Introduction

Genetic analysis is widely used in domestic animals for the identification and comparison of genetic variations within a population. It is a vital component of techniques such as DNA sequencing, structural variation, genotyping, gene expression and identifying regulatory or functional elements. Techniques for genetic analysis typically require large amounts of highly pure DNA. Genetic source material (e.g. follicle tissue, saliva, blood, cartilage, semen) determines the methods most appropriate for genetic analysis¹.

Unlike DNA extraction from somatic cells, with semen there are a number of difficulties in the purification methodology; these involve the physical and chemical characteristics of spermatozoid nuclear compaction, composition of the ejaculate's non-cellular fraction, and the diluents used in the preservation of frozen sperm^1,2,3. Spermatozoids contain specialized, low-molecular weight nuclear proteins called protamines. These generate a chromatin that is at least six times denser than the histones of somatic cells and that maintain the DNA cells condensed in the acrosome. In addition, their plasmatic membrane is linked by disulfide bonds, making it resistant to the chemical agents used in trADNtional somatic cell DNA extraction methods^4,5. The spermatozoids' acrosome contains hyaluronidase, an enzyme that attacks hyaluronic acid when it contacts the ovule. In DNA extraction, however, hyaluronidase is rel eased wh en th e pl asm atic membrane ruptures, possibly degrADNng the DNA. Another possible way DNA can be damaged is when spermatozoid connector mitochondria are lysed and released because they preserve their oxidative activity and can damage any DNA in the medium^6,7.

The non-cellular fraction of the ejaculate contains a number of minerals, such as zinc and copper, which originate in the prostate. If these are not removed from the final DNA sample, they interfere in the polymerase chain reaction (PCR). Moreover, glycogen and some lipids in the ejaculate can function as energy sources^6,7. Finally, when semen is frozen, the diluents added to preserve it simulate the composition of the non-cellular fraction. They contain proteins, lipids and minerals (e.g. Cr, Fe, Zn, Cu, Cl, K, P, Ca, Mg, Na, and S), making it necessary to remove them completely from samples before DNA extraction to prevent any interference with the PCR^7,8,9.

The present proposed technique is based on the guanidinium thiocyanate method, and other techniques for extracting DNA from frozen semen, which result in DNA samples of the necessary quality and quantity for current genetic identification techniques. It completely eliminates the need to mechanically separate the remaining protein; a required step in existing techniques because this protein can lead to widely varying reADNngs between samples if not removed^7,10,11. The present study objective was to develop and evaluate a technique based on the guanidinium thiocyanate method that can efficiently extract high-molecular weight genomic DNA from frozen bull semen for use in genetic identification techniques that require high purity and operate within values of 1.8 to 2.0 in the 260/280 wavelength range, and 2.0 to 2.2 in the 260/230 range.

Material and methods

All analyses were performed in the Laboratorio de Genética Molecular of the Departamento de Zootecnia, Universidad Autónoma Chapingo.

Results and discussion

The least means squares analysis identified differences (P<0.0001) between methods in the 260/280 wavelength range and between sample concentrations (Table 1). This confirms that the modified method extracted a higher quality and quantity of DNA compared to the methods of Hossain et al¹⁰ and Birren et al¹². Absence of difference in the 260/230 range indicates total cleansing of RNA from the samples was attained in all three extraction methods. These results are supported by the electrophoresis analysis results of the DNA samples extracted with each evaluated method (Figure 1).

Table 1 Least means squares (± standard error) results for quality and quantity of DNA extracted from frozen bull semen using three methods 

† Birren et al, 1997; ¶ Hossain et al, 1997; § Developed in present study.

^abc Least squares means with different letters in the same column are different (P<0.05).

Phenol-chloroform (lanes 1 to 4); guanidinium thiocyanate (lanes 5 to 8); and modified guanidinium thiocyanate (lanes 9 to 12). "M" lanes are 1 kb molecular weight markers.

Figure 1 1% gel electrophoresis of bovine semen prepared with one of three DNA extraction methods 

When extracted with the phenol-chloroform method, DNA samples exhibited low quality (1.47 ± 0.11 at 260/280; and -0.41 ± 0.41 at 260/230), and low concentrations. Electrophoresis demonstrated an absence of DNA, possibly due to degradation and loss from total washing during the protocol. This coincides with previous studies^1,10 in which DNA degradation was extensive or complete when usi ng the phenol-chloroform method¹². However, in these same studies DNA quality surpassed 1.8 at 260/280, possibly due to the presence of RNA and the remains of low-molecular weight proteins.

The guanidinium-thiocyanate method produced good quality DNA samples with higher concentrations than in the phenol-chloroform method. Nonetheless, electrophoresis showed slight DNA degradation in the samples and considerable presence of protein in the gels' upper fraction (Figure 1, rows 5 to 8). Slight DNA degradation and the presence of protein were also observed in samples in a previous study¹⁰, partially due to the need for DNA separati on by protei n tracti on , causin g incomplete purification of the nucleic acids.

The modified guanidinium-thiocyanate method proposed here produced high quality and quantity DNA samples. Nano-spectrophotometry showed quality to be between 1.8 and 2.2 in both wavelength ranges (i.e. 260/230 and 260/ 280) (Table 1). Little or no DNA degradation was apparent in the electrophoresis analysis, with a total absence of proteins and other contaminating factors (Figure 1, rows 9 to 12). The samples provided optimum conditions for use in genetic analysis.

Extraction efficiency from frozen bovine semen samples using the modified method proposed here was compared to the efficiency reported in previous studies^10,12. In the proposed method, a second washing with phosphate buffer solution was added to the previous methods to increase DNA purity. This was done even though livestock species normally produce more ejaculate than humans and its density is 0.2 to 30.0 times greater⁶. In addition, bull, pig and horse ejaculates have Cl, K and Ca concentrations 1.7 to 3.4 times higher than human ejaculate. If these ions are not removed from semen samples they can cause erroneous results during genetic analyses and even inhibit PCR amplification of DNA^7,9.

In the proposed method, two washings with organic solvents followed by high velocity centrifuging were used even though use of organic solvents is reported to make isolation of non-degraded DN A more difficult¹⁴. However, this modification helped to completely remove the protein that had not degraded in previous steps. It also aided in preserving DNA integrity by consolidating it into a pellet, thus preventing chemical damage by completely separating it from the organic solvent, any compounds in the ejaculate, and any diluents added for semen preservation. Addition of the phenol:chloroform:isoamyl alcohol washing step removed high-molecular weight proteins and residual chelating agents in the samples while the subsequent chloroform:isoamyl alcohol washing removed low-molecular weight proteins and any chelant traces remaining after the first washing^7,9.

Since Hossain et al¹⁰ reported that RNA material was preserved in some samples, an RNAase incubation was added in the modified method. This ensured the DNA in the samples was genetically pure, thus avoiding imprecise values when measuring concentrations by nano-spectrophotometry. If values are not exact, DNA sample dilution calculations can contain errors which affect PCR amplification results⁷.

Finally, use of 3 M sodium acetate in DNA incubation with isopropanol at -20 °C made DNA precipitation more efficient by augmenting solution ionic strength and removing organic solvents and diluent medications⁸. Centrifuging, decanting and washing with 70% ethanol removed all sodium acetate and sarkosyl remnants.

Conclusions and implications

The modified DNA extraction method proposed here for use with frozen bovine semen produced highly pure DNA samples free of degradation and with extremely low or null protein content. It is also simple and low-cost, and the resulting sample DNA quality and quantity are appropriate for genetic analyses, such as SNPs and microsatellites, that are commonly used in mammals.