In the studied herd, some of the cows were initially inseminated with semen imported from Bulgaria (from bull I-1 or II-2). According to the records, despite the fact that these two bulls were from the same Bulgarian stud, they were not related, at least up to two generations, but both grandfathers came from India, indicating a small chance of a previous relationship between them. Unfortunately, we have no information concerning the occurrence of albinism in this Bulgarian stud. During a 12-year period, 1.6% (16/970) of the calves produced were born with albinism. The pedigree of the calves with albinism is presented in Figure . With the exception of X-257, which shared kinship with only one of the Bulgarian sires (I-1), all of the albino animals born into the herd shared kinship with both of the imported sires (I-1 and II-2) whose imported semen was used on the farm. The calculated frequency for the recessive allele was 0.0552 and the frequency of albinism in the herd was 16/970. Considering the active sires in 1996 as the founders of the herd, the mean consanguinity coefficient was 6.01% for the 84 animals born in 2009. Among the albino animals, the consanguinity was 14.9%, and only two of the albino animals showed no consanguinity (III-110 and X-257). The results of the segregation analysis showed that the assessed genealogical data agree with the hypothesis of autosomal recessive inheritance. When the numbers of albino offspring from sires considered unequivocally heterozygous mated with their own daughters or with daughters from heterozygous sires were compared using segregation analysis, we obtained a value of 12/73, indicating no significant difference (p>0.11) from the expected value (9.1/73), even ignoring the possibility that the cows could have received the recessive allele from their mothers.
Pedigree of the albino buffalo herd. Black graphics represent the affected individuals.
In the animals with albinism, the skin and the stratum corneum of the horns (Figure ) and hooves were white, and the mucosa appeared pink. In the periocular region, an absence of pigmentation was observed in the eyelashes, conjunctiva and iris (Figure ). The affected buffalo were able to avoid obstacles, but they manifested signs of photophobia by keeping their eyes only partially open, especially when exposed to sunlight.
An albino dam and her albino calf (with photophobia) are shown among normal buffalo.
Periocular region in an albino buffalo with non-pigmented eyelashes, conjunctiva, and iris.
The TYR mRNA sequence obtained from the wild-type buffalo was 1,958 base pairs (bp) in length (accession number JN_887462), including 39 bp in the 5’-untranslated region (UTR) and 326 bp in the 3’-UTR. The open reading frame of the TYR sequence was 1,593 bp long, and the corresponding peptide sequence consisted of 530 amino acids.
In comparisons of the buffalo TYR coding sequence with the TYR coding sequences from seven other mammalian species, we observed that the length did not vary, except compared to humans (one fewer amino acid) and mice (three additional amino acids). The TYR coding sequence of the wild-type buffalo shared a greater identity with the cattle (98%) and sheep (97%) sequences than with other mammalian TYR sequences deposited in GenBankTM.
The TYR gene sequence obtained for the OCA buffalo was deposited in GenBankTM (JN_887463). The sequence was then analyzed for polymorphisms between the OCA and wild-type buffalo. Comparison of the TYR coding regions in the OCA buffalo revealed a single-base substitution at nucleotide 1,431 (G to A), which causes the conversion of a tryptophan (TGG477) into a stop codon (TGA477). This premature stop codon produces a truncated TYR protein with 53 fewer amino acids than the wild-type TYR protein.
The SNP sequence responsible for OCA in the buffalo was confirmed by PCR using the primers ASBTYR-F3 and ASBTYR-R3. The wild-type, heterozygote and albino sequences obtained in this study were assembled, and the resulting chromatogram is shown in Figure .
Figure 4 A partial chromatogram obtained from the assembly of tyrosinase sequences from wild-type, heterozygote and albino buffalo. The normal G at the 11th nucleotide position shown in the picture was observed in the wild-type TYR sequence. A double peak (R: (more ...)