Agricultural and Food Science, Vol. 16 (2007): 222-231 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 222 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 223 © Agricultural and Food Science Manuscript received November 2006 Milk protein genotypes and milk coagulation properties of Estonian Native cattle Ivi Jõudu, Merike Henno, Sirje Värv, Tanel Kaart, Olav Kärt Estonian University of Life Sciences, Institute of Veterinary Medicine and Animal Sciences, Kreutzwaldi 62, 51014 Tartu, Estonia, e-mail: ivi.joudu@emu.ee Käde Kalamees Estonian Native Cattle Breed Society, Selja tee 1a, 85008 Sauga, Pärnumaa, Estonia The genetic variation of αs1-, β- and κ-caseins and b-lactoglobulin was determined and their effects on the rennet coagulation properties were examined using 335 milk samples from 118 Estonian Native (EN) cows. We found 16 aggregate casein genotypes (αs1-, β-, κ-caseins), of which four − namely, BB A 2A2 AA (21.2%), BB A1A2 AB (16.9%), BB A1A2 AA (14.4%), and BB A2A2 AB (10.2%) – occurred among nearly two-thirds of the analysed cows. Aggregate casein genotype had a significant overall effect on rennet coagulation parameters. Better rennet coagulation properties were found for aggregate casein genotypes CC A2A2 AB and BC A1A2 BB, among frequent genotypes for BB A1A2 AB. Of the cattle breeds raised in Estonia, milk from EN had the best coagulation properties and highest frequency of favourable κ-Cn B allele. Key-words: Estonian Native cattle, milk protein polymorphism, coagulation properties Introduction Up until the 19th century, the Estonian farmers raised indigenous cattle. Breeding of Estonian Native (EN) cattle was started in 1910 when the West-Finnish breed was accepted as a breeding component. In 1956–1961 and 1989–1992, Jersey bulls were used to reduce the level of inbreeding in EN cattle. Swedish Red Polled bulls were employed in the late 1990s to modify the breed composition of the local cattle population (Kalamees 2004). Estonian Native cattle are typically yellow- whitish red and hornless, with a medium wide chest and strong legs and hooves. Adult males weigh A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 222 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 223 on average 700 kg and females 436 kg, and their wither height is 134 cm and 128 cm, respectively. The breed is characterised by good longevity, ad- aptation to the local conditions, easy calving, and low feed consumption per production unit. Aver- age milk yield per cow per lactation was 4524 kg in 2005, i.e. lower than of other breeds in Estonia, whereas their milk fat (4.59%) and protein (3.44%) content were the highest. According to the Estonian Agricultural Reg- isters and Information Board, the total EN cattle population at the beginning of 2005 was 1,525 head including crossbreds (752 cows, 554 female calves, 143 young bulls and 76 bulls), of which 538 EN cows (including 420 purebreds) from 167 farms were included in milk recording. The share of native cattle has not decreased but remained at the same level, constituting about 0.5% of the to- tal cattle population in Estonia. Due to its small population size, the Estonian Native cattle was categorised as an endangered breed by FAO in 1993. At present the breed has the risk status of an endangered-maintained breed (World Watch List for Domestic Animal Diversity 2000). Since the most sustainable conservation strat- egy is to promote self-supporting, productive popu- lations, it would be beneficial to establish a well- functioning selection programme for the breed. Ge- netic improvement could concentrate on maintain- ing or increasing the profitability of production in traits for which the breed still possesses a competi- tive edge (Toro and Mäki-Tanila 1999). Suitability of milk for cheese production could be one such trait. A preliminary comparison of milk coagulation properties among Estonian dairy breeds in an ear- lier study showed certain advantages of milk from EN cows, despite the limited number of EN cows in the study (Kübarsepp et al. 2005a). Moreover, once its suitability for cheese production is con- firmed, milk from EN cows can be used for the production of Protected Denomination of Origin (PDO) cheeses. The PDO cheese-making process requires milk with good renneting properties from specific (local) breeds. Bertoni et al. (2005) found that the PDO cheeses have gained increasing value, not only in economic but also in cultural terms, particularly in some European countries. Besides showing certain organoleptic characteristics, these cheeses also represent a production system that is traditional and environmentally friendly. The objective of this study was to examine the genetic variation of different milk proteins in milk from EN cows, and to determine the genotypic distributions and their effects on milk coagulation properties. To this end, we studied the rennet co- agulation properties of milk among EN cows to assess its suitability for cheese production, and thereby to increase public interest in the breed and offer EN breeders better opportunities to maintain their EN herds. Materials and methods Cows and sampling Milk samples (n=335) were collected from 112 cows on six farms recommended by the Estonian Native Cattle Breed Society, once every two months from March through November 2004, and from 6 cows on the Põlula Research Farm once a month throughout 2004. The sample represents more than 21% of the total EN cows included in milk recording. Samples were taken simultaneously with the monthly milk recording using in-line milk meters at two consecutive milkings, and preserved with Bronopol® for an analysis of milk composition and renneting. Samples for determing milk protein genotypes were collected from the cows in March 2004, preserved with sodium azide and transported without delay to Freising, Germany. Laboratory analyses Concentrations of fat and protein were measured from each milk sample at the Milk Analysis Labo- ratory of Estonian Animal Recording Centre using an automated infrared milk analyser (System 4000, Foss Electric). A G R I C U L T U R A L A N D F O O D S C I E N C E Jõudu I. et al. Milk protein genotypes and milk coagulation properties of EN cattle 224 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 225 Milk protein genotypes (αs1-casein, β-casein, κ-casein and β-lactoglobulin) were analysed at the Laboratory of Raw Milk, Munich University of Technology, Freising, Germany, by an isoelectric focusing/electrophoresis technique (Baranyi et al. 1993). Milk rennet coagulation properties were deter- mined on the day after milking at the Laboratory of Milk Quality, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, by a Formagraph method (Kübarsepp et al. 2005b). Two milk coagulation parameters were measured: milk coagulation time (RCT = time in minutes from rennet addition to milk until the be- ginning of coagulation) and curd firmness (E30 = diagram width in mm 30 min after rennet addition). If diagram width was less than 20 mm, the samples were classified as milk with poor rennet coagula- tion properties (NK20). In commercial cheese pro- duction such poorly coagulating milk would not reach the firmness needed to properly cut the curd. For samples that did not coagulate at all, it was only possible to record curd firmness (E30=0), and these samples were classified as noncoagulated milk (NCM). Statistical analysis The statistical analysis was performed on a dataset of altogether 335 milk samples from 118 Estonian Native cows (Table 1). Information on the birth, calving and pedi- gree of the cows was obtained from the Estonian Animal Recording Centre. The pedigree data used for statistical analysis covered two to four genera- tions, amounting to a total of 606 animals in the pedigree file. The cows in the dataset had calved 1 to 12 times, and parity was grouped into four classes: 1, 2, 3 to 4, and ≥ 5 parities. Lactation stage was grouped into 11 classes of 30-day intervals, except for the last class, which covered the days from the 301st day after calving to the end of lacta- tion. Rennet coagulation time was logarithmically transformed to obtain a normal distribution. Results were evaluated statistically using a general linear mixed model assuming a first-order autoregressive variance structure of repeated meas- urements from the individual cows (SAS Inst. Inc. 2006). In order to estimate the effects of different factors on the milk coagulation, compositional pa- rameters, the following models were used: yijklmof = μ + parityi + lactmonthj + αβκ_Cnk + β_Lgl + ao + pef + εijklmof, where: yijklmnop = milk coagulation (log RCT, E30), production (daily milk yield) or compositional trait (milk fat and protein contents), μ = general mean, parityi = fixed effect of parity class i (i = 1 to 4), lactmonthj = fixed effect of month of lactation j (j = 1 to 11), αβκ_Cnk = fixed effect of aggregate αs1-, β- and κ-Cn genotypes k (k = 1 to 15), β_Lgl = fixed effect of β-Lg genotype l, (l = 1 to 3); ao = random additive genetic effect of animal o, N(0, Aσ2a); pef = random permanent environmental effect of farm f, N(0, Iσ2pe); ε = random residual effect with spatial power covariance structure, N(0, R). Interaction of genotypes at the α-, β- and κ-Cn DMYa, kg Fat, % Protein, % RCT, min log RCT E30, mm Mean 15.8 4.67 3.57 7.3 0.83 33.0 SDb 6.34 0.870 0.422 3.05 0.169 12.96 Min 3.6 2.20 2.50 2.5 0.40 0 Max 39.5 7.25 4.72 23.0 1.36 57.0 Count 335 335 335 316 316 335 aDaily milk yield, bStandard deviation Table 1. Descriptive statistics of daily milk production, compositional and rennet coagulation parameters RCT in 118 Estonian Native cows. A G R I C U L T U R A L A N D F O O D S C I E N C E Jõudu I. et al. Milk protein genotypes and milk coagulation properties of EN cattle 224 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 225 loci were considered because of their close genetic linkage. The basic genetic variability of milk proteins was analysed by applying the Arlequine software package for population genetics (Excoffier et al. 2005). An estimation of gene diversities – expect- ed (HE) and observed (HO) heterozygosity – and a probability test for detecting genotypic deviations from Hardy-Weinberg equilibrium were performed. Genotypic disequilibrium was tested under the null hypothesis (genotypes at one locus are independ- ent from those at another locus). A Markov chain method was used to obtain P-value estimates us- ing the Genepop computer program (Raymond and Russout 1995). Allele and genotype frequencies were com- puted by direct counts. Results Effects of systematic environmental factors on studied traits Parity did not have any significant overall effect on the studied milk rennet coagulation parameters, daily milk yield (DMY), and milk protein and fat content (Table 2). However, compared to the later parities, there were more noncoagulated and poorly coagulated milk samples in the first parity when milk protein content was lowest. Milk formed the firmer curd in the second to fourth parity when milk fat and protein contents were higher. Daily milk yield exhibited a tendency to improve with increasing number of lactation. Lactation month had a significant effect on both the studied rennet coagulation traits (P<0.001) as well as on DMY and milk protein and fat content (P<0.0001). Milk coagulation properties were at their best in a very early stage and curd firmness also improved in the second half of lactation (Fig. 1). The proportions of noncoagulated and poorly coagulated milk were at their lowest at the begin- ning of lactation and clearly at their highest during midlactation. Daily milk yield declined during lac- tation. Also, milk fat and protein content decreased over the first three or four months of lactation and then started to increase again during midlactation when the coagulation properties were at their poor- est. Milk fat and protein content rose steeply in the second part of lactation. Genetic variability of milk proteins All of the analysed proteins showed genetic poly- morphism. Two to three alleles per locus were de- tected by isoelectrophoretic separation of milk (Table Trait Parity 1 2 3–4 ≥5 P value Number of samples 125 63 106 40 Daily milk yield, kg 0a 0.72±1.03ab 1.50±0.88b 2.20±1.06b 0.1605 Fat, % 0a 0.50±0.19b 0.31±0.16b 0.19±0.19ab 0.0665 Protein, % 0a 0.13±0.09a 0.07±0.08a 0.05±0.09a 0.5461 log RCT 0a –0.02±0.04ab –0.06±0.03b –0.02±0.04ab 0.2798 1E30, mm 0 ab 0.92±2.32a 0.98±1.97a –3.47±2.31b 0.2321 NCM, % 10.4 6.3 1.9 – NK20, % 10.4 6.3 5.6 5.0 1Estimates of curd firmness of coagulating (E30>0 mm) milk samples a,bEstimates within row with differing letters in superscript are significantly different (P<0.05) Table 2. Estimates ±SE of effect of parity on studied traits (zero refers to class of comparison) and percentages of non- coagulated (NCM) and poorly (E30<20 mm) coagulated (NK20) milk samples of all samples in respective parity class. A G R I C U L T U R A L A N D F O O D S C I E N C E Jõudu I. et al. Milk protein genotypes and milk coagulation properties of EN cattle 226 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 227 3). Milk protein gene diversities varied between HO=0.152 (αs1-Cn) and 0.525 (β-Cn), the average heterozygosity being 0.374. Allele frequencies ranged from 0.038 (β-Cn B allele) to 0.915 (αs1-Cn B allele) as shown in Table 4. The κ-Cn E allele was not detected in EN in current sampling. In αs1-Cn a single genotype (αs1-Cn BB) was prevalent, found in 83.9% of the studied EN cows (Table 5). Also β-Cn A2A2 (42.4%) and κ-Cn AB (52.5%) were frequent genotypes. Of the 16 de- tected aggregate genotypes, 11 genotypes occurred in more than one individual, and four of these (αs1-β-κ-Cn) BB A 2A2 AA (21.2%), BB A1A2 AB (16.9%), BB A1A2 AA (14.4%) and BBA2A2AB (10.2%) were found among nearly two thirds of the analysed cows. β-lactoglobulin genotype AA, AB and BB frequencies were 9.9, 42.2 and 47.9%, re- spectively. The frequencies of heterozygotes were consistent with the observed allele frequencies (Ta- ble 3), except for κ-Cn which showed a significant deviation from Hardy-Weinberg equilibrium, with an increased frequency of heterozygotes from both homozygote genotypes. Among the three possible pairs of casein loci we found disequilibrium between β-Cn and κ-Cn at a significance level of 0.05, where the κ-Cn BB genotype combined with β-Cn genotypes contain- ing A1 as well as A2, but was never observed with the homozygote A2A2 (Table 5). On the other hand, κ-Cn AA was most frequently found in combination with β-Cn A2A2. The αs1-Cn BC genotype combina- tions occurred only with β-Cn A1A2 and A2A2. The rare αs1-Cn CC genotype was observed only in one individual, in combination with β-Cn A2A2. 0 2 4 6 8 10 12 14 1 2 3 4 5 6 7 8 9 10 >10 kg -1.0 -0.8 -0.6 -0.4 -0.2 0.0 % DMY Fat Protein 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 10 >10 % NCM NK20 -8 -6 -4 -2 0 2 4 1 2 3 4 5 6 7 8 9 10 >10 E30, mm -0.14 -0.12 -0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 log RCT E30 log RCT Figure 1. Estimates of effect of lactation month on milk traits (log RCT – logarithmically transformed rennet coagulation time, E30 – curd firmness of coagulating milk samples, DMY – daily milk yield, Fat – milk fat content, Protein – milk protein content) and percent- ages of noncoagulated (NCM; E30=0 mm) and poorly coagulated (NK20; 0 mm0 mm) a,b,c,dEstimates within milk coagulation trait and aggregate casein or β-Lg genotype with differing letters in super- script are significantly different (P<0.05) Table 6. Estimates ±SE of effects of aggregate casein and β-Lg genotypes on milk coagulation parameters (zero refers to class of comparison) and percentages of noncoagulated (NCM) and poorly (NK20) coagulated milk samples within respective genotype A G R I C U L T U R A L A N D F O O D S C I E N C E Jõudu I. et al. Milk protein genotypes and milk coagulation properties of EN cattle 228 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 229 results about the influence of parity on milk rennet coagulation properties. Schaar (1984) found a favour- able effect of increasing parity number on coagulation properties, but in some other studies parity had either no significant effect (Davoli et al. 1990, Ikonen et al. 2004, Tyrisevä, et al. 2004) or coagulation properties were deteriorating with increasing number of lactation (Tyrisevä et al. 2003). In genetic terms, the Estonian Native belongs to the Nordic cattle breeds, with a close relationship to Western Finncattle, as revealed by DNA microsatel- lites in a recent analysis (Tapio et al. 2006). Our results regarding the casein allele frequen- cies further support the genetic relationship of EN with Western Finncattle. The observed difference between EN and Finncattle in the frequency of β-Lg variants in the current study probably results from genetic material introduced into EN by Jersey bulls and/or, on a smaller scale, Holstein and/or red breeds. Despite belonging to one genetic cluster with other old indigenous breeds (Tapio et al. 2006), the EN breed showed very similar distribution of milk pro- tein aggregate genotypes with common commercial dairy breeds (Eenennaam and Medrano 1991, Lien et al. 1999). A comparison of milk protein allele frequencies between EN and the breeds used for improvement (Finncattle, Danish Jersey) revealed similarities between breeds. A predominance of αs1-Cn B (or its monomorphism) has also been observed in the common dairy breeds in Europe (Tervala et al. 1983, Ikonen et al. 1996, Lundén et al. 1997, Erhard et al. 1998, Lien et al. 1999). The same predominant variants in κ- and β-Cn loci have been found in most dairy cattle: κ-Cn A, except for Finncattle, Jersey and Brown Swiss, where B-allele is widespread, and al- leles A1 and A2 at β-Cn (Tervala et al. 1983, Ikonen et al.1996, Freyer et al. 1999). The frequencies of β-Lg A and B alleles were similar in EN and in Jersey as well as in the dairy breeds of adjacent countries (Bech and Kristiansen 1990, Velmala et al. 1993, Ikonen et al.1996, Lundén et al. 1997). A comparison of milk protein allele frequencies between the EN breed and the other dairy breeds raised in Estonia, namely the Estonian Holstein (EHF) and Estonian Red (EPK), showed that EN’s frequency of favourable κ-Cn B allele resembled that of EPK, but was higher than for EHF (Kübarsepp et al. 2005a). Unfavourable κ-Cn E allele (Jakob and Puhan 1992, Buchberger and Dovč 2000) was found both among the commercial breeds EHF and EPK (Kübarsepp et al. 2006), but not among EN cows in the current sampling. Most of the genotypes in our sample followed the Hardy-Weinberg equilibrium. Only one protein, κ-Cn, displayed significantly higher observed hetero- zygosity than would have been expected by the allele frequencies. The excess of heterozygote κ-Cn geno- types probably reflects the occurrence of individuals with crossbred ancestors in the study. Freyer et al. (1999) presumed that the heterozygous κ-Cn geno- type might have a heterotic effect on the milk yield. The occurrence of linkage disequilibrium between alleles at the different casein loci in our data indicates a relatively recent introduction of genetic material carrying specific casein haplotypes. The most com- mon aggregate genotype was the homozygous com- bination BB A2A2 AA, reflecting a high frequency of the haplotype BA2A in the breed. We observed similar effects of casein genotypes on coagulation properties to those reported for other breeds by several research groups (Jakob and Puhan 1992, Van den Berg et al. 1992, Ikonen and Ojala 1995, Lodes et al. 1996, Ng-Kwai-Hang 1998, Buch- berger and Dovč 2000). Due to the close linkage of four Cn genes in chro- mosome 6 within a region of about 250kb in cattle (Rijnkels 2002) segregation of the αsn-Cn, b-Cn, and κ-Cn variants occurs nonindependently (Aleandri et al. 1990, Eenennaam and Medrano 1991). Because of this close linkage of Cn genes, the use of casein aggregate genotypes is a more appropriate way to estimate the effect of Cn polymorphism on milk pro- duction traits than the use of individual Cn genotypes (Ikonen et al. 1999). Aggregate genotypes, similar to those of Estonian Native breed, have been frequent in Swedish Red and White and in Swedish Holstein breed (Lundén et al. 1997). Aggregate casein geno- type had statistically significant (P<0.05) effect on curd firmness and rennet coagulation time. Most noncoagulated milk samples originated from cows possessing κ-Cn AA genotype. Although the number of cows sampled was a considerable as a proportion (>21%) of the EN popu- lation, the size of the data was statistically speaking A G R I C U L T U R A L A N D F O O D S C I E N C E Jõudu I. et al. Milk protein genotypes and milk coagulation properties of EN cattle 230 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 16 (2007): 222-231 231 small (Table 6). Although the overall effect of ag- gregate genotypes on milk rennet coagulation char- acteristics was significant the differences between genotypes were mostly not significant probably due to the high standard error values resulting from the small number of animals and samples representing each genotype. As the number of animals in the study was relatively small, the statistical analysis described by Hallén et al. (2007) was also carried out (results not shown). The results were no different however, and it was not possible to verify the superiority of any casein locus. According to Kübarsepp et al. (2005a) milk from EN cows form a stronger curd (E30 = 33 mm) than milk from the other Estonian breed, EHF (E30 = 27.6 mm) and EPK (E30 = 31.1 mm). Also the percent- age of poorly coagulated and noncoagulated milk samples (E30<20 mm) was lowest for EN, 13.1%, while the percentages for EHF and EPK were 19.5 and 17.5%, respectively (Kübarsepp et al. 2005a). Several earlier studies (Tervala et al. 1983, Mache- boeuf et al. 1993, Auldist et al. 2002) also asserted better renneting properties among native breeds as compared with the Holstein. Differences in milk co- agulation properties between breeds may be due to differences in milk composition that is attributable to variation in other parts of genome. The studies mentioned above associated the better milk coagu- lation properties among native breeds with a higher frequency of κ-Cn B allele. A positive effect of this allele was shown also in the present study on EN cows, which also showed a comparatively high fre- quency of the allele. Conclusions Our present findings confirm previously observed relationships between genetic milk protein variants and milk properties for cheese-making. In contrast to com- mon commercial dairy cattle breeds, Estonian Native cattle breed showed a relatively high frequency of the favourable κ-Cn B allele, although predominantly in heterozygote combination with the A allele, whereas no unfavourable κ-Cn E alleles were detected in EN in current study. On the other hand, favourable ag- gregate casein genotypes (containing κ-Cn BB, αs1-Cn BC or CC genotype) for improving the conversion of milk protein into cheese were rarely observed in EN. Noncoagulated milk originated mainly from cows possessing κ-Cn AA genotype. If we compare the milk coagulation properties among the cattle breeds raised in Estonia, based on our current and previous results, the best milk for cheese-making comes from Estonian Native cattle. In order to apply the genetic information obtained from this study in EN breeding programmes, we need to conduct additional determination of milk protein genotypes for all breeding bulls. 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