Untitled - Faculdade de Medicina Veterinária e Zootecnia - Unesp

draft performance of Chinese swamp buffalo is not enough to satisfy the needs of people living in a
commercialized era. By means of new genetic and reproductive technology, it becomes an urgent
task for current Chinese government to convert Chinese swamp buffalo to milk animal.
Establishment of buffalo sperm sorting technology (Lu et al., 2006, 2007, 2010; Liang et al., 2008)
provides a good technical basis to accelerate this process of change. The application of sex control
technology, combined with other reproductive biotechnologies as IVF, AI, embryo transfer, and so
on, could produce about the double number of milking buffaloes in the same period of time as in
traditional reproductive technology alone, which could hopefully resolve the shortage of milking
buffaloes in China and promote the development of buffalo dairy industry in southern China.
IDENTIFICATION OF X- AND Y-CHROMOSOME BEARING BUFFALO SPERM
The sperm sexing technology using flow cytometry involves staining of the sperm with
DNA-binding fluorochrome (Hoechst 33342). Flow cytometric analysis is subsequently used to
quantify the difference in DNA content between the X- and Y-chromosome bearing sperm, based
on their differences in fluorescence intensity (Johnson and Welch, 1999). The difference in DNA
content between X and Y-sperm of many animals has been quantified (Johnson, 1992, 2000;
Parrilla et al., 2004; O’Brien et al., 2005). However, no data on difference of DNA content in
buffalo sperm is available until 2006 reported by Lu et al (2006) from our laboratory.
Sperm sorting and analysis
The procedures for sperm sorting and analysis were those reported by Lu, et al (2006). Briefly,
buffalo semen collected from six fertile Murrah buffaloes and six fertile Nili-Ravi buffaloes at
Livestock and Poultry Breeding Station of Guangxi, China was extended in modified TALP
solution and then stained with Hoechst 33342 at a concentration of 5 mg/ml and incubated at 35 o C
for 60 min. The stained sperm were sonicated for 2–5s to remove the tails for more desirable
orientation when passing through the orifice of the flow cytometer nozzle.
When the sperm passed through the flow cytometer the fluorescence emitted by the sperm was
detected both by the 0◦ (forward) and the 90◦ (sideward) detectors and stored as frequency
distributions. Only those sperm with proper orientation were gated and subsequently analyzed for
the difference in DNA content between X- and Y-sperm populations. The nuclei of goat sperm were
used as control sample to evaluate the alignment of the cytometer before the buffalo sperm nuclei
were analyzed. A minimum of 50,000 signals of sperm nuclei per sample were recorded. Six
replicates from each bull were performed. The methods used for calculation and analysis of the
difference (%) of the DNA content between the X- and Y-sperm populations was those reported by
Garner et al., 1983.
RESULTS
The analysis of the mean values of the fluorescent intensity revealed that the difference in DNA
content was 3.59±0.11% for Murrah buffalo and 3.55±0.14% for Nili-Ravi buffalo (Table 1).
Differences were found among males (within breed) (p < 0.05) but not between breeds (p > 0.05).