concentrations of nonesterified fatty acids and lower transcription of genes involved in the oxidation of fatty acids, indicating reduced release and processing of fatty acids. Cows milked 1× produced 20% less milk, and although milk fat and protein concentrations were increased relative to cows milked 2×, yields of fat and protein were 14 and 17% less, respectively. The reduction in milk production with 1× milking (14.1 vs. 16.8kg/cow per d energy-corrected milk) was accompanied by increases in blood concentrations of glucose and insulin, with a concurrent decrease in the transcription of the insulin receptor and gluconeogenic genes. These results indicate a coordinated response to reduce glucose production due to decreased mammary demand. Expression of 2 genes linked to inflammation and adipokine signaling was reduced in cows milked 1× and may indicate a lower inflammatory state in the liver of cows milked 1× in late lactation. No effect was found of milking frequency during late lactation on milk production in the subsequent lactation. In summary, although 1× milking tended to reduce DMI and increase BCS in late lactation, these effects were lower than what is commonly supposed in pasture-based dairy systems. The modest BCS gains need to be considered with the reduced milk production when adopting 1× milking as a management strategy.

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts.

Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants’ udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).

Lactation traits associated with short- and long-term once-daily milking performance in New Zealand crossbred dairy cattle

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 (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.

DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

Expression of key lipid metabolism genes in adipose tissue is not altered by once-daily milking during a feed restriction of grazing dairy cows

The objective of this study was to investigate the effect of reduced milking frequency, at 2 feeding levels, on gene expression in adipose tissue of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) were grazed on pasture and milked twice daily (2×) from calving to 34±6d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2×2 factorial arrangement. Treatments consisted of 2 milking frequencies (2× or once daily; 1×) and 2 feeding levels for 3wk: adequately fed (AF), consuming 14.3kg of dry matter/cow per day, or underfed (UF), consuming 8.3kg of dry matter/cow per day. After the treatment period, all cows were fed to target grazing residuals ≥1,600kg of DM/cow per day and milked 2× for 20wk. Adipose tissue was collected from 12 cows per treatment by subcutaneous biopsy at −1, 3, and 5wk relative to treatment start, RNA was extracted, and transcript abundance of genes involved in lipid metabolism was quantified using a linear mixed model. At the end of the 3-wk treatment period, transcript abundance of genes involved in fatty acid (FA) uptake into adipose tissue (LPL), FA synthesis [FA synthase (FASN) and stearoyl-coenzyme A desaturase (SCD)], FA oxidation [acyl-coenzyme A synthetase long-chain family member 1 (ACSL1) and carnitine palmitoyltransferase 2 (CPT2)], glyceroneogenesis [glycerol-3-phosphate dehydrogenase 1 (GPD1) and pyruvate carboxylase (PC)], and triacylglyceride synthesis [diacylglycerol O-acyltransferase 2 (DGAT2)] were greater in AF1× cows compared with all other treatments. However, when cows were underfed, no effects of milking frequency were observed on transcript abundance of genes involved in adipose lipid metabolism. Despite increases in plasma NEFA concentrations in UF cows, no effects of underfeeding were observed on the transcription of lipolytic genes. At 5wk, after cows were returned to 2× milking and standard feed allowance, transcript abundances of genes involved in FA synthesis [acetyl-coenzyme A carboxylase α (ACACA) and SCD)] were increased in cows previously UF. Expression of ACSL1 was decreased in UF1× cows relative to UF2× cows and CPT2 expression was greater in AF1× cows compared with AF2× cows. In conclusion, after 3wk of reduced milking frequency during a feed restriction, transcription of genes involved in lipid metabolism in adipose tissue were not altered, possibly due to the reduced milk production in these animals. However, 3wk of 1× milking in AF cows increased transcription of genes involved in FA synthesis, oxidation, and triacylglyceride synthesis.

Once-daily milking during a feed deficit decreases milk production but improves energy status in early lactating grazing dairy cows

The objective of this study was to investigate the effect of milking frequency (MF) at 2 feeding levels (FL) on milk production, body condition score, and metabolic indicators of energy status in grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) grazed pasture and were milked twice daily (2×) from calving until 34±6d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2×2 factorial arrangement. Treatments consisted of 2FL: adequately fed [AF; 14.3kg dry matter intake (DMI)/cow per d] or underfed (UF; 8.3kg of DMI/cow per d) and 2 MF: 2× or once daily (1×). Treatments were imposed for 3wk. After the treatment period, all cows were offered a generous pasture allowance (grazing residuals >1,600kg of dry matter/ha) and milked 2×. During the 3-wk treatment period, we observed an interaction between FL and MF for energy-corrected milk (ECM), such that the decrease due to 1× milking was greater in AF than in UF cows (20 and 14% decrease, respectively). No interactions were found posttreatment. Cows previously UF produced 7% less ECM than AF cows during wk 4 to 12; however, no subsequent effect was observed of the previous underfeeding. Cows previously milked 1× produced 5% less ECM during wk 4 to 12, and differences remained during wk 13 to 23. During the 3-wk treatment period, UF cows lost 0.2 body condition score units (1–10 scale) and this was not affected by 1× milking. During the treatment period, UF cows had lower plasma glucose, insulin, and insulin-like growth factor I, and greater nonesterified fatty acids and β-hydroxybutyrate concentrations than AF cows. Cows milked 1× had greater plasma glucose, insulin, and insulin-like growth factor I, and lower nonesterified fatty acids and β-hydroxybutyrate concentrations compared with cows milked 2×. In conclusion, energy status was improved by 1× milking; however, when UF cows were milked 1×, milk production was reduced by more than underfeeding alone. The immediate and residual responses to 1× milking need to be considered when using this management strategy during a feed deficit.

The decrease in milk yield during once daily milking is due to regulation of synthetic activity rather than apoptosis of mammary epithelial cells in goats

Once daily milking (ODM) is a management practice that can improve working conditions and reduce production costs in dairy farming compared with twice daily milking (TDM). However, ODM is associated with a decrease in milk yield. Previous research indicated that disruption of tight junctions in the mammary gland may be one of the regulatory factors involved in the milk yield decrease observed during ODM. The aim of this study was to investigate the involvement of mammary epithelium disruption in the regulation of the activity and dynamics of mammary epithelial cells (MEC) during 5 weeks of ODM in goats. Twelve alpine goats (producing 3.67 ± 0.64 kg/day and 47 ± 1.6 days in milk) were assigned to two groups that were milked once or twice a day during 5 weeks and then switched back to TDM. Mammary biopsies were collected before and on days 2 and 16 of both ODM and TDM switchback periods. Milk purified epithelial cells were collected before and on days 1, 7, 21 and 28 during ODM as well on days 1 and 7 of the TDM switchback period. The mRNA levels of genes involved in the regulation of synthetic activity and apoptosis were analysed by RT-PCR in milk MEC and mammary biopsies. ODM decreased yields of milk (−23%), lactose (−23%) and casein (−16%). Lactose synthesis was regulated at the transcriptional level by downregulation of α-lactalbumin mRNA levels in both biopsy samples (−30%) and milk MEC (−74%). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining of mammary gland biopsies did not show any increase in cell apoptosis after 2 and 16 days of ODM (0.8% and 1%, respectively) despite upregulation of Bax mRNA levels in milk MEC. This suggests that the decrease in milk yield observed during ODM is attributable to a decrease in synthetic activity rather than to induction of MEC cell death. ODM induced the disruption of tight junctions in the mammary gland only on the first day of the treatment as indicated by increased blood lactose concentration. This indicates that the decrease in MEC activity observed over the 5 weeks of ODM was not due to disruption of the mammary gland tight junctions. There was no carryover effect of 5 weeks of ODM on milk production. Therefore, it appears that the decrease in milk yield that occurs during ODM in goats is due to regulation of synthetic activity rather than to apoptosis of MEC.

Effect of reduction of milking frequency and supplementation of vitamin E and selenium above requirements on milk yield and composition in Assaf ewes

The aim of this research was to study the effect of milking frequency and supplementation with a vitaminmineral complex above requirements on intake, body weight (BW), and milk yield and composition in highyielding Assaf ewes. Sixteen lactating Assaf ewes were used in this study, separated into 4 groups of 4 ewes each. Animals in 2 of the groups (control groups) did not receive any extra vitamin-mineral supplement, whereas animals in the other 2 groups (supplement groups) received daily an oral dose of 1g of vitamin E (1,000IU, dl-α-tocopherol acetate) and 0.4mg of selenium (sodium selenite anhydrous). The experiment consisted of 2 consecutive periods of 3 wk (twice-daily milking in both mammary glands) and 8 wk (once-daily milking in one mammary gland and twice-daily milking in the other gland). Intake, BW, and milk composition were controlled weekly, and milk production was recorded 3 times a week. Administration of the vitamin-mineral supplement had no effect on dry matter intake, BW, or milk production and composition. The reduction of milking from twice to once a day caused a decrease in milk production and lactose concentration and a significant increase in protein concentration, total solids, and somatic cell count, without affecting the fat content. Administration of a vitamin E and Se supplement at the doses used in the present study does not seem to exert, in the short term, a noticeable effect on the mammary gland when milking frequency is reduced.

Alteration of the nutrient uptake by the udder over an extended milking interval in dairy cows

Little is known about modifications of the mammary utilization of nutrients circulating in blood plasma when milk yield is strongly decreased by once-daily milking. A trial was carried out to describe the mammary nutritional adjustments linked to the downregulation of milk synthesis as milk accumulated over an extended milking interval in the bovine udder. Three Holstein dairy cows yielding 34.0kg/d of milk were fitted with an ultrasound flow probe around the left external pudic artery and with catheters inserted into the left carotid and milk vein to estimate mammary blood flow (MBF) and mammary uptake of acetate, β-hydroxybutyrate, nonesterified fatty acids, glycerol, glucose, O2, and CO2 release. The trial was carried out over 2 consecutive weeks, with wk 2 repeating wk 1. Cows were milked twice daily at 12-h milking intervals. On d 3, cows were milked at 0630h and were not milked for 36h until d 4 at 1830h. Over the following days, twice-daily milking was resumed using 12-h milking intervals. Each half-udder was milked separately. Secretion rates of milk and milk proteins decreased 67% during the 12-to-36-h interval of milk accumulation, whereas that of milk fat fell 30%. Timing of changes in MBF and lactose levels in blood plasma was concomitant and significant after 19.5 and 21.5h of milk accumulation in the udder, respectively. The MBF decreased, most likely because the usual increases in MBF no longer occurred when the udder was full of milk. After 24h of milk accumulation, MBF did not increase further when cows lay down, and did not increase as usual 3h after a meal, suggesting a possible physical effect of milk accumulated in the udder on MBF, complementing metabolic regulation. Mammary uptake or release of nutrients was lowered before 24h for glucose, acetate, and β-hydroxybutyrate and after 24h for total glycerol, O2, and CO2, mostly associated with the impaired MBF. However, these decreases ranged from 12 to 17%, and cannot entirely explain the −45 and −20% decreases in milk secretion rates observed during the entire 36h of milk accumulation, thus confirming the primary role of intramammary metabolic regulation in the downregulation of milk secretion. The larger amount of nutrients taken up by the udder could explain the enhanced milk fat levels, involving a strongly modified metabolic fate of nutrients.

Lactose in blood plasma and the ability of dairy cows to tolerate once-daily milking in terms of milk loss and milk recovery

This experiment described the variability among cows with the aim of studying the ability of dairy cows to tolerate once-daily milking (ODM) in terms of milk losses and milk recoveries observed when cows are switched from twice- to once-daily milking and then back to twice-daily milking (TDM). It also aimed to investigate whether or not lactose in blood plasma, which indicates the mammary epithelium permeability, is correlated with milk losses and recoveries and, consequently, a potential candidate assessor of dairy cow tolerance to ODM. The study used 86 crossbred dairy cows (Holstein × Normande) split into 5 groups over 2 yr. The trial consisted of 3 successive periods: a 1-wk control period of TDM, then 3 wk of ODM, followed by 2 wk of TDM. Blood samples were collected 1 to 1.5h before the morning milking and 5 to 6h after milking on d −3, 0, 1, 3, 7, 21, 23, and 28, where d 0 is the last d of the control period. Milk losses measured as kilograms per day were higher in cows with highest control-period milk yields (r=−0.66). When expressed in relative terms (%), milk losses were weakly correlated with the control-period milk yield (r=−0.22). When switched back to TDM, cows recovered only 47% of the milk lost. Milk recovery (kg/d) was weakly correlated with control milk yield (r=0.34) and not correlated with ODM milk yield. Milk recovery was correlated with milk yield losses: the higher the milk yield losses (kg/d or %), the higher the milk recovery (kg/d; r=−0.59 and −0.52, respectively). Rate of milk recovery expressed as the quantity of milk recovered per kilogram of milk lost, was not correlated with milk loss expressed as a percentage. This means that to be considered well-adapted to ODM scheduling, a cow must be well adapted to milk loss, and be well adapted to milk recovery. Blood plasma lactose concentration (log10-transformed data) was not correlated with milk production levels in either control, ODM, or back-to-TDM periods. It was poorly correlated with milk loss but was positively correlated with milk recovery (kg/d) and rate of milk recovery. We conclude that blood plasma lactose needs to be coupled with other indicators for evaluating dairy cow tolerance to ODM.

Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats

Generally, once-daily milking (ODM) decreases milk yield. This effect may be the consequence of a decrease in mammary epithelial cell (MEC) activity or a reduction in their number. The aim of this study was to determine the effect of ODM on the synthetic activity and rate of apoptosis of MEC using a non-invasive method. Eight Alpine goats were subjected to ODM or twice-daily milking for two 5-week periods. MECs were purified by centrifugation and immunocytochemical binding in milk after 1 and 5 weeks of each period. mRNA levels of some proteins involved in lactose and milk protein synthesis and in apoptosis were evaluated using real-time PCR. Isolation of MEC from milk was a useful method to investigate transcriptional regulation in a timeline study. ODM induced greater decreases in milk, lactose and protein yields after 1 week than after 5 weeks. This suggests an adaptation of the mammary gland to ODM, which reduces the inhibitory effect of this practice. Reductions in milk component yields were associated with lower α-lactalbumin transcripts, suggesting a transcriptional decrease of lactose synthesis during ODM. Glucose transporter GLUT1 transcripts were downregulated under ODM, suggesting that lactose precursor uptake by MEC might be involved in the regulation of lactose synthesis. κ-Casein mRNA levels tended to be lower during ODM. ODM increased levels of the pro-apoptotic transcript Bax after both 1 and 5 weeks, but no variation was observed in the Bax/Bcl-2 ratio. ODM affected cell synthetic activity through transcriptional regulation and may have induced apoptosis. The reduction of the negative effect of ODM on milk yield suggests that Alpine goats are able to adapt to ODM. Further studies are needed to investigate the effect of ODM on MEC turnover.

The combined effects of milking frequency and feeding level on dairy goat welfare and milk emission characteristics in late lactation

This study investigated the combined effects of once-daily milking and feeding level ( ad libitum or adjusted) on goat welfare and milk emission characteristics in late lactation. Forty dairy goats housed in individual pens were managed under twice-daily milking and fed ad libitum during a pre-experimental period (2 weeks). The experiment was then divided into two successive periods: a 1-week period of milking frequency reduction and a 3-week period of feed adjustment. During the first period, half the goats were managed under once-daily milking. During the second period, half the goats of each type of milking treatment was fed ad libitum, whereas the other half received a quantity of feed adjusted weekly to milk production. The following measurements were taken for all goats: milk yield, dry matter intake (DMI), somatic cell count (SCC), plasma cortisol concentrations before morning milking, milk flow parameters [latency (i.e., the time from teat cup attachment to first measurement of the milk with a jar), the maximum milk flow rate (MAXFR) and the time taken to reach this maximum (TMAX)] and goat behaviour at morning milking (number of goats ruminating, back hunching, foot moving or kicking at milking). In addition, the time–budget (time spent drinking, eating, standing, lying or climbing) of 16 goats was measured by video recording. Milk yield was reduced by once-daily milking (18 and 21% in the first and the second period, respectively) but not by feed adjustment. DMI was reduced by once-daily milking (12%) only during the second period or by feed adjustment (28%) during the same period. Both SCC and latency were not modified by once-daily milking nor feed adjustment. MAXFR was increased by once-daily milking but not modified by feed adjustment. TMAX was increased by once-daily milking only during the first period and reduced by feed adjustment during the second one. The time spent eating was reduced by once-daily milking or feed adjustment during the second period, in agreement with the reduction in DMI in this period. There were no effects of milking frequency or feeding level on plasma cortisol concentrations, the behaviour at morning milking or the time spent drinking, standing, lying or climbing. No welfare impairment was demonstrated under our experimental conditions in late lactation dairy goats under a once-daily milking management strategy whatever the feeding level.