Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Effects of dietary folic acid supplementation on lactation performance and mammary epithelial cell development of dairy cows and its regulatory mechanism.

The experiment investigated the impacts of FA on the proliferation of bovine mammary gland epithelial cells (BMECs) and to investigate the underlying mechanisms. Supplementation of 10 µM FA elevated the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin A2 and cyclin D1, and protein expression of PCNA and Cyclin A1. The mRNA and protein expression of B-cell lymphoma-2 (BCL2) and the BCL2 to BCL2 associated X 4 (BAX4) ratio elevated, while that of BAX, Caspase-3 and Caspase-9 reduced by FA. Both Akt and mTOR signaling pathways were activated by FA. Moreover, the stimulation of BMECs proliferation, the alteration of proliferative genes and protein expression, the change of apoptotic genes and protein expression, and the activation of mTOR signaling pathway caused by FA were obstructed by Akt inhibitor. Suppression of mTOR with Rapamycin reversed the FA-modulated promotion of BMECs proliferation and change of proliferous genes and protein expression, with no impact on mRNA or proteins expression related to apoptosis and FA-activated Akt signaling pathway. Supplementation of rumen-protected FA in cow diets evaluated milk yields and serum insulin-like growth factor-1 and estradiol levels. The results implied that the proliferation of BMECs was stimulated by FA through the Akt-mTOR signaling pathway.

Human Breast Milk microRNAs, Potential Players in the Regulation of Nervous System.

Human milk is the biological fluid with the highest exosome amount and is rich in microRNAs (miRNAs). These are key regulators of gene expression networks in both normal physiologic and disease contexts, miRNAs can influence many biological processes and have also shown promise as biomarkers for disease. One of the key aspects in the regeneration of the nervous system is that there are practically no molecules that can be used as potential drugs. In the first weeks of lactation, we know that human breast milk must contain the mechanisms to transmit molecular and biological information for brain development. For this reason, our objective is to identify new modulators of the nervous system that can be used to investigate neurodevelopmental functions based on miRNAs. To do this, we collected human breast milk samples according to the time of delivery and milk states: mature milk and colostrum at term; moderate and very preterm mature milk and colostrum; and late preterm mature milk. We extracted exosomes and miRNAs and realized the miRNA functional assays and target prediction. Our results demonstrate that miRNAs are abundant in human milk and likely play significant roles in neurodevelopment and normal function. We found 132 different miRNAs were identified across all samples. Sixty-nine miRNAs had significant differential expression after paired group comparison. These miRNAs are implicated in gene regulation of dopaminergic/glutamatergic synapses and neurotransmitter secretion and are related to the biological process that regulates neuron projection morphogenesis and synaptic vesicle transport. We observed differences according to the delivery time and with less clarity according to the milk type. Our data demonstrate that miRNAs are abundant in human milk and likely play significant roles in neurodevelopment and normal function.

Linseed oil supplementation and DGAT1 K232A polymorphism affect the triacylglycerol composition and crystallization of milk fat.

We studied the effect of dietary linseed oil (LSO) supplementation and DGAT1 K232A (DGAT1) polymorphism on the triacylglycerol composition and crystallization of bovine milk fat. LSO supplementation increased unsaturated triacylglycerols, notably in the C52-C54 carbon range, while reducing the saturated C29-C49 triacylglycerols. These changes were associated with an increase in the low-melting fraction and the crystal lamellar thickness, as well as a reduction in the medium and high-melting fractions and the formation of the most abundant crystal type at 20 °C (ß'-2 polymorph). Furthermore, DGAT1 KK was associated with higher levels of odd-chain saturated triacylglycerols than DGAT1 AA, and it was also associated with an increase in the high-melting fraction and the endset melting temperature. An interaction between diet and DGAT1 for the unsaturated C54 triacylglycerols accentuated the effects of LSO supplementation with DGAT1 AA. These findings show that genetic polymorphism and cows' diet can have considerable effects on milk fat properties.

Genetic aspects of Wood's lactation curve parameters in Jersey crossbred cattle using Bayesian approach.

The study was undertaken to estimate the genetic parameters of lactation curve parameters of Wood's function in Jersey crossbred cattle using the Bayesian approach. Data on 33,906 fortnightly test day milk yields of 1,718 lactation records of Jersey crossbred cows, maintained at the ICAR-National Dairy Research Institute in West Bengal, were collected over a period of 40 years. The lactation curve parameters including 'a' (initial milk yield after calving), 'b' (ascending slope up to peak yield) and 'c' (descending slope after peak yield) and lactation curve traits, peak yield (ymax), time of peak yield (tmax) and persistency of milk yield (P) of individual cow for each lactation were estimated using the incomplete gamma function (Wood's model) by fitting the Gauss-Newton algorithm as an iteration method using PROC NLIN procedure of SAS 9.3. Variance components and genetic parameters of lactation curve parameters/traits were estimated by a repeatability animal model using the Bayesian approach. Estimates of heritabilities were found to be 0.18 ± 0.05, 0.09 ± 0.03 and 0.11 ± 0.04 for parameters 'a', 'b' and 'c', respectively and 0.24 ± 0.05, 0.12 ± 0.04, and 0.15 ± 0.05 for ymax, tmax and P, respectively. Repeatability estimates were 0.31 ± 0.03, 0.21 ± 0.04 and 0.30 ± 0.04 for parameters 'a', 'b' and 'c' respectively and 0.39 ± 0.03, 0.24 ± 0.03 and 0.37 ± 0.03 for ymax, tmax and p, respectively. Genetic correlations among lactation curve parameters/traits ranged from -0.75 to 0.95. Existence of genetic correlations among lactation curve parameters/traits indicated substantial genetic and physiological relationships among lactation curve parameters/traits of Jersey crossbred cattle.

Identification of Candidate Immune System MicroRNAs Differentially Found in Colostrum and Milk Exosomes.

BACKGROUND: The fetus grows in a sterile womb environment. After birth, the newborn immune system has two immediate hurdles to clear. First immediate suppression of the womb compatible immune system and turn on the immune system of the newborn that can counter the antigenic world. The underlying mechanism of immune fluctuation by milk microRNAs (miRNAs) can be crucial for the treatment of critical or premature newborn. METHODS: We collected fourteen samples of each colostrum and mature milk from lactating mothers, four samples of each were used for microarray analysis, and the other ten were used for miRNA expression profiling by real-time PCR. RESULTS: From the microarray, 154 differentially expressed miRNAs were identified, whereas 49 miRNAs were revealed as immune-related miRNAs based on a literature study. Among the 49 miRNAs, 33 were already shown as strongly validated immune-related miRNAs (validated by qPCR, Western Blot, and Luciferase assay) and were considered for further analysis. Twenty-two miRNA expressions were analysed by real-time PCR as their Ct values were within considerable limits. Twelve numbers of miRNAs were significantly downregulated in mature milk compared to colostrum, which were again subjected to bioinformatics analysis to predict the biological mechanisms behind the differentially expressed miRNAs. CONCLUSION: This study shed light on the human milk exosome miRNA expression dynamics during lactation and their possible role in the gradual skewing of the newborns' immune system. The information is crucial for the development and onset of sepsis in premature newborns in the NICU.

Differential hepatic mitochondrial function and gluconeogenic gene expression in 2 Holstein strains in a pasture-based system.

The objective of this study was to assess hepatic ATP synthesis in Holstein cows of North American and New Zealand origins and the gluconeogenic pathway, one of the pathways with the highest ATP demands in the ruminant liver. Autumn-calving Holstein cows of New Zealand and North American origins were managed in a pasture-based system with supplementation of concentrate that represented approximately 33% of the predicted dry matter intake during 2017, 2018, and 2019, and hepatic biopsies were taken during mid-lactation at 174 ± 23 days in milk. Cows of both strains produced similar levels of solids-corrected milk, and no differences in body condition score were found. Plasma glucose concentrations were higher for cows of New Zealand versus North American origin. Hepatic mitochondrial function evaluated measuring oxygen consumption rates showed that mitochondrial parameters related to ATP synthesis and maximum respiratory rate were increased for cows of New Zealand compared with North American origin. However, hepatic gene expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase was increased in North American compared with New Zealand cows. These results altogether suggest an increased activity of the tricarboxylic cycle in New Zealand cows, leading to increased ATP synthesis, whereas North American cows pull tricarboxylic cycle intermediates toward gluconeogenesis. The fact that this occurs during mid-lactation could account for the increased persistency of North American cows, especially in a pasture-based system. In addition, we observed an augmented mitochondrial density in New Zealand cows, which could be related to feed efficiency mechanisms. In sum, our results contribute to the elucidation of hepatic molecular mechanisms in dairy cows in production systems with higher inclusion of pastures.

When Two plus Two Is More than Four: Evidence for a Synergistic Effect of Fatty Acids on Peroxisome Proliferator-Activated Receptor Activity in a Bovine Hepatic Model.

The inclusion of fat in livestock diets represents a valuable and cost-effective way to increase the animal's caloric intake. Beyond their caloric value, fatty acids can be understood in terms of their bioactivity, via the modulation of the ligand-dependent nuclear peroxisome proliferator-activated receptors (PPAR). Isotypes of PPAR regulate important metabolic processes in both monogastric and ruminant animals, including the metabolism of fatty acids (FA), the production of milk fat, and the immune response; however, information on the modulation of bovine PPAR by fatty acids is limited. The objective of this study was to expand our understanding on modulation of bovine PPAR by FA, both when used individually and in combination, in an immortalized cell culture model of bovine liver. Of the 10 FA included in the study, the greatest activation of the PPAR reporter was detected with saturated FA C12:0, C16:0, and C18:0, as well as phytanic acid, and the unsaturated FA C16:1 and C18:1. When supplemented in mixtures of 2 FA, the most effective combination was C12:0 + C16:0, while in mixtures of 3 FA, the greatest activation was caused by combinations of C12:0 with C16:0 and either C18:0, C16:1, or C18:1. Some mixtures display a synergistic effect that leads to PPAR activation greater than the sum of their parts, which may be explained by structural dynamics within the PPAR ligand-binding pocket. Our results provide fundamental information for the development of tailored dietary plans that focus on the use of FA mixtures for nutrigenomic purposes.

Expression patterns of major genes in fatty acid synthesis, inflammation, oxidative stress pathways from colostrum to milk in Damascus goats.

The molecular regulation of milk secretion and quality in the transition period from colostrum to milk in goats is largely unknown. In the present study, mammary gland secretion of goats was collected in 0th, 4th, 7th, 14th and 28th days after parturition. In addition to composition and fatty acid profile of colostrum or milk, FASN, SCD, ACACA, COX-2, NRF2, TLR2, NF-kB, LTF and PTX3 genes expression patterns were determined from milk somatic cells. While somatic cell count (SCC), malondialdehyde (MDA), fat, fat-free dry matter, protein and lactose were highest as expression levels of the oxidative and inflammatory genes, freezing point and electrical conductivity were lowest in colostrum. With the continuation of lactation, most of the fatty acids, n3 ratio, and odour index increased but C14:0 and C16:0 decreased. While FASN was upregulated almost threefolds in 14th day, ACACA was upregulated more than fivefolds in 7th and 14th days. Separately, the major genes in fatty acid synthesis, inflammation and oxidative stress were significantly associated with each other due to being positively correlated. MDA was positively correlated with SCC and some of the genes related inflammation and oxidative stress. Furthermore, significant negative correlations were determined between SCC and fatty acid synthesis related genes. With this study, transition period of mammary secretion was particularly clarified at the molecular levels in Damascus goats.

Methionine promotes crop milk protein synthesis through the JAK2-STAT5 signaling during lactation of domestic pigeons (Columba livia).

Crop milk is the sole source of nutrition that sustains young pigeons (squabs) throughout growth and development. Protein accounts for approximately 55% of the nutrients in crop milk; however, its regulation mechanism remains unclear. In our study, three experiments were conducted to investigate the possible underlying mechanism of crop milk protein synthesis and nutritional interventions. Isobaric tagging for relative and absolute quantification (iTRAQ) analysis found that the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway was significantly up-regulated in breeding pigeons during lactation compared to non-breeding pigeons. Moreover, the serum prolactin (PRL) levels increased, and the protein expression of the PRL receptor (PRLR)/JAK2/STAT5 pathway was significantly up-regulated during lactation. The serum PRL, the PRLR/JAK2/STAT5 pathway, the crop milk protein synthesis, and the squab growth performance were inhibited by bromocriptine mesylate injection, a PRL-specific inhibitor. In addition, dietary supplementation with 0.30% dl-methionine or dl-methionine-dl-methionine (especially 0.30% dl-methionine-dl-methionine), significantly increased serum PRL levels and PRLR/JAK2/STAT5 activity, and improved the crop milk protein synthesis. In conclusion, our results demonstrated that the PRL-induced PRLR/JAK2/STAT5 signaling pathway plays a vital regulatory role in crop milk protein synthesis, and 0.30% dl-methionine-dl-methionine is superior to dl-methionine in promoting crop milk protein synthesis.

Restriction and hyperlipidic diets during pregnancy, lactation and adult life modified the expression of dopaminergic system related genes both in female mice and their adult offspring.

The neurocircuitry underlying hunger, satiety, motivation to eat and food reward is complex, however a lot of mechanisms are still unknown. Two main cerebral areas are responsible for controlling feeding through hunger and food reward: the hypothalamus (HPT) and the ventral tegmental area (VTA), respectively. The dopaminergic system modulates both these areas and is essential to control food ingestion. Therefore, we aim to evaluate the effects of restrictive and hyperlipidic diets during pregnancy, lactation and during adult life of the offspring, on the expression of dopaminergic system genes in VTA and HPT of mice dams and their adult male offspring. We also measured diets' effect in locomotor activity in the open field (OF) test. Female mice were divided into control (CONT), restriction (RD) and hyperlipidic (HD) dietary groups, and mated with isogenic male mice. On the 9th postpartum day (PPD), dams were tested in the OF, and on the 22nd PPD cerebral areas were collected. After weaning, the offspring also were divided into one of three diet groups, independently of the diets provided to their dams. In the 80th PPD, the offspring was tested in the OF, and at 100th PPD, VTA and HPT were collected. Gene expression was analyzed by quantitative reverse transcription real-time polymerase chain reaction. The correlation between gene expression and locomotor activity was also assessed. In dams' VTA, both diets upregulated the expression of Th, Slc6a3/Dat1, Drd1 and Drd2 genes. In opposition, in the offspring the maternal diet was associated with a reduction in Th and Ddc gene expression. In the HPT, mice dams that received restriction or hyperlipidic diets had increased Th mRNA levels, but reduced the expression of Drd4 gene. The offspring diet had no effect on the expression of the studied genes in their adult lives. Both diets increased mice dam's locomotion in the OF, however none of them altered the offspring locomotor activity. We detected a positive correlation between the duration of total locomotion in the OF and Slc6a3/Dat1 gene expression in VTA of mice dams. In the HPT, a negative correlation of locomotion and Drd4 mRNA levels, and a positive correlation with Th gene expression was observed. Our results show that restriction and hyperlipidic diets alter mice dams' locomotor activity in the OF and modify the expression of dopaminergic system genes in VTA and HPT of mice dams and in VTA of the offspring.

Effects of 25-hydroxycholecalciferol supplementation in maternal diets on reproductive performance and the expression of genes that regulate lactation in sows.

One hundred Yorkshire × Landrace sows were randomly assigned to one of two dietary treatments (diet ND: 6,000 IU vitamin D3 /d feed; diet 25-D: 200 µg/day 25OHD3 feed). The experiment began on d 90 of gestation and continued until weaning on day 21 of lactation. In sows that received 25OHD3 , the growth rate of the piglets before weaning was significantly accelerated (0.266 kg/day, p < .05). Sow serum was collected after weaning, and those in the 25OHD3 group were found to have significantly higher serum calcium (CA) and phosphorus (PI) levels (p < .05). Interestingly, the oestrus cycle of sows fed 25OHD3 was significantly shortened (p < .05), the oestrus time was concentrated on the fifth day after weaning, and the piglets were born with a higher degree of uniformity (p < .05). Colostrum was collected on the day of delivery, and the colostrum of sows fed 25OHD3 contained higher milk fat content than the control group (p < .05). 25OHD3 supplementation increased the mRNA and protein expression of INSIG1 and SREBP1, which regulate milk fat synthesis, in the mammary gland of lactating sows (p < .05). In conclusion, 25OHD3 supplementation in maternal diets improved reproductive performance, milk fat content and the mRNA and protein levels of genes regulating milk fat synthesis in lactating sows.

Identification and comparison of exosomal microRNAs in the milk and colostrum of two different cow breeds.

Bovine milk and colostrum provide essential nutrients and immunologically active factors that are beneficial to a newborn calf. Milk-and-colostrum-derived exosomes are known as the most important for cellular communication. Exosomes also contain non-coding RNA, such as microRNA. However, there is limited information about exosomal miRNA derived from the milk and colostrum of Holstein and DAK cattle. This study aimed to identify and characterize the exosomal microRNA in the milk and colostrum of Holstein and Dogu Anadolu Kirmizisi (DAK) cows. For this purpose, total RNA isolation was carried out on the milk and colostrum samples that were collected from the Holstein and DAK cattle breeds. The RNA samples were subjected to RNA sequencing and the microRNAs were determined. Lastly, gene ontology analysis was performed for target genes. A total of 795 miRNAs that were expressed differently were identified. A total of 545 of these were known miRNAs and 260 were found to be novel miRNAs. In the functional enrichment analysis, the miRNAs expressed in Holstein milk were mostly associated with milk synthesis, and those in colostrum were mostly involved in the immunity pathways. It was also observed that the miRNAs expressed in DAK milk regulated milk fat and protein metabolism, and there were miRNAs that regulated immune pathways in the colostrum. In addition to this, many novel miRNAs were defined in DAK colostrum. When the target genes of exosomal miRNA in Holstein and DAK milk and colostrum were compared, it was suggested that the DAK breed had genes that were mostly associated with the immune system. As a result, the data obtained from this study will provide beneficial contributions to potential miRNA biomarker studies for milk yield and mastitis.

Identification and verification of differentially expressed genes in yak mammary tissue during the lactation cycle.

Yaks (Bos grunniens) live primarily in the Qinghai-Tibetan plateau (altitude: 2000-5000 m). Their milk presents unusual characteristics, containing large amounts of solids including fat and protein, and it is, therefore, important to understand the genetic makeup of the yak. To identify potentially critical genes playing a role in yak mammary tissue from colostrum to mature milk phase of lactogenesis, the early lactation (colostrum) stage (ELS; day 1 after parturition) and mature lactation (milk) stage (MLS; day 15) were chosen for comparison. An ELS-specific cDNA library was established by suppression subtractive hybridization and 25 expressed sequence tags at ELS were identified by sequencing and alignment. To further confirm our results the expression levels of 21 genes during the lactation cycle were measured using quantitative real-time RT-PCR (qRT-PCR). The qRT-PCR results confirmed 9 significantly up-regulated genes at ELS vs. MLS in yak mammary tissue, in which the l-amino acid oxidase 1 (LAO1) and collagen, type I, alpha I (COL1A1) were the most significantly up-regulated. During the lactation cycle, the highest expression of some milk fat genes (i.e., XDH and FABP3) in yak mammary tissue appears earlier than that in dairy cow. Our data also indicate MYC potentially playing a central role through putative regulation of COL1A1, CD44, SPARC, FASN and GPAM.

Associations between postpartum phenotypes, cow factors, genetic traits, and reproductive performance in seasonal-calving, pasture-based lactating dairy cows.

The objective of this study was to evaluate associations between corpus luteum (CL) status, uterine health, body condition score (BCS), metabolic status, parity, genetic merit for fertility traits, and reproductive performance in pasture-based dairy cows managed for seasonal reproduction. First- and second-lactation (n = 2,600) spring-calving dairy cows from 35 dairy farms located in Ireland were enrolled in the study. Farms were visited every 2 wk, and animals that were at wk 3 (range: 14-27 d in milk) and wk 7 (range: 42-55 d in milk) postpartum were examined. Body condition score was measured using a 1-to-5 scale in 0.25-point increments. Transrectal ultrasound examination was performed at wk 3 and 7 postpartum to determine presence or absence of CL and ultrasound reproductive tract score (scale of G1-G4). Blood samples were collected at each visit, and the concentrations of glucose, ß-hydroxybutyrate (BHB), and fatty acids (FA) were analyzed using enzymatic colorimetry. Animals were grouped into 3 BCS categories [low (≤2.5), target (2.75-3.25), and high (≥3.5)], 2 CL categories (present or absent), 2 uterine health status categories (normal or abnormal), and 3 metabolic status categories [good (high glucose, low FA and BHB), poor (low glucose, high FA and BHB), and moderate (all other combinations)]. Fisher's exact test was used to test for associations between variables and was supplemented by logistic regression. More cows with a CL at wk 7 were served during the first 21 d of the breeding period compared with cows without a CL. Cows classified as having a uterine score of G3 or G4 at wk 3 and 7 had lower odds of pregnancy establishment during the breeding period compared with animals with a uterine score of G1 or G2. Animals with low BCS at wk 7 had lower odds of pregnancy establishment than cows with a target BCS. Cows classified as having good metabolic status at both wk 3 and wk 7 had greater odds of pregnancy establishment during the first 21 d of the breeding season than those classified as having poor metabolic status. Overall, primiparous cows had greater reproductive performance than second-parity cows. Animals in the quartiles with the best predicted transmitting ability for survival and calving interval had better reproductive performance compared with animals in the other quartiles. Cows that had better genetic merit for fertility traits and good metabolic status, achieved target BCS, and had a favorable ultrasound reproductive tract score and a CL present at wk 7 postpartum had superior reproductive performance.

Genetic parameters of colostrum traits in Holstein dairy cows.

The main objective of this study was to assess the genetic background of colostrum yield and quality traits after calving in Holstein dairy cows. The secondary objective was to investigate genetic and phenotypic correlations among laboratory-based and on-farm-measured colostrum traits. The study was conducted in 10 commercial dairy herds located in northern Greece. A total of 1,074 healthy Holstein cows with detailed pedigree information were examined from February 2015 to September 2016. All cows were clinically examined on the day of calving and scored for body condition. All 4 quarters were machine-milked, and a representative and composite colostrum sample was collected and examined. Colostrum total solids (TS) content was determined on-farm using a digital Brix refractometer. Colostrum fat, protein, and lactose contents were determined using an infrared milk analyzer, and energy content was calculated using National Research Council (2001) equations. Dry period length (for cows of parity ≥2), milk yield of previous 305-d lactation (for cows of parity ≥2), age at calving, parity number, season of calving, time interval between calving and first colostrum milking, and milk yield were recorded. Each trait (colostrum yield and quality traits) was analyzed with a univariate mixed model, including fixed effects of previously mentioned factors and the random animal additive genetic effect. All available pedigrees were included in the analysis, bringing the total animal number to 5,662. Estimates of (co)variance components were used to calculate heritability for each trait. Correlations among colostrum traits were estimated with bivariate analysis using the same model. Mean percentage (±SD) colostrum TS, fat, protein, and lactose contents were 25.8 ± 4.7, 6.4 ± 3.3, 17.8 ± 4.0, and 2.2 ± 0.7%, respectively; mean energy content was 1.35 ± 0.3 Mcal/kg and mean colostrum yield was 6.18 ± 3.77 kg. Heritability estimates for the above colostrum traits were 0.27, 0.21, 0.19, 0.15, 0.22, and 0.04, respectively. Several significant genetic and phenotypic correlations were derived. The genetic correlation of TS content measured on-farm with colostrum protein was practically unity, whereas the correlation with energy content was moderate (0.61). Fat content had no genetic correlation with TS content; their phenotypic correlation was positive and low. Colostrum yield was not correlated genetically with any of the other traits. In conclusion, colostrum quality traits are heritable and can be amended with genetic selection.

The impact of organic vs. inorganic selenium on dairy goat productivity and expression of selected genes in milk somatic cells.

The aim of this study was to determine the effect of diet supplemented with selenized yeast (Se-yeast) on milk yield and milk composition of goats and expression of casein and mammary-gland-immune system genes in milk somatic cells (MSC). Twenty-four dairy goats in their second to fourth lactations were divided into control and experimental groups, balanced according to lactation number and breed (Polish White or Fawn Improved). Morning milk and blood samples were collected four times during lactation (on the 21st, 70th, 120th, 180th day after kidding). The control and experimental groups were fed diets with 0.7 mg inorganic Se/goat/day (sodium selenite) or 0.6 mg organic Se/goat/day (selenized yeast), respectively. Milk, fat and protein yields during lactation as well as average somatic cell count, fat, protein and lactose contents in milk were evaluated. Microelements in milk and blood serum and biochemical parameters in blood serum were determined at the beginning and the end of the experiment. The expression levels of the genes encoding αS1-casein (CSN1S1), αS2-casein (CSN1S2), κ-casein (CSN3), interleukin 8 (IL-8), serum amyloid A3 (SAA3), interleukin 1ß (IL-1ß), bactenecin 7.5 (BAC7.5), bactenecin 5 (BAC5), ß2-defensin (GBD2), hepcidin (HAMP), chemokine 4 (CCL4), tumour necrosis factor α (TNFα), toll-like receptor 2 (TLR2), cathelicidin-7 (MAP34) and cathelicidin-6 (MAP28) were determined in MSC. Milk, fat, and protein yields were higher and somatic cell count (SCC expressed as natural logarithm) was lower in the milk of goats fed organic Se. The Se concentration in milk was twice as high in the organic vs. inorganic treatment groups at the end of the experiment, while there were no differences in studied biochemical parameters between groups. The transcript levels of CSN1S2 and BAC7.5 were higher and IL-8 was lower in MSC of Se-yeast treated groups. Such results may indicate better health status of mammary glands of goats treated with organic Se as well as positive impact of selenized yeast on the goat's milk composition. Differences in the IL-1ß and IL-8 transcript levels were also noted between the stages of lactation, with the highest expression at the peak of lactation (day 70), highlighting the metabolic burden at this time. We concluded that the Se-yeast supplementation improved the productivity and health status of goats and could have significant economic impact on farmer's income.

Association of bovine major histocompatibility complex (BoLA) gene polymorphism with colostrum and milk microbiota of dairy cows during the first week of lactation.

BACKGROUND: The interplay between host genotype and commensal microbiota at different body sites can have important implications for health and disease. In dairy cows, polymorphism of bovine major histocompatibility complex (BoLA) gene has been associated with susceptibility to several infectious diseases, most importantly mastitis. However, mechanisms underlying this association are yet poorly understood. In the present study, we sought to explore the association of BoLA gene polymorphism with the dynamics of mammary microbiota during the first week of lactation. RESULTS: Colostrum and milk samples were collected from multiparous Holstein dairy cows at the day of calving and days 1 and 6 after calving. Microbiota profiling was performed using high-throughput sequencing of the V1-V2 regions of the bacterial 16S rRNA genes and ITS2 region of the fungal ribosomal DNA. Polymorphism of BoLA genes was determined using PCR-RFLP of exon 2 of the BoLA-DRB3. In general, transition from colostrum to milk resulted in increased species richness and diversity of both bacterial and fungal communities. The most dominant members of intramammary microbiota included Staphylococcus, Ruminococcaceae, and Clostridiales within the bacterial community and Alternaria, Aspergillus, Candida, and Cryptococcus within the fungal community. Comparing the composition of intramammary microbiota between identified BoLA-DRB3.2 variants (n = 2) revealed distinct clustering pattern on day 0, whereas this effect was not significant on the microbiota of milk samples collected on subsequent days. On day 0, proportions of several non-aureus Staphylococcus (NAS) OTUs, including those aligned to Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus sciuri, and Staphylococcus haemolyticus, were enriched within the microbiota of one of the BoLA-DRB3.2 variants, whereas lactic acid bacteria (LAB) including Lactobacillus and Enterococcus were enriched within the colostrum microbiota of the other variant. CONCLUSION: Our results suggest a potential role for BoLA-gene polymorphism in modulating the composition of colostrum microbiota in dairy cows. Determining whether BoLA-mediated shifts in the composition of colostrum microbiota are regulated directly by immune system or indirectly by microbiota-derived colonization resistant can have important implications for future development of preventive/therapeutic strategies for controlling mastitis.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Maternal intake of grape seed procyanidins during lactation induces insulin resistance and an adiponectin resistance-like phenotype in rat offspring.

Previously, we demonstrated that a grape seed procyanidin extract (GSPE) supplementation in pregnant and lactating rats exerted both healthy and deleterious programming effects on their offspring. Here, we evaluated whether the administration of GSPE during lactation (100 mg.kg-1.day-1) in rats elicited beneficial effects in their normoweight (STD-GSPE group) and cafeteria-fed obese (CAF-GSPE group) adult male offspring. STD-GSPE and CAF-GSPE offspring showed increased energy expenditure and circulating total and high-molecular-weight adiponectin. However, these rats showed hyperinsulinemia, decreased insulin sensitivity, increased insulin resistance, down-regulated mRNA levels of adiponectin receptors in inguinal white adipose tissue (Adipor1 and Adipor2) and soleus muscle (Adipor2), and decreased levels of phosphorylated AMPK, the downstream post-receptor target of adiponectin, in the soleus muscle. These deleterious effects could be related to an increased lipid transfer to the pups through the milk, since GSPE-supplemented dams displayed decreased fat content and increased expression of lipogenic genes in their mammary glands, in addition to increased circulating total adiponectin and non-esterified free fatty acids. In conclusion, maternal intake of GSPE during lactation induced insulin resistance and an adiponectin resistance-like phenotype in their normoweight and obese offspring. These findings raise concerns about the possibility of using GSPE as a nutraceutical supplement during this period.