Cryopreservation of buffaloes (Bubalus bubalis) sperm in Tes-Tris diluter containing lowdensity lipoproteins in replacement of egg yolk

The objective of this research was to test different concentrations of low density lipoproteins (LDL) in replacement of egg yolk in Tes-Tris extender for cryopreservation of buffalo spermatozoa. The experiment was conducted at the Bufalloes, Experimental Farm on the UFMG, in Pedro Leopoldo, MG. Six Murrah bulls, aged 29-36 months, were used to collect semen (through artificial vagina) 3 ejaculates from each animal, performing a total of 18 ejaculates were collected. Total motility was analyzed by a Sperm Class Analyzer-SCA® v.4.0. Sperm concentration was evaluated by Neubauer chamber, sperm morphology analysis (phase contrast microscope) and the hypo-osmotic test (Host) was performed in fresh semen samples. The ejaculates were fractioned and each fraction was diluted in different extender. Dilution was performed to reach a final concentration of 50x106 spermatozoa mL-1. Treatments were: control (20% egg yolk) and 2, 4, 8 and 14% (v/v) LDL. Extender semen was packaged into 0.25 mL straws starting there after the cooling process using -0.25oC/min cooling rate (computer assisted cooling machine TK 4000). Samples were maintained in equilibrium at 5°C for 4 hours. Straws were frozen in liquid nitrogen vapor, 5 cm above the liquid surface, for 20 minutes. There after straws were immersed in liquid nitrogen and stored for a minimum of one week. After thawing (37oC for 30 seconds) straws contents were submitted to a thermal resistance test (TRT/120 minutes) evaluated by a CASA. Sperm cells were evaluated by a hypo-osmotic swelling test and integrity of the membranes by fluorescent probes (CFDA/Pi). Results were compared by the Tukey test (P<0.05). Pre-cooling sperm motility was less in samples diluted in the control extender containing egg yolk than in 2 and 4% LDL extenders. Post-thaw sperm motility was higher (56.53±9.73%) in the control extender than in all extender containing LDL (47.16±11.06%, 45.64±7.46%, 45.71±6.56 %, 43.33±5.97%, for 2, 4, 8 and 14% LDL extenders, respectively). However, during the TRT sperm motilities were similar between all extenders. Values of LDL, VCL, VSL, VAP, LIN and BCF were lower in sperm diluted in the control extender than in extenders containing LDL, during the pre-cooling phase, post-thaw and throughout the TRT. There was no difference between treatments regarding the integrity of sperm membranes (38.75 to 43.14% intact) and the reaction to the Host (49.39 to 55.39% reacted). It can be concluded that 2, 4 and 8% LDL concentrations in Tes-Tris extender, were more effective than Tes-Tris containing 20% of egg yolk for increase the kinetics characteristics of buffalo sperm cells.