Short communication: Milk protein genetic variation and casein haplotype structure in the Original Pinzgauer cattle

Milk protein genetic polymorphisms are often used for characterizing domesticated mammalian species and breeds, and for studying associations with economic traits. The aim of this work was to analyze milk protein genetic variation in the Original Pinzgauer, a dual-purpose (dairy and beef) cattle breed of European origin that was influenced in the past by human movements from different regions as well as by crossbreeding with Red Holstein. A total of 485 milk samples from Original Pinzgauer from Austria (n = 275) and Germany (n = 210) were typed at milk proteins α_S1-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin by isoelectrofocusing to analyze the genetic variation affecting the protein amino acid charge. The Original Pinzgauer breed is characterized by a rather high genetic variation affecting the amino acid charge of milk proteins, with a total of 15 alleles, 12 of which were found at a frequency >0.05. The most polymorphic protein was β-casein with 4 alleles detected. The prevalent alleles were CSN1S1*B, CSN2*A², CSN1S2*A, CSN3*A, LGB*A, and LAA*B. A relatively high frequency of CSN1S2*B (0.202 in the whole data set) was found, mainly occurring within the C-A²-B-A haplotype (in the order CSN1S1-CSN2-CSN1S2-CSN3), which seems to be peculiar to the Original Pinzgauer, possibly because the survival of an ancestral haplotype or the introgression of Bos indicus.     

Characterization of the casein gene complex in West African goats and description of a new alpha(s1)-casein polymorphism

The analysis of casein polymorphisms was carried out in West Africa goat populations: Red Sokoto (n = 57), West African Dwarf Nigeria (n = 27), West African Dwarf Cameroon (n = 39), and Borno (n = 37). The 4 casein genes α_s1 (CSN1S1), β (CSN2), α_s2 (CSN1S2), and κ (CSN3) were typed at the DNA level. No null alleles were found in any of the genes analyzed. A PCR single-strand conformation polymorphism method was implemented for the identification of CSN1S1*F allele simultaneously with A/0₁, B/E, N and the new allele. The allele differed from CSN1S1*B by a synonymous transversion TCG→TCT in the codon corresponding to Ser₆₆ of the mature protein. The new allele, named CSN1S1*B′, occurred at a high frequency in all the populations, ranging from 0.295 (West African Dwarf Cameroon) to 0.405 (Borno). A greater frequency was found for alleles associated with high α_s1-casein quantity, as has already been observed in the goat populations from the Mediterranean area. The intermediate E allele occurred only in the Red Sokoto and at a low frequency. The faint F allele occurred in 3 populations at frequencies lower than 0.03. Linkage disequilibrium occurred in all the populations, with highly significant differences in Borno, Red Sokoto, and West Africa Dwarf Nigeria, and significant differences in West Africa Dwarf Cameroon. Only 10 haplotypes showed frequencies ≥0.05 in at least 1 of the 4 populations considered, and the overall frequency was >0.1 only for 4 haplotypes: BAAB, B′ACA, ACAB, and BACA (in the order CSN1S1-CSN2-CSN1S2-CSN3). Haplotype BAAB, postulated as an ancestral haplotype in previous studies, was the most common haplotype in all breeds except Borno, where B′ACA was predominant. The results obtained are of considerable significance given that very little information exists on the subject for African goats. The high frequency of strong alleles in the calcium-sensitive caseins as well as the high linkage disequilibrium found among the casein genes in the African breeds analyzed may suggest that specific casein haplotypes have already been selected due to their advantages for nutrition. Haplotypes providing greater protein and casein content would increase the energy content of milk, thus resulting in more favorable growth and survival of young goats and humans consuming the milk.     

Influence of residual milk-clotting enzyme on alpha(s1) casein hydrolysis during ripening of Reggianito Argentino cheese

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on alpha(s1)-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on alpha(s1)-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60 degrees C) on milk-clotting enzyme residual activity and alpha(s1)-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of alpha(s1)-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60 degrees C- and 52 degrees C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52 degrees C-cooked cheeses. Cheese curds that were cooked at 45 degrees C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of alpha(s1)-casein was detected early in cheeses made at 45 degrees C, and later in those made at higher temperatures. The peptide alpha(s1)-I was not detected in 60 degrees C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from alpha(s1)-casein in hard cheeses may be at least, partially due to this proteolytic agent.     

Lactation response and nitrogen,calcium, and phosphorus utilization of dairy goats differing by the genotype for alpha S1-casein in milk,and fed diets varying in crude protein concentration

Twenty-four dairy goats were used in a preliminary trial to evaluate the effect of the genotype for alpha S1-casein (alpha S1-CN) in milk [homozygous variant A/A (n = 12) or F/F (n = 12)] on milk yield and composition for 2 wk from kidding. After this period, the main trial aimed at determining the effects of the genotype for alpha S1-CN in milk, the dietary crude protein concentration on milk yield and composition, and utilization of N, Ca and P. The goats within each genotype were allocated to a 3 x 3 Latin square for 14 wk with three crude protein concentrations in the total mixed ration (13.2, 16.8, and 19.8% of dry matter) and three periods (wk 3 to 6, wk 8 to 11, and wk 13 to 16 postpartum) as factors. Balances of N, Ca, and P were determined in the last week of each period. Two wk after kidding, the alpha S1-CN A/A goats had higher percentage and yield of protein and lower body weight than the alpha S1-CN F/F goats. During the main trial, yields of protein and fat, as well as percentages of fat and protein in milk were higher for the alpha S1-CN A/A goats than for the alpha S1-CN F/F goats, independent of dietary CP concentration and period. Efficiency of N digestion for milk N was higher for the alpha S1-CN A/A goats than for the alpha S1-CN F/F goats. Urinary N as a percentage of digested N, and total N excretion expressed relative to milk N were lower for the alpha S1-CN A/A goats than for the alpha S1-CN F/F goats. Neither the apparent absorption of calcium or phosphorus was affected by the genotype for alpha S1-CN. Goats fed the low crude protein diet had lower milk yield and lower yields of fat and protein than those fed the other diets. Increasing dietary crude protein concentration increased urinary N, milk N, and N excretion relative to milk N; it also decreased the efficiency of digested N for milk N. In conclusion, selection of goats with a genetically higher yield of casein and fed with diets formulated to reduce N excretion improves the cheese-making properties of goat milk and reduces concerns about N wastes in the environment.     

Contributions of terminal peptides to the associative behavior of alphas1-casein

The N- and C-terminal segments of bovine α_s1-casein-B (f1-23 and f136-196) were characterized under conditions that promoted or inhibited self-association to determine the relative contributions of each fragment to the interaction of α_s1-casein with itself or with other caseins. In earlier studies of f1-23, nuclear magnetic resonance (NMR) data and circular dichroism (CD) spectra showed that its conformation was thermostable between 10° and 25°C. In contrast, NMR studies of f136-196 indicated temperature sensitivity between 10 and 60°C, as did near-UV and far-UV CD data, suggesting a molten globule-like structure at higher temperatures. To compare the effects of temperature on conformational attributes of α_s1-casein and its terminal peptides, additional CD studies were conducted over a broader temperature range (10 to 70°C). The far-UV CD spectra indicated little temperature sensitivity for α_s1-casein, and the N-terminal peptide remained thermostable. During molecular dynamics simulations, the N-terminal peptide conformation did not change significantly, but the conformation of the C-terminal peptide (f136-196) was dramatically altered. These changes are correlated with the thermal instability observed by both CD and NMR in f136-196. Analytical ultracentrifugation studies of the self-association reactions of genetic variants A, B, and C of α_s1-casein showed that at 37°C the associative state is primarily dimeric; the amounts of higher order polymers significantly decreased when temperature was increased from 20 to 37°C. In all 3 genetic variants, the C-terminal portion of the whole molecule showed thermal instability with respect to aggregation to higher polymers, confirming the predictions of CD data and molecular dynamics simulations. The temperature dependency of these conformational changes suggests a possible function for α_s1-casein in facilitating casein-casein interactions in casein micelle formation.     

Goat milk allergenicity as a function of αs₁-casein genetic polymorphism

Cow milk allergy is the most frequent allergy in the first years of life. Milk from other mammalian species has been suggested as a possible nutritional alternative to cow milk, but in several cases, the clinical studies showed a high risk of cross-reactivity with cow milk. In the goat species, α_S1-casein (α_S1-CN), coded by the CSN1S1 gene, is characterized by extensive qualitative and quantitative polymorphisms. Some alleles are associated with null (i.e., CSN1S1*0₁) or reduced (i.e., CSN1S1*F) expression of the specific protein. The aim of this work was to obtain new information on goat milk and to evaluate its suitability for allergic subjects, depending on the genetic variation at α_s1-CN. Individual milk samples from 25 goats with different CSN1S1 genotypes were analyzed by sodium dodecyl sulfate PAGE and immunoblotting, using monoclonal antibodies specific for bovine α-CN and sera from children allergic to cow milk. A lower reaction was observed to 2 goat milk samples characterized by the CSN1S1* 0₁0₁ and 0₁F genotypes. Moreover, a fresh food skin prick test, carried out on 6 allergic children, showed the lack of positive reaction to the 0₁0₁ milk sample and only one weak reactivity to the 0₁F sample. The risk of cross-reactivity between cow and goat milk proteins suggests the need for caution before using goat milk for infant formulas. However, we hypothesize that it can be used successfully in the preparation of modified formulas for selected groups of allergic patients. The importance of taking the individual goat CN genetic variation into account in further experimental studies is evident from the results of the present work.     

Short communication: extraction of beta-casein from goat milk

Peptides derived from milk β-casein have potential biological activities, such as antihypertensive and immunostimulating properties. These biological properties increase the demand for the production of specific bioactive peptides. β-Casein can be isolated directly from renneted skim milk, based on the preferential solubilization of β-casein at low temperature. This study was conducted to compare the recovery and purity of β-casein extracted from goat and cow milks. Rennet casein was prepared from both milks, heat treated, and dispersed in demineralized water at various temperatures. β-Casein recovery in the soluble phase increased with decreasing incubation temperature. Concentration of β-casein was 43% higher in goat milk than in cow milk, which had a direct effect on β-casein recovery. Furthermore, β-casein was extracted more efficiently from goat rennet casein. As a result, the extraction yield of β-casein was 53% higher in goat milk than in cow milk. The purity of β-casein extracted from both milks reached approximately 90% after incubation at 0°C.     

Short communication: Effect of αS1-casein (CSN1S1) and κ-casein (CSN3) genotypes on milk composition in Murciano-Granadina goats

The effects of the caprine α_S1-casein (CSN1S1) polymorphisms on milk quality have been widely demonstrated. However, much less is known about the consequences of the κ-casein (CSN3) genotype on milk composition in goats. Moreover, the occurrence of interactions between CSN3 and CSN1S1 genotypes has not been investigated. In this study, an association analysis between CSN1S1 and CSN3 genotypes and milk quality traits was performed in 89 Murciano-Granadina goats. Total milk yield as well as total protein, fat, solids-not-fat, lactose, α_S1-casein (CSN1S1), and α_S2-casein (CSN1S2) contents were recorded every other month during a whole lactation (316 observations). Data analysis using a linear mixed model for repeated observations revealed no interaction between the CSN1S1 and CSN3 genotypes. With regard to the effect of the CSN3 locus, AB and BB genotypes were significantly associated with higher levels of total casein and protein content compared with the AA CSN3 genotype. In strong contrast with French breeds, the CSN1S1 genotype did not affect protein, casein, and fat concentrations in Murciano-Granadina goats. These results highlight the importance of taking into consideration the CSN3 genotype when performing selection for milk composition in dairy goats.     

Short communication: milk fat composition of 4 cattle breeds in the Netherlands

Milk fatty acid (FA) composition was compared among 4 cattle breeds in the Netherlands: Dutch Friesian (DF; 47 animals/3 farms), Meuse-Rhine-Yssel (MRY; 52/3), Groningen White Headed (GWH; 45/3), and Jersey (JER; 46/3). Each cow was sampled once between December 2008 and March 2009 during the indoor housing season, and samples were analyzed using gas chromatography. Significant breed differences were found for all traits including fat and protein contents, 13 major individual FA, 9 groups of FA, and 5 indices. The saturated fatty acid proportion, which is supposed to be unfavorable for human health, was smaller for GWH (68.9%) compared with DF (74.1%), MRY (72.3%), and JER (74.3%) breeds. The proportion of conjugated linoleic acid and the unsaturation index, which are associated positively with human health, were both highest for GWH. Differences in milk fat composition can be used in strategies to breed for milk with a FA profile more favorable for human health. Our results support the relevance of safeguarding the local Dutch breeds.     

Frequency and effect of the bovine acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism in Swedish dairy cattle

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol synthesis in the mammary gland, and the corresponding gene has emerged as a strong candidate for the variation in milk fat percentage. In this study, the allele frequencies and effects of the DGAT1 K232A variants in the Swedish dairy breeds Swedish Red and Swedish Holstein were investigated. A total of 239 cows, 143 of the Swedish Red breed and 96 of the Swedish Holstein breed, in the experimental herd at the Swedish University of Agricultural Sciences were genotyped for the DGAT1 polymorphism. The Swedish Red cows in the herd belonged to 1 of 2 selection lines with high or low milk fat percentage, respectively, but with similar high total milk energy production. The frequency of the K variant was found to be significantly greater in the high-fat line than in the low-fat line. The average frequency of the K variant in the 2 lines of the Swedish Red cows was 0.09 compared with 0.12 among the Swedish Holstein cows. Mixed model analysis was used to estimate the effect of the DGAT1 K232A polymorphism based on 16,866 test-day records for milk production traits. In accordance with previous studies, the most pronounced effects were found for fat and protein percentages and milk yield; and the K variant was associated with an increase in milk fat and protein percentages but less milk yield compared with the A variant. Less pronounced effects were found for yields of fat and protein for which the K variant was associated with greater fat yield but less protein yield.     

Short communication: evidence for a major gene by polygene interaction for milk production traits in German Holstein dairy cattle

The present study investigated putative interaction effects between the DGAT1 K232A mutation and the polygenic term (i.e., all genes except DGAT1) for 5 milk production traits in the German Holstein dairy cattle population. Mixed models were used, and the test for interaction relied on the comparison of polygenic variance components depending on the sire's genotypes at DGAT1 K232A. Substitution effects were highly significant for all traits. Significant interaction effects were found for milk fat and protein percentage.     

Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression

Polymorphisms in 5′-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine α_s1-casein encoding gene (CSN1S1), which is affected by an A→G exchange at −175 bp (CSN1S1^−175bp). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1^−175bp binding site. Further investigations comprised genotyping of the variable CSN1S1^−175bp activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1^−175bp*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1^−175bp*G showed higher content (%) as well as quantity (g/d) of α_s1-casein than CSN1S1^−175bp*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1^−175bp*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.     

Short communication: predominance of beta-casein (CSN2) C allele in goat breeds reared in Italy

A protocol for the rapid and simultaneous genotyping of A, C, and 0 'CSN2 alleles in goat was developed by single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) technique. Screening the CSN2 variability in 7 goat breeds reared in Italy validated the genotyping test. The SSCP-PCR technique was also suitable for monitoring CSN2 polymorphism. In particular, the discrimination between CSN2*A and CSN2*C is important because the 2 corresponding protein variants cannot be separated by standard typing techniques. The monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In most breeds, CSN2*C occurred with the highest frequency, except in Saanen where CSN2*A and CSN2*C showed similar frequencies. Variant CSN2*C occurred with a frequency of 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica). The alignment among the mature CSN2 sequences of different species suggests that CSN2*A is the ancestral allele compared with CSN2*C. Interestingly, the CSN2*A goat variant showed higher frequencies in selected breeds (Saanen and Camosciata).     

Short communication: Heritability of susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in Holstein cattle

Johne's disease in cattle is the result of infection of the small intestine by Mycobacterium avium ssp. paratuberculosis (MAP), leading to an incurable inflammatory bowel disease (Johne's disease or paratuberculosis). The disease is a concern both for its direct cost to dairy producers and for its zoonotic potential. The objective of this study was to estimate the heritability for susceptibility to infection of cattle by MAP using Johne's testing records (ELISA test for presence of antibodies to MAP in milk or blood) from US Holstein cattle from 2009 to 2016. Data sets were edited to include records from herds with 100 or more total records and sires with 50 or more daughters. Data sets were further edited to include (1) only herds with at least 1 positive test, (2) herds with at least 2.5% positive test results, and (3) herds with at least 5% positive test results to examine the effect of data from herds with higher proportions of positive tests, and presumably higher pathogen exposure, on heritability estimates. Two models were used in this study, a linear sire model and a binary threshold-probit sire model. Both were mixed models considering fixed effects of herd and age at test, the latter as a covariate accounting for linear and quadratic effects; random effects included sire and residual. Analyses were conducted using a restricted maximum likelihood method. Heritability estimates (±standard error) from the linear model were 0.041 ± 0.004, 0.050 ± 0.004, and 0.062 ± 0.007 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the threshold model were 0.157 ± 0.014, 0.174 ± 0.016, and 0.186 ± 0.021 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the linear model were affected by population incidence for positive tests, in contrast to estimates from the threshold model, likely accounting for the difference in magnitude of heritability estimates between models and suggesting that the threshold model analysis is the better choice. Heritability estimates increased as data were restricted to herds with presumed higher MAP exposure for both linear model and threshold model analyses. These estimates are similar to previous estimates in other dairy cattle populations and suggest the potential for selection to lessen susceptibility to MAP infection.     

Identification of the critical amino acid residues of immunoglobulin E and immunoglobulin G epitopes on αs1-casein by alanine scanning analysis

α_s1-Casein represents one of the major allergens causing cow milk allergy. Few studies have clearly evaluated immunological relationships between IgE- and IgG-binding epitopes of α_s1-casein. This study aimed to map IgE- and IgG-binding epitopes of α_s1-casein by the serology method, and identify the critical amino acids of α_s1-casein by alanine scanning analysis. Our initial data revealed IgE-binding epitopes located in the sequences of AA 126 to 140, AA 6 to 20, AA 171 to 185, and AA 11 to 25. The sequences at AA 21 to 35, AA 56 to 70, and AA 161 to 175 were recognized by IgG antibodies. The alanine scanning analysis showed that IgE- and IgG-binding epitopes share the same critical AA: arginine at position 22 and phenylalanine at position 23. Results obtained from this study will provide necessary information to alter the cDNA to encode a protein with reduced IgE- or IgG-binding capacity.     

Short communication: characterization of a new genetic variant in the caprine kappa-casein gene

A new polymorphism has been identified in the goat kappa-casein gene by evaluating genomic DNA from the Montefalcone breed in Italy. The polymorphic site consists of a single nucleotide substitution A to G at position 242 of the exon 4 and produces an amino acid substitution Asp/Gly. A polymerase chain reaction-restriction fragment length polymorphism protocol for rapid genotyping of the variant has been developed, using the HaeIII enzyme. Animals from Italian, Spanish, and French breeds have been analyzed to investigate the occurrence of the allele in other populations. The allele appears to be exclusive to the Montefalcone breed.     

Short communication: Simultaneous identification of five kappa-casein (CSN3) alleles in domestic goat by polymerase chain reaction-single strand conformation polymorphism

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the κ-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants.     

Effects of milk fat composition, DGAT1, and SCD1 on fertility traits in Dutch Holstein cattle

Recently, selective breeding was proposed as a means of changing the fatty acid composition of milk to improve its nutritional quality. Before implementing such breeding objectives, effects on other economically important traits should be investigated. The objectives of this study were to examine 1) the effect of milk fat composition, and 2) the effect of polymorphisms of DGAT1 and SCD1 genes on female fertility in commercial Dutch Holstein-Friesian cattle. Data on 1,745 first-lactation cows were analyzed by fitting linear mixed models. We found that higher concentrations of trans fatty acids within total milk fat negatively affected reproductive performance. Furthermore, results suggested a potential effect of the DGAT1 polymorphism on nonreturn rates for insemination 28 and 56 d after the first service. Our results can be used to assess the correlated effects of breeding for improved milk fat composition on reproduction, thereby allowing for better evaluation of breeding programs before implementation.     

Short communication: characterization of DRB3 alleles in the MHC of Japanese shorthorn cattle by polymerase chain reaction-sequence-based typing

A study was made of exon 2 of the bovine leukocyte antigen BoLA-DRB3 gene of 176 Japanese Shorthorn cattle at six farms in Japan using polymerase chain reaction-sequence-based typing (PCR-SBT). An initial round of PCR using conserved locus-specific primers, a second round of PCR using a locus-specific primer, and at least one sequence-specific primer (SSP), followed by direct sequencing of products of PCR with SSP were conducted. Twenty-one BoLA-DRB3 alleles were identified with frequencies ranging from 0.3 to 19.6% in 176 individuals, and two of these alleles were new alleles that have not been reported previously. The three most frequently observed alleles (DRB3*1201, *0301, and *0801) accounted for 43.8% of the alleles in the population of these herds. Next, we tested the products of amplification by PCR of BoLA-DRB3 exon 2 with RsaI, BstYI, and HaeIII, and identified 18 previously described PCR-restriction fragment length polymorphism (RFLP) types. The PCR-RFLP types reflected the results of PCR-SBT exactly. Our results indicate that exon 2 of the BoLA-DRB3 gene is highly polymorphic in Japanese Shorthorn cattle.