Performance of dairy cows milked twice daily at contrasting intervals

The time constraints of the classic twice-daily milking routine are less easily endured by individual dairy farmers, because of their impact on quality of life. Our aim was to evaluate milk production responses by dairy cows milked twice daily at contrasting intervals. In experiments 1 (20 cows) and 2 (28 cows), four milking regimes were compared during a 3-week period beginning after the peak of lactation. Three groups of five cows were milked twice daily (TDM) with milking intervals of 11 : 13, 7 : 17 and 3 : 21 h in experiment 1, and three groups of seven cows at 11 : 13, 5 : 19 and 2.5 : 21.5 h in experiment 2. One group (five and seven cows respectively) was milked once daily (ODM) in each experiment. In experiment 3 (three groups, 12 cows per group), one group was milked at 10 : 14 h and one at 5 : 19 h, and the third group once daily. Milking treatments began during the second week of lactation and continued for an average of 23 weeks. In experiments 1 and 2, daily milk yields were reduced by 4.1%, 11.5% and 28%, for the 5 : 19, 3 : 21 and ODM milking treatments compared with the 11 : 13 h interval. In experiment 3, the decrease in daily milk yields for 5 : 19 h and ODM was 10% and 40% compared with the 10 : 14 h time interval. In the average daily milk, fat and protein contents and somatic cell counts were not different between the TDM groups, and the ODM group had (or tended to have) a higher fat and protein content. For a given milking, milk fat content decreased from about 60 to 32 g/kg as the preceding milking interval increased from 2.5 to 3 h up to 12 h. It then levelled out and even increased, mainly after 18 to 20 h. Somatic cell count showed a similar trend, and protein content did not change steadily. Dry matter intake, body weight and body condition score were not affected by contrasting milking intervals. After resumption of TDM with conventional intervals, productions of milk, fat and protein no longer differed between the TDM groups. Milk yield of previously ODM cows remained lower by 2 kg/day (P = 0.15) in experiments 1 and 2, and by 7 kg/day (P < 0.05) in experiment 3. These results suggest that TDM at contrasting intervals up to 5 : 19 h is feasible as it decreases milk yield only moderately, especially if implemented from peak of lactation.     

Endocrine responses and milk emission characteristics in high yielding Alpine dairy goats under once daily milking management

This study was done to verify if the lack of increase of milk fat content observed in Alpine dairy goats under once daily milking (ODM) compared to twice daily milking (TDM), results from disturbances of animals and/or milk ejection reflex. In this respect, we determined the milk yield and composition in the same 12 multiparous Alpine dairy goats when they were managed first under TDM (period 1: P1) and then under ODM (period 2: P2). Furthermore, oxytocin (OT) and cortisol (CORT) releases and milk emission kinetic of these goats were measured at morning milking 2 and 4 times in P1 and P2, respectively.ODM compared to TDM, caused 18 and 23% reductions, respectively in daily milk yield and milk fat content without milk protein content modification.Although baseline concentration of blood OT was lower under ODM than TDM management (7.0 pg/mL vs 17.8 ± 2.0 pg/mL, respectively), ODM did not modify the total amount of OT released during milking (15,484 pg/mL/32 min vs 18,996 ± 2865.3 pg/mL/32 min, respectively), the peak concentration of OT (57.2 pg/mL vs 73.6 ± 10.5 pg/mL, respectively) or the time to reach it (3.0 min vs 2.0 ± 0.5 min, respectively) by comparison to TDM.ODM compared to TDM, never modified the baseline concentrations of CORT (6.1 ng/mL vs 7.1 ± 1.1 ng/mL, respectively), the total amount of CORT released (11,727 ng/mL/32 min vs 10,073 ± 1522.2 ng/mL/32 min, respectively), the peak concentrations of CORT (16.1 ng/mL vs 15.1 ± 2.1 ng/mL, respectively) and the time to reach it (13.1 min vs 11.0 ± 1.5 min).During ODM, milk flow latency was reduced (−61%) while the mean flow rate was increased (+28%) by comparison to TDM. ODM compared to TDM management, did not modify the maximum flow rate (1.6 L/min vs 1.6 ± 0.2 L/min, respectively) or the time to reach this maximum (103.0 s vs 95.0 ± 10.0 s, respectively). The total milking duration at morning milking was not different (250.0 s vs 221.0 ± 22.0 s, respectively) although the morning milk yield was significantly higher under ODM management (2.63 kg/d vs 1.87 ± 0.1 kg/d, respectively).This results show that milk emission is improved under ODM management in Alpine goats without inhibition or disturbance of neuro-hypophyseal OT release pattern and lack of activation of the hypothalamic–pituitary–adrenal axis and plasma cortisol increase, disproving our initial hypotheses of a disturbance of animals and/or incomplete milk ejection to explain the low fat content of milk under ODM in this breed.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypomethylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.Key words: cord blood, birth weight, folic acid, homocysteine, BeadArray, hierarchical clustering, Illumina     

Folic Acid Supplementation during Pregnancy Induces Sex-Specific Changes in Methylation and Expression of Placental 11β-Hydroxysteroid Dehydrogenase 2 in Rats

In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (−378 to −275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypo-methylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.     

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.     

Maternal psychosocial stress during pregnancy alters the epigenetic signature of the glucocorticoid receptor gene promoter in their offspring: a meta-analysis

Prenatal stress has been widely associated with a number of short- and long-term pathological outcomes. Epigenetic mechanisms are thought to partially mediate these environmental insults into the fetal physiology. One of the main targets of developmental programming is the hypothalamic-pituitary-adrenal (HPA) axis as it is the main regulator of the stress response. Accordingly, an increasing number of researchers have recently focused on the putative association between DNA methylation at the glucocorticoid receptor gene (NR3C1) and prenatal stress, among other types of psychosocial stress. The current study aims to systematically review and meta-analyze the existing evidence linking several forms of prenatal stress with DNA methylation at the region 1_F of the NR3C1 gene. The inclusion of relevant articles allowed combining empirical evidence from 977 individuals by meta-analytic techniques, whose methylation assessments showed overlap across 5 consecutive CpG sites (GRCh37/hg19 chr5:142,783,607-142,783,639). From this information, methylation levels at CpG site 36 displayed a significant correlation to prenatal stress (r = 0.14, 95% CI: 0.05–0.23, P = 0.002). This result supports the proposed association between a specific CpG site located at the NR3C1 promoter and prenatal stress. Several confounders, such as gender, methylation at other glucocorticoid-related genes, and adjustment for pharmacological treatments during pregnancy, should be taken into account in further studies.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3–6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.     

Conditional repression of STAT5 expression during lactation reveals its exclusive roles in mammary gland morphology, milk-protein gene expression, and neonate growth

The role of Stat5 in maintaining adequate lactation was studied in Stat5a(-/-) mice expressing a conditionally suppressed transgenic STAT5 in their mammary glands. This system enables distinguishing STAT5's effects on lactation from its contribution to mammary development during gestation. Females were allowed to express STAT5 during their first pregnancy. After delivery, STAT5 levels were manipulated by doxycycline administration and withdrawal. In two lines of genetically modified mice, the absence of STAT5 expression during the first 10 days of lactation resulted in a decrease of 29% or 41% in newborn weight gain. The STAT5-dependent decrease in growth was recoverable, but not completely reversible, particularly when STAT5 expression was omitted for the first 4 days of lactation. Within the first 10 days of STAT5-omitted lactation, alveolar occupancy regressed by 50% compared to that measured at delivery. By Day 10, only 18% of the fat-pad area was involved in milk production. The alveolar regression caused by 4 days of STAT5 deficiency was reversible, but neonate growth remained delayed. STAT5 deficiency resulted in reduced estrogen receptor α and connexin 32 gene expression, accompanied by delayed induction of both anti- and pro-apoptotic Bcl-2 family members. An increase in Gata-3 expression may reflect an attempt to maintain alveolar progenitors. A decrease of 39% and 23% in WAP and α-lactalbumin expression, respectively, with no associated effects on β-casein, also resulted from lack of STAT5 expression in the first 10 days of lactation. This deficiency enhances the major effect of alveolar regression on delayed weight gain in newborns.     

Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis

DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to our understanding of the reprogramming of imprinted genes during bovine oogenesis.