Bovine Milk Proteins Research Articles

Looking Inside of the Intestinal Permeability Regulation by Protein-Derivatives from Bovine Milk.

The prime function of the epithelium is to regulate the intestinal permeability; the latter is a quantitative parameter that refers to the measurement of the rate of passage of solutes through the epithelial monolayer. Function of epithelial monolayer depends on the expression of protein complexes known as tight junction proteins; whose function and expression can be disrupted under conditions of inflammation including irritable bowel disease (IBD), intestinal infections, and high-fat diets, among others. This manuscript is focused to outline the effects of bovine milk protein derivatives on the intestinal permeability addressed mostly in animal models in which the intestinal barrier is disrupted. At present, the properties of bovine milk protein derivatives on intestinal permeability have been scarcely documented in humans, but evidence raised from clinical trials provides promising findings of potential application of colostrum to control of the intestinal permeability in critically ill patients, users of non-steroid anti-inflammatory drugs, like athletes and militia members.

Deciphering the conformational changes induced by high-risk nsSNPs in β-lactoglobulin

Whey protein from bovine milk is highly valued in the food and pharmaceutical industries because of its high protein content and abundance of essential amino acids. The relationship between whey protein and the β-lactoglobulin (BLG) gene has been extensively discussed because BLG is the most abundant whey protein, making up approximately 50 % of the total whey protein in bovine milk. In recent years, researchers have been interested in this gene because of its critical role in healthy milk production, and any genetic polymorphism in this gene may deteriorate the milk quality. In the current study, we identified several deleterious and damaging non-synonymous single nucleotide polymorphisms (nsSNPs) in BLG and analyzed their destabilizing effects using different computational algorithms. Cumulative results from all tools and evolutionary conservation profiles of BLG suggested that four nsSNPs, G17A, W19C, F136S, and C119R, were the most deleterious and could affect the structural integrity of the protein. Detailed molecular dynamics simulation analysis revealed that all variants induced major structural alterations, that affected the ability of the protein to interact with natural and synthetic ligands. Particularly, the G17A, F136S, and C119R variants induced large conformational changes in the EF loop and main α-helix of BLG, which may affect the access of natural and synthetic ligands to the central calyx of BLG. We hope that the suggested nsSNPs will guide future studies and assist researchers in improving the quality of bovine milk.

Detection of intact bovine milk proteins after simulated gastrointestinal infant digestion using UHPLC − HRMS

Detection of intact bovine milk proteins after simulated gastrointestinal infant digestion using UHPLC − HRMS

Genetic variation in bovine milk proteins: Implications for functional and nutritional properties

The six major bovine milk proteins are encoded by highly polymorphic chromosomes. Genetic variation has become a topic of interest and controversy in recent years due to supposed adverse health effects, beginning with the A2 variant of β‐casein. The functional implications of genetic variants have been well characterised, specifically heat stability and coagulation (i.e. rennet and acid) properties of milk, which have found that the B variant of β‐casein, κ‐casein and β‐lactoglobulin are the most beneficial. Genetic variants of milk proteins provide considerable potential to create new dairy products to add value to milk.

Two major bovine milk whey proteins induce distinct responses in IEC-6 intestinal cells

Abstract α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Detection of Antimicrobial Proteins/Peptides and Bacterial Proteins Involved in Antimicrobial Resistance in Raw Cow's Milk from Different Breeds.

Proteins involved in antibiotic resistance (resistome) and with antimicrobial activity are present in biological specimens. This study aims to explore the presence and abundance of antimicrobial peptides (AMPs) and resistome proteins in bovine milk from diverse breeds and from intensive (Pezzata rossa, Bruna alpina, and Frisona) and non-intensive farming (Podolica breeds). Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) profiling, bottom-up proteomics, and metaproteomics were used to comprehensively analyze milk samples from various bovine breeds in order to identify and characterize AMPs and to investigate resistome proteins. LAP-MALDI MS coupled with linear discriminant analysis (LDA) machine learning was employed as a rapid classification method for Podolica milk recognition against the milk of other bovine species. The results of the LAP-MALDI MS analysis of milk coupled with the linear discriminant analysis (LDA) demonstrate the potential of distinguishing between Podolica and control milk samples based on MS profiles. The classification accuracy achieved in the training set is 86% while it reaches 98.4% in the test set. Bottom-up proteomics revealed approximately 220 quantified bovine proteins (identified using the Bos taurus database), with cathelicidins and annexins exhibiting higher abundance levels in control cows (intensive farming breeds). On the other hand, the metaproteomics analysis highlighted the diversity within the milk's microbial ecosystem with interesting results that may reflect the diverse environmental variables. The bottom-up proteomics data analysis using the Comprehensive Antibiotic Resistance Database (CARD) revealed beta-lactamases and tetracycline resistance proteins in both control and Podolica milk samples, with no relevant breed-specific differences observed.

Electrophoresis analysis and specific IgE-binding capacity of C18 unsaturated fatty acid-milk protein complexes during in vitro gastrointestinal digestion

The main whey allergenic proteins in bovine milk are α-lactalbumin (BLA) and β-lactoglobulin (BLG). When people consume bovine milk, BLA and BLG bind to C18 unsaturated fatty acids (UFA) in the matrix and form fatty acid-milk protein complexes. In this study, we explored the digestive stability of fatty acid-milk protein complexes by simulating infant and adult gastrointestinal digestion conditions and assessed the effects of C18 UFA on milk protein allergenicity. Our findings revealed that the optimal pH digestion for milk proteins in infants and adults was pH 3.0 and pH 2.0, respectively. The tricine-SDS-PAGE and IgE-binding capacity results revealed that the complexes were not easily degraded during digestion resistance, and the digestion products had strong IgE-binding capacity. Therefore, C18 UFA make milk proteins resistant to digestion, thereby increasing their allergenicity.

Urinary opioid peptides in children with autism spectrum disorders. A Pilot Study

BACKGROUND: Circulating β-casomorphins (BCM) and gluten exorphins (GE), which are exogenous opioid peptides originating from bovine milk protein casein and cereal protein gluten, respectively, have been proposed as potential trigger factor to symptoms observed in children with autism. OBJECTIVE: Given the debate surrounding the detectability of these opioid peptides in body fluids, particularly using highly sensitive and specific mass spectrometry (HPLC-MS) techniques, we aimed to investigate their presence in urine samples of autistic subjects. METHODS: We employed an HPLC-MS method for peptide detection. RESULTS: The presence of several BCMs and GEs in the urine of both autistic children (ASD) and healthy controls (HC) was documented. The detection of dietary opioid peptides even at very low concentrations underscores the sensitivity of this novel HPLC-MS method. BCM-8 was more often detected in the ASD group compared to the HC group. A higher prevalence of gastrointestinal (GI) symptoms were also observed in the ASD group. CONCLUSIONS: This pilot study supports the presence of BCMs and GEs in urine samples in subjects with autism as well as healthy controls which was the main goal of this pilot study. Prolonged exposure to bovine BCMs and GEs may play a role in the manifestation of core and GI symptoms in subgroups of autism. Further research is warranted to investigate this phenomenon thoroughly.

Rational development of functional hydrophilic polymer to characterize site-specific glycan differences between bovine milk and colostrum

As vastly modified on secreted proteins, N-glycosylation is found on milk proteins and undergo dynamic changes during lactation, characterizing milk protein glycosylation would benefit the elucidation of glycosylation pattern differences between samples. However, their low abundance required specific enrichment. Herein, through rational design and controllable synthesis, we developed a novel multi-functional polymer for the isolation of protein glycosylation. It efficiently separated glycopeptides from complex background inferences with mutual efforts of hydrophilic interaction chromatography (HILIC), metal ion affinity and ion exchange. By fine-tuning Ca2+ as regulators of aldehyde hyaluronic acid (HA) conformation, the grafting density of HA was remarkably improved. Moreover, grafting Ti4+ further enhanced the enrichment performance. Application of this material to characterize bovine milk and colostrum proteins yields 479 and 611 intact glycopeptides, respectively. Comparative analysis unraveled the distinct glycosylation pattern as well the different distribution of glycoprotein abundances between the two samples, offering insights for functional food development.

Reducing the Allergenicity of β-Lactoglobulin by Covalent Modification with Different Contents of Epigallocatechin Gallate (EGCG): In Vitro and In Vivo Studies.

β-Lactoglobulin (βLG) is a major allergen in bovine milk protein. This study was designed to investigate changes in βLG structure, digestibility, and allergenicity induced by covalent binding modification with different contents of (-)-epigallocatechin 3-gallate (EGCG). The reaction of EGCG conjugation with βLG reached saturation at a molar ratio of 1:60 βLG:EGCG. Conjugation with EGCG altered the βLG structure, decreased IgE-binding capacity, and increased digestibility in a dose-dependent manner. In vivo studies showed that covalent conjugation with EGCG can reduce βLG-induced allergic symptoms with reducing levels of IgE, histamine, and mast cell protease-1 (mMCP-1) and the percentage of sensitized mast cells. Allergenicity was reduced more effectively in saturated βLG-EGCG conjugates compared to semisaturated conjugates. Observed changes in IFN-γ, IL-4, IL-5, IL-10, and TGF-β levels suggested that βLG-EGCG conjugates were able to promote Th1/Th2 immune balance. These findings further our understanding of the relationship between the degree of polyphenol conjugation and the allergenicity of food allergens.

Synthesis and dual sensing of vanadium and copper ions using protein capped selenium nanoparticles

Highly homogenous and spherical shaped Selenium nanoparticles with an average size of 20 nm were synthesized from an eco-friendly and environmentally safe approach using bovine milk protein casein as stabilizing agent (named as CasSeNPs). Protein functionalization provided excellent stability at variable temperatures (25–60 °C), pH (8–10), ionic strength (0.05–2 M), and storage period for more than five months. Synthesized CasSeNPs were extensively characterized using various spectroscopic (UV–visible, Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy), particle sizing (dynamic light scattering (DLS) and zeta potential), microscopic (scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM/EDS), atomic force microscopy (AFM)), and X-ray diffraction (XRD) measurements. Moreover, CasSeNPs was established as highly sensitive and selective sensor for the simultaneous detection of vanadium (V+2) and copper (Cu+2) ions with significant linearity (0.5–10 and 0.1–1.0 µM, respectively), detection limits (LOD = 0.03 and 0.067 µM, respectively), and percent recoveries (94–105 %) in real environmental (tap water) and biological (human plasma and urine) samples with adequate test performance. Cumulatively, obtained results revealed that the established very simple, biocompatible and economical nanosensor has great potential for applications in analytical chemistry as well as for biological investigations.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

Comparative analysis of goat and bovine milk proteins in the improvement of sarcopenia based on host-microbial interactions

Comparative analysis of goat and bovine milk proteins in the improvement of sarcopenia based on host-microbial interactions

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased β-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

2′-FL glycosylation of bovine milk protein matrices alters protein structure and reduces antibody affinity

Three glycosylated proteins were prepared from 2′-fucosyllactose (2′-FL) with α-lactalbumins, β-lactoglobulin and whey protein isolate, respectively, and their protein structures, glycosylation sites, digestion and transport capacities, and antibody binding capacities were determined. The findings indicate that following heating at 68 °C for 90 min, 2′-FL can form stable bonds with amino acid residues in milk proteins, leading to enhanced antioxidant properties, reduced hydrophobicity, and modified secondary structure content of the proteins. The glycosylation sites were mainly located in the amino acid sequence of β-lactoglobulin (K47, K91 and K135) and significantly reduce the affinity of its specific antibodies. 2′-FL glycosylation did not have a significant effect on the digestibility of cow's milk proteins, but it could significantly reduce the affinity of their specific antibody, indicating that it could be used in producing hypoallergenic dairy product.

Phosphoproteomics Revealed Differentially Expressed Sites and Function of the Bovine Milk Fat Globule Membrane in Colostrum and Mature Milk.

One type of large and intricate post-translational modification of milk proteins that has significant biological implications is phosphorylation. The characterization of phosphoproteins found in the bovine milk fat globule membrane (MFGM) is still mostly unknown. Here, label-free phosphoproteomics was used to identify 94 phosphorylation sites from 54 MFGM phosphoproteins in bovine colostrum (BC) and 136 phosphorylation sites from 91 MFGM phosphoproteins in bovine mature milk (BM). αs1-Casein and β-casein were the most phosphorylated proteins in bovine colostrum. In bovine mature milk, perilipin-2 was the protein with the greatest number of phosphorylation sites. The results show that bovine colostrum MFGM phosphoproteins were mainly involved in immune function, whereas bovine mature MFGM phosphoproteins were mainly involved in metabolic function. Plasminogen and osteopontin were the most strongly interacting proteins in colostrum, whereas perilipin-2 was the most strongly interacting protein in bovine mature milk. This work demonstrates the unique alterations in the phosphorylation manner of the bovine MFGM protein during lactation and further expands our knowledge of the site characteristics of bovine MFGM phosphoproteins. This result confirms the value of MFGM as a reference ingredient for infant formula during different stages.

Comparing physicochemical properties related to thermal stability of caprine and bovine milk protein concentrate dispersions

Thermal stability and physicochemical properties as a function of heating temperature, pH and salt addition was compared between caprine and bovine milk protein concentrate dispersions. Caprine milk protein concentrate (MPC) had a lower amount of heat coagulation time and a higher percentage of sediments than bovine MPC. Calcium ion activity of caprine MPC was higher than that of bovine. The amount of κ-casein in the serum of caprine MPC dispersions was higher than that of bovine, and increased with increasing temperatures, pH and the addition of NaCl and citrate. The amount of whey protein in serum was also higher in heated caprine MPC compared with bovine. Particle size of caprine MPC was larger than bovine MPC and was more susceptible to induced aggregation. The lower thermal stability of caprine MPC might be associated with the higher calcium ion activity and the higher amount of κ-casein dissociation promoted by whey protein denaturation and complexation in serum.

Mining Bovine Milk Proteins for DPP-4 Inhibitory Peptides Using Machine Learning and Virtual Proteolysis.

Dipeptidyl peptidase-IV (DPP-4) enzyme inhibitors are a promising category of diabetes medications. Bioactive peptides, particularly those derived from bovine milk proteins, play crucial roles in inhibiting the DPP-4 enzyme. This study describes a comprehensive strategy for DPP-4 inhibitory peptide discovery and validation that combines machine learning and virtual proteolysis techniques. Five machine learning models, including GBDT, XGBoost, LightGBM, CatBoost, and RF, were trained. Notably, LightGBM demonstrated superior performance with an AUC value of 0.92 ± 0.01. Subsequently, LightGBM was employed to forecast the DPP-4 inhibitory potential of peptides generated through virtual proteolysis of milk proteins. Through a series of in silico screening process and in vitro experiments, GPVRGPF and HPHPHL were found to exhibit good DPP-4 inhibitory activity. Molecular docking and molecular dynamics simulations further confirmed the inhibitory mechanisms of these peptides. Through retracing the virtual proteolysis steps, it was found that GPVRGPF can be obtained from β-casein through enzymatic hydrolysis by chymotrypsin, while HPHPHL can be obtained from κ-casein through enzymatic hydrolysis by stem bromelain or papain. In summary, the integration of machine learning and virtual proteolysis techniques can aid in the preliminary determination of key hydrolysis parameters and facilitate the efficient screening of bioactive peptides.

The impact of heat-set milk protein gel textures modified by pH on circulating amino acid appearance and gastric function in healthy female adults: a randomised controlled trial.

Modification of dairy proteins during processing impacts structural assemblies, influencing textural and nutritional properties of dairy products, and release and availability of amino acids during digestion. By modifying only pH, acid heat-set bovine dairy gels with divergent textural properties were developed to alter protein digestion. In vitro assay confirmed faster digestion of protein from a firm gel (pH 5.65) versus a soft gel (pH 6.55). We hypothesised that firm gel (FIRM-G; pH 5.6) would result in greater indispensable amino acid (IAA) appearance in circulation over 5 h and corresponding differences in gastric myoelectrical activity relative to soft gel (SOFT-G; pH 6.2). In a randomised, single-blind cross-over trial, healthy females (n = 20) consumed 150 g of each gel; plasma amino acid appearance was assessed over 5 hours. Iso-nitrogenous, iso-caloric gels were prepared from identical mixtures of bovine milk and whey protein concentrates; providing 17.7 g (FIRM-G) and 18.9 g (SOFT-G) of protein per serving. Secondary outcomes included gastric myoelectrical activity measured by body surface gastric mapping, glycaemic, triglyceridaemic, and subjective appetite and digestive responses. Overall plasma IAA (area under the curve) did not differ between gels. However, plasma IAA concentrations were higher, and increased more rapidly over time after SOFT-G compared with FIRM-G (1455 ± 53 versus 1350 ± 62 μmol L-1 at 30 min, p = 0.024). Similarly, total, branched-chain and dispensable amino acids were higher at 30 min with SOFT-G than FIRM-G (total: 3939 ± 97 versus 3702 ± 127 μmol L-1, p = 0.014; branched-chain: 677 ± 30 versus 619 ± 34 μmol L-1, p = 0.047; dispensable: 2334 ± 53 versus 2210 ± 76 μmol L-1, p = 0.032). All other measured parameters were similar between gels. Peak postprandial aminoacidaemia was higher and faster following ingestion of SOFT-G. Customised plasma amino acid appearance from dairy is achievable by altering gel coagulum structure using pH during processing and may have minimal influence on related postprandial responses, with implications for targeting food design for optimal health. The Clinical Trial Registry number is ACTRN12622001418763 (https://www.anzctr.org.au) registered November 7, 2022.

Analysis of β-casein gene for A1 and A2 allelic variants in Holstein-Local crossbred cattle of Bangladesh using allele-specific PCR

Beta-casein (CSN2) is one of the important bovine milk proteins synthesized by β-casein gene. The aim of this study was to investigate the A1 and A2 allelic variants of CSN2 gene in Holstein-Friesian and Local (HF×L) crossbred cattle of Bangladesh. Allele specific polymerase chain reaction (AS-PCR) was carried out for genotyping of allelic variants. A total of 390 blood samples of HF×L crossbred cattle was included from govt., military and private dairy farms. Six different crossbred genotypes were identified in the studied population as 50%HF×50%L, 62.5%HF×37.5%L, 68.75%HF×31.25%L, 75%HF×25%L, 81.25%HF×18.75%L and 87.5%HF×12.5%L. The mean frequencies of the A1A1, A1A2, and A2A2 genotypes were 0.11, 0.48, and 0.41, respectively, while the corresponding frequencies of the A1 and A2 alleles were 0.34 and 0.66, respectively. The frequency of desirable A2A2 genotype reduced from 0.60 (50%HF×50%L) to 0.38 (87.5%HF×12.5%L) with the advancement of exotic HF inheritance from 50 to 87.5%. In contrast, the frequency of A1A1 and A1A2 genotypes increased proportionately with the progression of HF inheritance level. The undesirable A1A1 genotype frequency was found highest in the HF crossbreds of private dairy farms (0.13) compared to government (0.08) and military dairy farm (0.07) that signifies the availability of upper graded cows in the private dairy farms. Altogether, this study provides current scenario of A1 and A2 allelic variants of HF×L crossbreds of Bangladesh that could be utilized to design suitable breeding plans in crossbreeding programs for production of premium quality milk.