Donkey Milk Study: κ-Casein Gene from Milk Cells (Singh 2024)

DNA was obtained from 10 mL of milk from ten Halari donkeys; a 235 bp fragment of the κ-casein gene (CSN3) was detected in all samples. The sequence matched the reference by 99%.

The study in brief

• Original title: Milk somatic cell DNA isolation and characterization of κ-casein gene in Halari donkey milk
• Authors: Singh P., Bhardwaj A., Nayan V. et al.
• Journal: Heliyon, 10(2), e24991 (2024)
• Country: India · Study type: Laboratory study

How it was investigated

From ten lactating Halari donkeys of the donkey milk unit of the ICAR-National Research Centre on Equines in Hisar (India), kept under identical housing and feeding conditions, approximately 10 mL of milk was hand-milked, filtered, and stored for 24 h at 4 °C before processing. The somatic milk cells were obtained by centrifugation (400 × g), washed, and the DNA isolated using a lysis and isopropanol precipitation method. A 235 bp fragment of the κ-casein gene (CSN3) was amplified by PCR (35 cycles, annealing at 57 °C), and GAPDH served as a control gene.

Important figures

• 10 – Halari donkeys, one milk sample each (biological replicates)
• 10 mL – Milk volume per sample for DNA extraction
• 24 h at 4 °C – Storage of the milk until DNA extraction
• 400 × g – Centrifugation to separate the somatic milk cells (10 min, 4 °C)
• 235 bp – Length of the amplified CSN3 fragment (κ-casein gene)
• 100 bp – Fragment of the GAPDH control gene

Results

Genomic DNA was obtained from all ten milk samples, which appeared as a single, clear band in gel electrophoresis. The authors thus evaluate the milk as a non-invasive DNA source for molecular investigations.

The PCR yielded a single band of 235 bp for the κ-casein gene (CSN3) in all samples, as well as the 100-bp fragment of the GAPDH control gene; according to the authors, no non-specific products occurred.

Sanger sequencing of the fragment showed a 99% match with the genomic reference sequence. Thus, the CSN3 gene has been detected in the DNA obtained from the milk cells of the Halari donkey.

Limitations of the study

• Contradiction in the abstract: The abstract contains the sentence that the κ-casein gene was not amplified from the DNA of the milk cells, and claims the opposite a few sentences later. The results and figures confirm the successful amplification; the sentence is probably an editorial error.
• Gene detection, no protein measurement: Only investigated whether a 235 bp long gene fragment is present (qualitative PCR). This says nothing about the amount or properties of κ-casein in the milk. The authors' conclusion that properties more similar to humans would result from the pedigree is not supported by the data.
• Origin of the pedigree sequences: According to the methods section, all amino acid sequences, including those of the donkey, come from a public database; the figure legend, however, refers to the sequence of the Halari donkey. No complete protein can be derived from the own fragment, which is only 235 bp long.
• Blood comparison hardly described: Neither the number nor the origin of the blood samples is stated for the comparison with blood DNA. With 100% detection in both groups, the chi-square test with P > 0.99 is not very meaningful.
• Incomplete information: The statement on conflicts of interest announces information but does not provide any. The primers and conditions for the GAPDH control gene are missing, and "99% complementarity" is phrased imprecisely for a sequence match.

Level of evidence: Basic research (laboratory) – describes properties of the milk, no statement about effects in humans.

Our assessment

The study is a methodological laboratory work: It shows that DNA for genetic analyses can be obtained from a few milliliters of donkey milk. Statements about the κ-casein content of the milk, its tolerability, or its effects in humans cannot be derived from it.

Source: Singh P., Bhardwaj A., Nayan V., Legha R.A., Pal Y., Soni S., Giri S.K., Bhattacharya T.K. (2024). Milk somatic cell DNA isolation and characterization of κ-casein gene in Halari donkey milk. Heliyon. DOI: 10.1016/j.heliyon.2024.e24991 · Summarized by the DON DONKEY'S editorial team, as of October 2026.

End of the study summary. The study was conducted independently of DON DONKEY'S® and does not refer to our products. Our donkey milk is a food.