High salt method: a simple and rapid procedure for isolation of genomic DNA from buffalo (Bubalus bubalis) white blood cells.

Three different methods, namely guanidine hydrochloride, phenol-chloroform extraction and high salt method, were compared in order to develop a simple method for the extraction of high yield of DNA from buffalo blood suitable for genome analysis. Both phenol-chloroform and high salt methods produced good yields of high molecular weight DNA as determined by agarose gel electrophoresis. The yield and quality of DNA extracted by high salt method was comparable to that of phenol-chloroform method. The mean yields of DNA from 10 ml of whole blood extracted by either the phenol-chloroform or the high salt methods were 446.16 micrograms (SE = 26.68) and 432.83 micrograms (SE = 19.34) respectively. The DNA obtained from both methods was suitable for conventional as well as polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) studies. Extraction using the guanidine hydrochloride method resulted in a gelatinous material that failed to resuspend in TE buffer. The high salt method is quick and reliable and can be routinely used for the extraction of DNA from buffalo samples instead of phenol-chloroform extraction which is hazardous and time-consuming.

Induction of acrosome reaction in fresh and frozen-thawed bovine spermatozoa by platelet activating factor.

In vitro capacitation has been induced in fresh and frozen spermatozoa of Karan Swiss (KS) and Karan Fries (KF) crossbred cattle by using a phospholipid-platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The capacitation was monitored by examining acrosome reaction (AR) and motility at various levels of platelet activating factor (PAF) and at the end of each incubation time. On an average, with the increase in incubation time, there was a gradual decrease in motility and increase in acrosome reaction in both fresh as well as frozen spermatozoa. As PAF level increased, the motility of fresh sperms decreased and their acrosome reaction increased dramatically. However, in frozen-thawed semen, the motility remained almost the same and increase in AR of frozen spermatozoa was not pronounced. PAF level of about 100 microM was observed to be most optimum as at this level AR improved significantly without much loss of motility.