Protein requirement of Japanese quail

Analele IBNA vol. 27, 2011

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Selection and breeding of Bulgarian Rhodopean cattle with respect to milk proteins polymorphism Denitsa Teofanova1, G. Radoslavov1, I. Mehandzhiyski2, Аneliya Yoveva1, L. Zagorchev3, P. Hristov1† 1 Institute of Biodiversity and Ecosystem Research – Bulgarian Academy of Sciences, 25 ”Acad. Georgy Bonchev” Str., 1113 Sofia, Bulgaria; 2 Agricultural and Stockbreeding Experimental Station – Agricultural Аcademy, 2 “Nevyastata” Str., 4700 Smolyan, Bulgaria; 3 Sofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Biochemistry, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria

SUMMARY Allelic variants of milk protein genes have significant role in genetic improvement of dairy cattle breeds. Present investigation shows the influence of the genetic variants of kappa-casein and beta-lactoglobulin gene on milk production and milk quality of Bulgarian Rhodopean cattle. Through PCR-RFLP assay 63 animals of that breed were genotyped for kappa-casein gene and 86 for beta-lactoglobulin gene. Results for kappa-casein genotyping show superiority of heterozygous AB genotype with respect to milk production and milk butter and domination of homozygous AA genotype as it concerns higher protein and fat content. In contrast with kappa-casein data, B allele of betalactoglobulin gene was associated with higher values of milk production, butter milk and fat content. Higher protein content in the milk was determined by AA genotype. Analysis of kappa-casein and beta-lactoglobulin gene polymorphism accentuates the influence of the genetic variants on milk quantitative and qualitative traits. That gives opportunity to control selection and breeding of dairy cattle with regards to definite milk features and to preserve Bulgarian Rhodopean cattle gene fund. Keywords: beta-lactoglobulin, breeding, Bulgarian Rhodopean cattle, genetic polymorphism, каppa-casein, selection

INTRODUCTION Selection and reproduction of the dairy cattle breeds in Republic of Bulgaria are determined by searching of an economically efficient way of increasing milk production and definite milk quality. During the last years †

Corresponding author e-mail: [email protected]

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Denitsa Teofanova et al.

together with traditionally methods for breeding the genetic polymorphism of milk protein genes has been used as an additional selection criteria (Bovenhuis et al., 1992). Regarding that most researches have focused on αs1-and kappafrom the group of caseins and β-lactoglobulin from whey proteins. The mentioned proteins possess great effect on milk production and milk constituents (Erhardt, 1996). The genetic variants of kappa-casein gene (CSN3) have a dominant role due to the protein influence on formation, structure and stabilization of the casein micelles with respect to milk technological properties (Farrell et al., 1996). The CSN3 gene is situated on sixth chromosome and recently 12 genetic variants have been determined (Formaggioni et al., 1999; Holt et al., 2003). A and B alleles are most frequently distributed among all species of genus Bos (Holt et al., 2003). β-Lactoglobulin (LGB) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. The biological functions of this protein are still not known. It could have a role in metabolism of phosphate in the mammary gland and the transport of retinol and fatty acids in the gut (Hill et al., 1997). The LGB gene is situated on bovine chromosome 11 (Eggen and Fries, 1995) and a total of 15 alleles are known for it (Caroli et al., 2009). Analogically to CSN3 alleles for LGB A and B alleles are most frequently distributed among all species of genus Bos as well (Hill et al., 1997). The aim of recent research is to investigate the genetic variants of CSN3 and LGB gene influence on milk qualitative and quantitative traits of the Bulgarian Rhodopean cattle with regards to its genetic improvement by controlled selection and reproduction. The Bulgarian Rhodopean cattle breed is with long term of agricultural usage and unswayed by the environmental conditions. It is incomparable with any other breeds in Bulgaria with respect to milk yield, fat and protein content, viability, usage continuance and fertility. Because of all its features Bulgarian Rhodopean cattle breed is valuable gene fund for the country and its preservation and improvement is of higher significance for the recent investigation.

MATERIAL AND METHODS Sample collection 90 blood samples (5 ml) were obtained from v. jugularis into vaccum tube (BD Vacutainer®) from animals of Bulgarian Rhodopean cattle from Agricultural and Stockbreeding Experimental Station – Smolyan. Milk samples were collected from each cow monthly for milk production and milk quality measurements (MilkoScan 133-B, Foss Electric).


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DNA extraction and amplification Total genomic DNA was extracted using GeneJet™ Genomic DNA Purification Kit (Fermentas) according to the manufacturers protocol. PCR amplification of the polymorphic region of CSN3 gene (located between exon IV and intron IV) and LGB gene (located between exon IV and intron IV) was performed with primers described by Medrano and Cordova (1990a, 1990b). PCR was accomplished by LittleGenius thermocycler (BIOER Technology Co., Ltd) under the following conditions: initial denaturation 94ºС for 5 min.; 35 cycles (denaturation 94ºС for 30 sec.; primer anealing 50ºС for 30 sec.; extension 72ºС for 1 min.) and final extension 72ºС for 10 min. PCR products were visualized on 1% agarose gel with ethidium bromide under UV light. Fragment size was determined using GeneRuler™ 1kb Ladder Plus (Fermentas). RFLP assay Amplified fragments were 350 bp and 252 bp in length for CSN3 and LGB respectively. Digestion was performed with HinfI endonuclease (Fermentas) for CSN3 amplicons and with HaeIII restrictase for LGB amplicons on 37ºС for 1 hour. Restriction products for CSN3 were visualized on 2% agarose gel with ethidium bromide under UV light and these for LGB were visualized on 12% polyacrylamide gel after silver staining. Fragment size was determined using GeneRuler™ 100 bp Ladder Plus (Fermentas). According to restriction profile allelic variants of CSN3 and LGB genes were determined. Statistical analysis Milk productivity and qualitative traits data was analyzed by Statistical tool Descriptive statistics (Microsoft Excel, 2007). Calculated mean values (shown as mean value ± SEM) for milk productivity and qualitative traits were compared within different genotypes. Genotype and allele frequencies were determined. Validity of Hardy-Weinberg equilibrium for the population was evaluated using χ2 test (Preacher, 2001). Text

RESULTS CSN3 gene PCR-RFLP assay. 63 animals were genotyped. 34 were heterozygous individuals (AB) and they were with the highest prevalence (about 54%). Four electrophoretic bands characterize that genotype (266 bp; 134 bp; 132 bp and 84 bp). 20 animals (about 32%) were defined as homozygous on A allele (AA) which was visualized with three electrophoretic bands (134 bp; 132 bp and 84 bp). With least representatives (9 cows) were homozygous BB animals (about 14%),

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identified with two electrophoretic bands (266 bp and 84 bp). Obtained results are shown on Figure 1.

Figure 1. PCR-RFLP assay of CSN3 gene after restriction of the polymorphic region with HinfI restrictase. 1, 7 – АА genotype; 2, 5 – ВВ genotype; 3, 6 - АВ – genotype; 4 – DNA Ladder; The numbers in white show the size of restriction fragments

Genotype and allele frequencies Distribution of genotype and allele frequencies among the studied animals was presented on Table 1. Table 1. Genotype and allele frequencies for CSN3 and LGB genes of Bulgarian Rhodopean cattle Genotype frequencies Allele Gene Genotype χ2 frequencies Observed Expected АА 0.318 0.345 А – 0.587 a CSN3 ВВ 0.143 0.170 0.01 В – 0.413 AB 0.540 0.485 АА 0.395 0.439 А – 0.686 a LGB ВВ 0.023 0.099 0.13 В – 0.314 АВ 0.581 0.416

pvalue 0.99

0.94

a – non-significant differences

Qualitative and quantitative milk traits Milk productivity (300 days lactation) of heterozygous AB cows (4112±149,4 kg) is about 600 kg higher than that of homozygous BB animals (3495±290,4 kg) and about 300 kg than AA representatives (3838,2±160,2 kg). That result presents the superiority of A allele with respect to milk productivity. Similar tendency was observed for butter milk as well (AВ 191,45±6,97 kg; BВ -161,17±10,55 kg; АА -180,27±7,28 kg).


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Fat and protein content of cow milk were with similar mean values among the three genotypes. Nevertheless there was slight predominance of AA genotype. The results about qualitative and quantitative milk traits were summarized on Figure 2.

Figure 2. Influence of CSN3 gene polymorphism on milk production and milk quality traits in cows of Bulgarian Rhodopean cattle. АА, ВВ, АВ – genotypes

LGB gene PCR-RFLP assay 86 animals were genotyped. 50 animals were established to be heterozygous (AB) and they were with the highest prevalence (about 58%). AB genotype was characterized by four electrophoretic bands (144 bp; 108 bp; 74 bp and 70 bp). Homozygous on A allele (AA) animals (34 cows) were observed with frequency about 40% and electrophoretically that genotype was visualized with two bands (144 bp and 108 bp). With the lowest frequency (about 2%) were homozygous BB animals (2 cows) which were identified with three electrophoretic bands (108 bp; 74 bp and 70 bp). Obtained results are shown on Figure 3. Genotype and allele frequencies Distribution of genotype and allele frequencies among the studied animals was presented on Table 1.

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Figure 3. PCR-RFLP assay of LGB gene after restriction of the polymorphic region with HaeIII restrictase. 1, 2, 4 – АА genotype; 5, 6 – ВВ genotype; 3 - АВ genotype. The numbers above the bands show the size of restriction fragments

Figure 4. Influence of LGB gene polymorphism on milk production and milk quality traits in cows of Bulgarian Rhodopean cattle. АА, ВВ, АВ – genotypes


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Qualitative and quantitative milk traits With regards to the LGB polymorphism the results show that milk productivity (300 days lactation) was highest in homozygous BB animals (4240,5±33,5kg) and is about 660 kg higher than that of homozygous AA cows (3581,48±154,14 kg). Heterozygous AB genotype defines average milk production (3955,24±125,45 kg). That result presents the superiority of B allele with respect to quantitative milk traits. Similar tendency was observed for fat content and butter milk as well. The differences were more obvious for butter milk mean values (BB-203±1; AA-168,4±7,26; AB-184,30±5,57). Protein content of cow milk was with almost equal mean values among the three genotypes. Nevertheless there was slight predominance of AA genotype. The results about qualitative and quantitative milk traits were summarized on Figure 4.

DISCUSSION The results of recent studies allowed the opportunity to define animals with particular genotypes in relation with desirable qualitative and quantitative milk features. That enables controlled selection and reproduction of parental couples with these particular genotypes: Therefore milk protein genes polymorphism could be a reliable tool as an additional selection criterion for genetic improvement of Bulgarian Rhodopean cattle population. CSN3 and LGB allele and genotype frequencies. A genotype and allele frequency of both milk protein genes were shown on Table 1 and from this data is obvious that A allele frequency is predominant in comparison with that of B allele for both CSN3 and LGB genes. These findings are in agreement with previous researches which defines B allele with lower frequency in most cattle breeds (Tsiaras et al. 2005; Heck et al., 2009). Observed and expected genotype frequencies were with similar values which confirms validity of Hardy-Weinberg equilibrium for Bulgarian Rhodopean cattle population. Dominant frequency of A allele and heterozygous AB genotype for both examined genes allows the presumption that animals with AA and/or AB genotypes were used during the selection and reproduction of Bulgarian Rhodopean cattle breed. Extremely low frequency of homozygous BB individuals confirms the presumption mentioned above. Relationships between CSN3 and LGB genotypes and milk traits Available data for the relationships between different genotypes of milk protein genes and milk traits is contradictory and controversial question. Usually these relationships depend on cattle breed and origin country that are

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object of the studies. As it concerns CSN3 gene some studies claim that the BB genotype was associated with higher (Van Eenennaam and Medrano, 1991) or lower (Bovenhuis et al., 1992) milk yield, whereas other studies indicated no effect (Ng-Kwai-Hang et al., 1990a; Lundeń et al., 1997). Presented results supported the Bovenhuis et al. (1992) data according to 15% lower milk production of BB homozygous animals in comparison with AB heterozygous cows. According to the presented findings there was a slight difference between butter milk mean values of AB genotype in comparison with BB genotype of CSN3 gene which was with about 16% higher. Most of the authors (VanEenennaam and Medrano (1991) Lundeń et al. (1997)) previously reported insignificant differences between genotypes of CSN3 gene for that feature of cow milk or only slight prevalence of AA genotype (Miciński et al., 2007). With respect to fat and protein content differences between three genotypes were insignificant which allowed the presumption that CSN3 gene polymorphism has no effect on these two milk traits. There was only a slight prevalence of AA genotype as it concerns these milk traits – less than 1% for fat content and about 3% for protein content. Data about fat content was in contrast with literature findings where prevalence of AB genotype above AA and BB genotypes was shown, but confirms researchers’ results for protein content (Miciński et al., 2007). Obtained results for LGB gene polymorphism influence on milk traits were clearer then these for CSN3. In studies of LGB genotypes effects on milk production, several authors reported no significant associations (Lundeń et al., 1997; Ojala et al., 1997). Nevertheless there were reports for positive influence on milk quantity of all genotypes - AA (Aleandri et al., 1990; Bovenhuis et al., 1992), AB (Pupkova, 1980), or BB (Jairam and Nair, 1983). Results of the present research showed that BB genotype determines higher milk production. Published studies represented a prevalence of AA genotype of LGB on protein content (Aleandri et al., 1990; Bovenhuis et al., 1992), which is identical to described results. That is the only milk feature that is affected from AA genotype of LGB gene and coincides with data for CSN3 gene. Positive effects of the BB genotype on fat content (Aleandri et al., 1990; Bovenhuiset al., 1992; Hill, 1993) and of AA genotype on butter milk (Miciński et al., 2007) were reported. As it concerns the fat content and butter milk described results for Bulgarian Rhodopean cattle breed were similar and expressed favorable influence on these milk traits for BB homozygous genotype. The differences were far more significant with regards to butter milk in comparison with fat content. The distinction between fat content values that referred to BB and AB genotype was about 2% and between butter milk values that referred to BB and AA genotypes was 17%.


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Analysis of genetic polymorphism of CSN3 and LGB genes for Bulgarian Rhodopean cattle breed revealed its influence on quantitative and qualitative milk traits. That allows selection of proper animals (cows and bulls) with desirable genotypes and increasing the frequency of favorable alleles within the population. The aim of that kind of controlled selection and breeding of dairy cattle is improving milk composition and properties.

CONCLUSIONS Predominant frequency of A allele of CSN3 and LGB genes was established within the Bulgarian Rhodopean cattle population. Positive effect on milk productivity and butter milk had AB genotype of CSN3 gene and BB genotype of LGB gene. AA genotypes of both milk protein genes were found to be related with higher protein content. Favorable influence on fat content had AA genotype of CSN3 gene and BB genotype of LGB gene. Genetic variants of both studied genes could be utilized as additional markers for selection, reproduction and genetic improvement of Bulgarian Rhodopean cattle population with regards to preservation of gene fund of that breed.

ACKNOWLEDGMENTS This study was supported by grant YRG No 02/23 28.12.2009 from the National Science Fund of the Bulgarian Ministry of Education, Youth and Science, Sofia, Bulgaria.

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