Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen.

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10(6) motile spermatozoa mL(-1)) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.

Akhter S ，Ansari MS ，Rakha BA ，Andrabi SM ，Khalid M ，Ullah N ... -  《-》

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender.

This study was designed to compare commercially available extender Bioxcell with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 degrees C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 degrees C having 50 x 10(6) spermatozoa/ml) in tris-citric egg yolk or Bioxcell extender. Diluted semen was cooled to 4 degrees C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (-196 degrees C). After 24 hours of storage, semen straws were thawed at 37 degrees C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 +/- 1.1, 45.0 +/- 1.4), viability (66.2 +/- 1.1, 64.4 +/- 1.3) plasma membrane integrity (60.4 +/- 1.2, 59.2 +/- 1.4) and normal apical ridge (82.9 +/- 0.5, 80.7 +/- 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell extender, respectively. Similarly, sperm abnormalities of head (1.20 +/- 0.1, 1.20 +/- 0.1), mid piece (0.67 +/- 0.1, 0.87 +/- 0.1) and tail (11.7 +/- 0.2, 11.6 +/- 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.

Akhter S ，Ansari MS ，Rakha BA ，Andrabi SM ，Iqbal S ，Ullah N ... -  《-》

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa.

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.

Andrabi SM ，Ansari MS ，Ullah N ，Anwar M ，Mehmood A ，Akhter S ... -  《ANIMAL REPRODUCTION SCIENCE》

Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm.

Lower fertility in buffaloes with frozen-thawed semen is attributed to sperm damage that is believed to be due to formation of ice crystals during freeze/thaw process. It was hypothesized that antifreeze proteins in the extender may improve the post thaw quality of buffalo bull sperm. For this purpose, two separate experiments were conducted to evaluate antifreeze proteins III (AFP III) at 0 (control), 0.1, 1 and 10 μg/mL (Experiment I) and 0 (control), 0.01, 0.1 and 1 μg/mL (Experiment II) for its effect on post thaw quality of buffalo bull semen. Semen was collected from three Nili-Ravi buffalo (Bubalus bubalis) bulls with artificial vagina (42 °C) for three weeks (replicate) per experiment. For each experiment, qualifying ejaculates (6 ejaculates/bull) were divided into four aliquots and diluted (at 37 °C having 50 × 10(6) sperm/mL) in tris-citric acid extender containing above mentioned concentrations of AFP III. Diluted semen was cooled to 4 °C in 2 h, equilibrated for 4 h, filled in 0.5 mL straws, kept over liquid nitrogen vapors for 10 min and plunged in the liquid nitrogen. After 24 h of storage, semen straws were thawed at 37 °C for 30 s to assess sperm progressive motility (SM), plasma membrane integrity (PMI), viability (live sperm with intact acrosome) and normal epical ridge (NAR). In experiment I, improvement (P<0.05) in percentage SM and sperm PMI was recorded in extender containing 0.1 μg/mL AFP III compared to control, the higher concentrations (1 μg/mL and 10 μg/mL) being inefficient. While evaluating the lower concentration (experiment II), 0.01 μg/mL of AFP III in the extender it was found to be ineffective to improve semen quality parameters, while 0.1 μg/mL AFP III in extender was found better in terms of progressive motility and plasma membrane integrity of buffalo bull semen compared to control. Sperm viability and NAR remained similar (P>0.05) in extenders containing different concentrations of AFP III and control in both of experiments. In conclusion addition of AFP III in the extender at 0.1 μg/mL improved the progressive motility and plasma membrane integrity of cryopreserved buffalo bull semen.

Qadeer S ，Khan MA ，Ansari MS ，Rakha BA ，Ejaz R ，Husna AU ，Ashiq M ，Iqbal R ，Ullah N ，Akhter S ... -  《-》

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa.

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.

Akhter S ，Ansari MS ，Andrabi SM ，Rakha BA ，Ullah N ，Khalid M ... -  《-》