Lactation Biology Research Articles

Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome

Human milk is a complex mix of nutritional and bioactive components that provide complete nourishment for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we characterize relationships between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in up to 310 exclusively breastfeeding mother-infant pairs. We identified 482 genetic loci associated with milk gene expression unique to the lactating mammary gland and link these loci to breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with milk interleukin-6 (IL-6) concentration and the abundance of Bifidobacterium and Escherichia in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

Variations in Casein Genes Are Associated with Milk Protein and Fat Contents in Sarda Goats (Capra hircus), with an Important Role of CSN1S2 for Milk Yield.

This work aimed to assess the variability of casein genes in a population of 153 bucks and 825 lactating does of the Sarda breed, and to perform association analysis between polymorphic sites and milk yield and composition traits. To genotype the casein genes, we chose an SNP panel including 44 SNPs mapping to the four casein genes CSN1S1, CSN2, CSN1S2, and CSN3. Genotyping (made by KASP™ genotyping assay, based on competitive allele-specific PCR) revealed the high variability of the Sarda goat, and haplotype analysis revealed linkage disequilibrium (LD) between CSN1S1 and CSN2 genes, in addition to two LD blocks within the CSN1S2 and two LD blocks within the CSN3 gene, in bucks and does. Association analysis revealed that variability at all four casein genes was associated with milk protein content, total solids, and milk energy. The three Ca-sensitive casein genes were associated with lipid content, and CSN1S2 showed a unique pattern, with intron variants associated with milk yield, in addition to milk pH, NaCl, and SCS (Somatic Cell Score). This information might prove useful in selection schemes and in future investigations aiming to better understand the biology of lactation, and the direct link between genotype and phenotype.

Using QTL to Identify Genes and Pathways Underlying the Regulation and Production of Milk Components in Cattle.

Milk is a complex liquid, and the concentrations of many of its components are under genetic control. Many genes and pathways are known to regulate milk composition, and the purpose of this review is to highlight how the discoveries of quantitative trait loci (QTL) for milk phenotypes can elucidate these pathways. The main body of this review focuses primarily on QTL discovered in cattle (Bos taurus) as a model species for the biology of lactation, and there are occasional references to sheep genetics. The following section describes a range of techniques that can be used to help identify the causative genes underlying QTL when the underlying mechanism involves the regulation of gene expression. As genotype and phenotype databases continue to grow and diversify, new QTL will continue to be discovered, and although proving the causality of underlying genes and variants remains difficult, these new data sets will further enhance our understanding of the biology of lactation.

Mammary epithelial cell transcriptome reveals potential roles of lncRNAs in regulating milk synthesis pathways in Jersey and Kashmiri cattle

BackgroundLong noncoding RNAs (lncRNAs) are now proven as essential regulatory elements, playing diverse roles in many biological processes including mammary gland development. However, little is known about their roles in the bovine lactation process.ResultsTo identify and characterize the roles of lncRNAs in bovine lactation, high throughput RNA sequencing data from Jersey (high milk yield producer), and Kashmiri cattle (low milk yield producer) were utilized. Transcriptome data from three Kashmiri and three Jersey cattle throughout their lactation stages were utilized for differential expression analysis. At each stage (early, mid and late) three samples were taken from each breed. A total of 45 differentially expressed lncRNAs were identified between the three stages of lactation. The differentially expressed lncRNAs were found co-expressed with genes involved in the milk synthesis processes such as GPAM, LPL, and ABCG2 indicating their potential regulatory effects on milk quality genes. KEGG pathways analysis of potential cis and trans target genes of differentially expressed lncRNAs indicated that 27 and 48 pathways were significantly enriched between the three stages of lactation in Kashmiri and Jersey respectively, including mTOR signaling, PI3K-Akt signaling, and RAP1 signaling pathways. These pathways are known to play key roles in lactation biology and mammary gland development.ConclusionsExpression profiles of lncRNAs across different lactation stages in Jersey and Kashmiri cattle provide a valuable resource for the study of the regulatory mechanisms involved in the lactation process as well as facilitate understanding of the role of lncRNAs in bovine lactation biology.

A career in lactation biology

This introduction summarizes some of the contributions made by woman in the field of lactation biology and endocrine regulation of lactation. In particular, it highlights the career of Dr. Karen Plaut, who at the time of writing this introduction was Dean of the College of Agriculture at Purdue University. She was a trailblazer for women in lactation biology following in the footsteps on some influential women in the field. She describes some of the accomplishments made by women in the field and some of the opportunities for the future.

Resolving Human Lactation Heterogeneity Using Single Milk-Derived Cells, a Resource at the Ready.

Single cell RNA sequencing (scRNAseq) of human milk-derived cells (HMDCs) makes highly detailed analyses of the biology of human lactation possible. We explore this powerful application as an exciting tool to inspect the cellular composition of human milk. We point out some important challenges unique to this approach and highlight the importance of collaborations between biologists and well-trained bioinformaticians to utilize these data to their maximum potential. We extend this focus by discussing the first two such studies that describe HMDCs via scRNAseq and a variety of important questions in the field that warrant attention through further research. The stage is set to apply scRNAseq in human lactation biology, potentially leading to new insights regarding the molecular and cellular diversity of human secretory mammary epithelial cells.

Effect of season and temperature before and after calving on the future milk production of born heifers

Effect of season and temperature before and after calving on the future milk production of born heifers

Mucosal Immunity and Liver Metabolism in the Complex Condition of Lactation Insufficiency.

Lactation insufficiency is variously defined and includes the inability to produce milk, not producing enough milk to exclusively meet infant growth requirements, and pathological interruption of lactation (e.g., mastitis). Of women with intent-to-breastfeed, lactation insufficiency has been estimated to affect 38%-44% of newly postpartum women, likely contributing to the nearly 60% of infants that are not breastfed according to the World Health Organization's guidelines. To date, research and clinical practice aimed at improving feeding outcomes have focused on hospital lactation support and education, with laudable results. However, researchers' reports of recent rodent studies concerning fundamental lactation biology have suggested that the underlying pathologies of lactation insufficiency may be more nuanced than is currently appreciated. In this article, we identify mucosal biology of the breast and lactation-specific liver biology as two under-researched aspects of lactation physiology. Specifically, we argue that further scientific inquiry into reproductive state-dependent regulation of immunity in the human breast will reveal insights into novel immune based requirements for healthy lactation. Additionally, our synthesis of the literature supports the hypothesis that the liver is an essential player in lactation-highlighting the potential that pathologies of the liver may also be associated with lactation insufficiency. More research into these biologic underpinnings of lactation is anticipated to provide new avenues to understand and treat lactation insufficiency.

Immunology of Human Milk and Lactation: Historical Overview.

The development of the mammary glands and the process of lactation is an integral component of mammalian evolution, and suckling has been essential for the survival of the neonates of most mammalian species. The colostrum and milk, the major products of lactation, contain a wealth of biologically active products derived from the immunologic and microbiological experiences in the maternal circulation and in the maternal mucosal surfaces. These include major immunoglobulin isotypes in the maternal circulation, secretory IgA, a variety of soluble proteins, casein, nutritional components, hormones, a large number of cellular elements and their secreted functional products (cytokines and chemokines), several peptides, lipids, polysaccharides and oligosaccharides, and a diverse spectrum of microorganisms. During the past few decades, significant new information has become available about the evolutionary biology of mammalian lactation, the functional characterization of antibody and cellular immunologic products, the role of oligosaccharides and other proteins and peptides, and about the distribution and biologic functions of the microbiome observed in human products of lactation. This workshop explores this information in some detail in a series of presentations. A brief overview of the earlier observations on the immunologic aspects of lactation is presented here, and detailed reviews of more recent observations are reported in subsequent presentations in this workshop.

Single-step genome-wide association for longitudinal traits of Canadian Ayrshire, Holstein, and Jersey dairy cattle

Estimating single nucleotide polymorphism (SNP) effects over time is essential to identify and validate candidate genes (or quantitative trait loci) associated with time-dependent variation of economically important traits and to better understand the underlying mechanisms of lactation biology. Therefore, in this study, we aimed to estimate time-dependent effects of SNP and identifying candidate genes associated with milk (MY), fat (FY), and protein (PY) yields, and somatic cell score (SCS) in the first 3 lactations of Canadian Ayrshire, Holstein, and Jersey breeds, as well as suggest their potential pattern of phenotypic effect over time. Random regression coefficients for the additive direct genetic effect were estimated for each animal using single-step genomic BLUP, based on 2 random regression models: one considering MY, FY, and PY in the first 3 lactations and the other considering SCS in the first 3 lactations. Thereafter, SNP solutions were obtained for random regression coefficients, which were used to estimate the SNP effects over time (from 5 to 305 d in lactation). The top 1% of SNP that showed a high magnitude of SNP effect in at least 1 d in lactation were selected as relevant SNP for further analyses of candidate genes, and clustered according to the trajectory of their SNP effects over time. The majority of SNP selected for MY, FY, and PY increased the magnitude of their effects over time, for all breeds. In contrast, for SCS, most selected SNP decreased the magnitude of their effects over time, especially for the Holstein and Jersey breeds. In general, we identified a different set of candidate genes for each breed, and similar genes were found across different lactations for the same trait in the same breed. For some of the candidate genes, the suggested pattern of phenotypic effect changed among lactations. Among the lactations, candidate genes (and their suggested phenotypic effect over time) identified for the second and third lactations were more similar to each other than for the first lactation. Well-known candidate genes with major effects on milk production traits presented different suggested patterns of phenotypic effect across breeds, traits, and lactations in which they were identified. The candidate genes identified in this study can be used as target genes in studies of gene expression.

Meta-analysis of mammary RNA seq datasets reveals the molecular understanding of bovine lactation biology.

A better understanding of the biology of lactation, both in terms of gene expression and the identification of candidate genes for the production of milk and its components, is made possible by recent advances in RNA seq technology. The purpose of this study was to understand the synthesis of milk components and the molecular pathways involved, as well as to identify candidate genes for milk production traits within whole mammary transcriptomic datasets. We performed a meta-analysis of publically available RNA seq transcriptome datasets of mammary tissue/milk somatic cells. In total, 11 562 genes were commonly identified from all RNA seq based mammary gland transcriptomes. Functional annotation of commonly expressed genes revealed the molecular processes that contribute to the synthesis of fats, proteins, and lactose in mammary secretory cells and the molecular pathways responsible for milk synthesis. In addition, we identified several candidate genes responsible for milk production traits and constructed a gene regulatory network for RNA seq data. In conclusion, this study provides a basic understanding of the lactation biology of cows at the gene expression level.

Human Milk Science and Innovation Conference.

Breastfeeding MedicineVol. 14, No. S1 Proceedings from the 6th Annual International Conference on Human Milk Science and InnovationOpen AccessCreative Commons licenseHuman Milk Science and Innovation ConferenceArthur I. EidelmanArthur I. Eidelman—Arthur I. Eidelman, MD, Editor-in-ChiefSearch for more papers by this authorPublished Online:12 Apr 2019https://doi.org/10.1089/bfm.2019.29123.aieAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Breastfeeding Medicine, the official journal of the Academy of Breastfeeding Medicine, is once again honored and most pleased to publish the proceedings of the sixth Annual International Conference on “Human Milk Science and Innovation,” sponsored and supported by Prolacta Bioscience. This annual conference continues to serve as a major platform for the presentation both of the data from the most up-to-date research in the biology of human lactation and the outcome of clinical research programs that focus on the care and welfare of the maternal–infant dyad.The proceedings, as in previous years, are guest edited by William D. Rhine, MD, from the Department of Pediatrics (Neonatology) of the Stanford University School of Medicine, Palo Alto, California, and Lars Bode, PhD, from the Larsson-Rosenquist Foundation Mother-Milk-Infant Center of Research Excellence, University of California, San Diego, California.Presentations were from leading international experts from a wide variety of academic specialties. A special feature was the emphasis on the long-term health benefits of breastfeeding, both to the infant and the mother. The relationship of breastfeeding and the infant's microbiome was highlighted by more than one speaker.The conference also was the setting of the awarding Ruth A. Lawrence Investigator Award for Research in Human Milk Science to Steven D. Townsend, PhD, for his research on the effect of the ingestion of human milk oligosaccharides as a basic defense mechanism against pathogens The award honored both Dr. Townsend for his current academic achievements and Dr. Lawrence for her pioneering activities in the development and support of the study of the science of breastfeeding and human lactation. Dr. Lawrence was the founding editor of Breastfeeding Medicine some 14 years ago, so it is most appropriate that the award in her name be properly highlighted in the pages of Breastfeeding Medicine, an endeavor most dear to her professional career.Special thanks to Prolacta Bioscience for hosting the conference in September 2018 and in facilitating the publishing of the edited proceedings in Breastfeeding Medicine.FiguresReferencesRelatedDetails Volume 14Issue S1Apr 2019 Information© Arthur I. Eidelman, 2019; Published by Mary Ann Liebert, Inc.To cite this article:Arthur I. Eidelman.Human Milk Science and Innovation Conference.Breastfeeding Medicine.Apr 2019.S-1-S-1.http://doi.org/10.1089/bfm.2019.29123.aiecreative commons licensePublished in Volume: 14 Issue S1: April 12, 2019Open accessThis Open Access article is distributed under the terms of the Creative Commons License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.PDF download

Milk-derived mammary epithelial cells as non-invasive source to define stage-specific abundance of milk protein and fat synthesis transcripts in native Sahiwal cows and Murrah buffaloes.

The molecular physiology of milk production of two important dairy species; Sahiwal cows (Bos indicus) and Murrah buffaloes (Bubalus bubalis) are not fully understood due to constraints in obtaining mammary tissue samples because of sacred and ethical reasons. The present study suggests the use of milk-derived mammary epithelial cells (MECs) as a non-invasive method to understand molecular aspects of lactation biology in dairy animals. A total of 76 MECs were collected from five different lactation periods viz. colostrum (0-2), early (5-20), peak (30-50), mid (90-140) and late lactation (> 215 days) stages from Sahiwal cows and Murrah buffaloes to study the transcription kinetics of milk protein, fat synthesis, and their regulatory genes. Significant changes were observed in milk composition of both dairy species with lactation stages. High mRNA abundance of all milk protein and fat synthesis genes was observed in MECs of Murrah buffaloes as compared to Sahiwal cows. The mRNA abundance of caseins (CSN1S1, CSN1S2, CSN2, and CSN3) and whey protein (LALBA, LF) were higher in early lactation stage. Similarly, the expression of milk fat synthesis genes (SCD, BTN1A1, ACACA, GPAM, FAPB3, FASN) was also high in early lactation stage. The relative abundance of 4 regulatory genes (JAK2, STAT5, SREBF1 and EIF4BP41) remained high during early lactation indicating their regulatory roles in lactogenesis process. Overall, results suggested a significant effect of lactation stages on milk composition and transcription abundance of milk protein and fat synthesis genes. The present study establishes the fact that milk-derived MECs could be utilized as a valuable source to understand mammary gland functioning of native cows and buffaloes.

Human milk sIgA antibody in relation to maternal nutrition and infant vulnerability in northern Kenya.

BackgroundThe maternal buffering hypothesis posits that human lactation biology can buffer milk against the mild-to-moderate malnutrition that occurred routinely in evolutionary history through the mobilization of maternal body reserves. This perspective may provide insights for understanding human milk immune content variation, such as milk sIgA, which protects infants’ intestines from microbial colonization and prevents diarrheal disease.ObjectiveTo investigate how maternal delivery of sIgA to milk may vary in a way that can buffer milk against maternal malnutrition, while taking into consideration infants’ varying needs for immune protection across age or by sex.MethodologyA cross-sectional study analyzed archived milk specimens from breastfeeding mothers in Ariaal communities of northern Kenya surveyed during the 2006 Horn-of-Africa drought. Multiple regression models for ln-transformed sIgA were constructed using maternal nutrition, infant age/sex and their interactions as predictors. Maternal nutrition variables included iron-deficiency anemia (IDA), vitamin A deficiency (VAD) and mid-upper arm circumference (MUAC). Infant vulnerability was considered high in young age and/or male sex.Results and implicationsMilk sIgA did not significantly differ by maternal IDA. Milk sIgA increased with infant age and maternal MUAC (n = 202). Significant interactions were observed between infant age and maternal VAD and between infant sex and maternal MUAC, such that milk sIgA content was low for younger infants particularly among VAD mothers, while among mothers with low MUAC, sIgA was lower for male infants. Results imply that mothers’ ability to deliver/buffer milk sIgA may be lowered when nutritional stress is combined with high infant vulnerability to infection.Lay SummaryHuman milk sIgA antibody content was low for younger infants among vitamin A deficient mothers. Among mothers with small arm-circumference, milk sIgA was lower for sons. Double burden of raising young or male infants with high needs for immune protection and being malnourished, might lower maternal sIgA delivery to milk.

Integrated analysis of long noncoding RNA and mRNA expression profiles reveals the potential role of long noncoding RNA in different bovine lactation stages

Long noncoding RNA (lncRNA) play a critical role in mammary development and breast cancer biology. Despite their important role in the mammary gland, little is known of the roles of lncRNA in bovine lactation, particularly regarding the molecular processes underlying it. To characterize the role of lncRNA in bovine lactation, 4 samples of Holstein cow mammary gland tissue at peak and late lactation stages were examined after biopsy. We then profiled the transcriptome of the mammary gland using RNA sequencing technology. Further, functional lncRNA-mRNA coexpression pairs were constructed to infer the function of lncRNA using a generalized linear model, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. More than 1,000 putative lncRNA were identified, 117 of which were differentially expressed between peak and late lactation stages. Bovine lncRNA were shorter, with fewer exon numbers, and expressed at significantly lower levels than protein-coding genes. Seventy-two differentially expressed (DE) lncRNA were coexpressed with 340 different protein-coding genes. The KEGG pathway analysis showed that target mRNA for DE lncRNA were mainly related to lipid and glucose metabolism, including the peroxisome proliferator-activated receptors and 5' adenosine monophosphate-activated protein kinase signaling pathways. Further bioinformatics and integrative analyses revealed that 12 DE lncRNA potentially played important roles in bovine lactation. Our findings provide a valuable resource for future bovine transcriptome studies, facilitate the understanding of bovine lactation biology, and offer functional information for cattle lactation.

TRIENNIAL LACTATION BIOLOGY SYMPOSIUM/BOLFA: The biology of lactation- from genes to cells to milk.

TRIENNIAL LACTATION BIOLOGY SYMPOSIUM/BOLFA: The biology of lactation- from genes to cells to milk.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Late gestation heat stress of dairy cattle programs dam and daughter milk production.

Anticipated increases in the world population to 9 billion people will lead to increased demand for food. Dairy products represent one of the most sustainable animal sources of food protein because ruminants can utilize byproduct and forage feeds unsuitable for human consumption. Continued improvements in productivity will depend on deeper understanding of the biology of lactation, including developmental programming of tissues critical to that process. Although prenatal programming of postnatal phenotype is well documented for growth, behavior, and disease, there may also be instances of "programming" that last for a specific physiological stage (e.g., lactation). We distinguish between these 2 terms by the use of developmental programming to describe a permanent effect, whereas the more general term is used to describe nonpermanent impacts on the mammary gland. Despite this complexity, here we review the evidence that exposure to elevated temperature and humidity during late gestation can program reduced yields in the subsequent lactation, largely through effects at the mammary gland. Furthermore, we provide emerging evidence that adult capacity for milk synthesis can be programmed in the calf that dam is carrying by events during fetal life occurring 2 yr before. Specifically, calves born to dams that are heat stressed for the final 6 wk of gestation produce 19% less milk in lactation relative to calves from dams provided with evaporative cooling. Importantly, the increased milk yield in animals derived from dams under evaporative cooling occurred without a greater decline in BW that accompanies negative energy balance during early lactation. Therefore, the increase in milk production suggests an increase in the efficiency of conversion of feed to milk. These data indicate that a brief period of heat stress late in development reduces the physiological efficiency of the cow in a coordinated manner to result in a substantial decline in productivity. It is likely that this programming effect would be observed across genetic lines and result in poor sustainability of milk production. Milk will continue to be an important source of high-quality, human-edible food and technologies that improve the efficiency of production will be critical to enhance sustainability. These data provide compelling support for the concept that programming impacts on the dam and the developing fetus will play a role in optimizing the efficiency of production.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA:Historical perspectives of lactation biology in the late 20th and early 21st centuries.

The latter half of the 20th century and the early portion of the 21st century will be recognized as the "Golden Age" of lactation biology. This period corresponded with the rise of systemic, metabolomic, molecular, and genomic biology. It includes the discovery of the structure of DNA and ends with the sequencing of the complete genomes of humans and all major domestic animal species including the dairy cow. This included the ability to identify polymorphisms in the nucleic acid sequence, which can be tied to specific differences in cellular, tissue, and animal performance. Before this period, classical work using endocrine ablation and replacement studies identified the mammary gland as an endocrine-dependent organ. In the early 1960s, the development of RIA and radioreceptor assays permitted the study of the relationship between endocrine patterns and mammary function. The ability to measure nucleic acid content of tissues opened the door to study of the factors regulating mammary growth. The development of high-speed centrifugation in the 1960s allowed separation of specific cell organelles and their membranes. The development of transmission and scanning electron microscopy permitted the study of the relationship between structure and function in the mammary secretory cell. The availability of radiolabeled metabolites provided the opportunity to investigate the metabolic pathways and their regulation. The development of concepts regarding the coordination of metabolism to support lactation integrated our understanding of nutrient partitioning and homeostasis. The ability to produce recombinant molecules and organisms permitted enhancement of lactation in farm animal species and the production of milk containing proteins of value to human medicine. These discoveries and others contributed to vastly increased dairy farm productivity in the United States and worldwide. This review will include the discussion of the centers of excellence and scientists who labored in these fields to produce the harvest of knowledge we enjoy today.

Quantitative proteome analysis of bovine mammary gland reveals protein dynamic changes involved in peak and late lactation stages

Mammary gland is an important organ for milk synthesis and secretion. It undergoes dramatic physiological changes to adapt the shift from peak to late lactation stage. Protein plays a final very vital role in many life functions, and the protein changes during different lactation stages potentially reflect the biology of lactation and the functions of mammary gland in cows. In current study, we adopted tandem mass tags label-based quantitative analysis technique and to investigate proteome changes occurring in bovine mammary gland from peak to late lactation stages. A total of 3753 proteins from mammary tissues taken at two lactation points from four individual cows by biopsy were quantified, out of which 179 proteins were expressed differentially between two stages. We observed five new DEPs (AACS, DHCR7, GSTM3, SFRP1 and SFRP4) and nine functional well-studies known proteins (PLIN2, LPIN1, PLIN3, GSN, CD74, MMP2, SOD1, SOD3 and GPX3) related to milk performance and mammary morphology. Bioinformatics analyses of the DEPs showed a majority of the up-regulated proteins during late lactation stage were related to apoptosis and immune process, while the downregulated proteins were mainly involved in localization, lipid metabolic and transport process. This suggests that the mammary gland can adapt to different molecular functions according to the biological need of the animal. From the integrated analysis of the differentially expressed proteins with known quantitative trait loci and genome-wide association study data, we identified 95 proteins may potentially affect milking performance. We expect findings in this study could be a valuable resource for future studies investigating the bovine proteome and functional studies.

Osteopontin gene polymorphism association with milk traits and its expression analysis in milk of riverine buffalo.

Osteopontin gene is regarded as a plausible candidate in mammary gland differentiation and development, expressed by variety of cells, tissues, and biological fluids including milk. The current study was performed in two phases. In the first phase, Osteopontin gene polymorphisms were identified and associated with milk composition such as ash, milk fat, SNF, lactose, and protein. In the second phase, milk samples from five healthy mastitis-free Nili Ravi buffaloes were analyzed for expression of Osteopontin gene at transition (day 15), mid (day 90), and end (day 250) stage of their second lactation. Briefly, blood samples were collected from Nili Ravi buffalo to isolate the genomic DNA, specific primers were designed for PCR amplification. The amplified PCR products were sequenced bi-directionally. Six polymorphisms were identified in the coding region and four in the intronic region of the gene. The results showed that SNP g.38329758T>C causing substitution of valine to alanine (V127A) was associated with high milk protein. For mRNA expression analysis, somatic cells were separated from milk samples for RNA isolation. Analysis of differential gene expression data has permitted us to illustrate the expression pattern of osteopontin gene in lactating buffalo. The Osteopontin gene was found to be transcribed among all three lactation stages, but expression was observed with the highest value (fold change) in peak lactation and remained elevated till the end of lactation. Identified gene marker may be helpful for the prediction of superior animal for selection. The presented study also gave an insight into the genetic screening and lactation biology of riverine buffalo, offering direction for future research in lactating buffalo.