MATERIALS AND METHODS

Animals and semen collection: The experiment was conducted at the International Livestock Management training Center (ILMTC), Sakha belonging to the Animal Production Research Institute, Ministry of Agriculture, Egypt. A total of 72 ejaculates from six mature Egyptian buffalo bulls aged 3-4 years with known fertility were collected by an artificial vagina one weekly for 12 weeks during October-December (Winter season). The volume of ejaculates was measured in a conical tube graduated at 0.1 mL intervals and sperm concentration was determined by means of Neubaur Heamocytometer according to the procedures described by Sansone et al.⁷. Ejaculates fulfilling minimum standard of sperm motility (75%) and sperm morphology (80%) were processed for freezing. The ejaculates were pooled in order to have sufficient semen to make a replicates and to eliminate bull effect.

Semen processing: Immediately after collection, the ejaculates were immersed in warm water bath at 37°C until the laboratory evaluation. Semen assessment was performed within 10 min. The reference of cryopreservation extender was tris (Buffer oxford Lab-Chem., India)-egg yolk (20% EY, control) containing 20 mL of egg yolk, 3.025 g tris (hydroxyl methyl amino-methane), 1.675 g citric acid, 0.75 g glucose, 7 mL glycerol, 0.25 g lincomycin, 0.005 g streptomycin and completed with up to 100 mL bi-distilled water. The glutamine (Sigma Chem. Aldrich, USA) was added to the tris-egg yolk based extender at different concentrations (0 "Control", 20, 40, 60, 80 and 100 mM, respectively). Qualifying semen ejaculates were split into 6 aliquots and diluted with the different experimental extenders at 37°C in order to provide concentration of 80 million of motile spermatozoa per milliiter. The diluted semen was cooled slowly to 5°C within 2 h and equilibrated for 4 h at the same temperature. Semen was packed into 0.25 mL French straws using a semen filling machine and kept on liquid nitrogen vapors 4 cm above its surface for 10 min. Then, straws were plunged into the liquid nitrogen, stored for one month. For thawing after one month of storage, straws were dipped into a water bath at 37°C for 30 sec, then the percentages of sperm progressive motility, viability, abnormal morphology, plasma membrane and acrosome integrity were determined.

Semen evaluation: Sperm progressive motility was estimated using phase contrast microscope (×400 magnification) by placing semen sample on pre-warmed (37°C) glass slide and covered with a cover slip. Sperm plasma membrane integrity (%) was assessed using hypo-osmotic swelling test (HOST) as outlined by Jeyendran et al.¹³. One hundred spermatozoa were assessed and the percentage of spermatozoa with curled tail (swollen intact plasma membrane) was calculated. Sperm viability, abnormalities and acrosome integrity were determined by dual staining procedure¹⁴ using trypan blue (Sigma T 8154) with Giemsa stains (Sigma GS 500).

Fertility trial: The fertilizing ability of spermatozoa from each extender was calculated based on the rectal palpation at 45-60 days post insemination of 65 Egyptian buffalo cows. Buffalo cows were artificially inseminated with one insemination dose (20×10⁶) of frozen samples with different glutamine levels and control one.

Statistical analysis: The experimental data were statistically analyzed using the general model program¹⁵. Data of semen parameters was subjected to repeated measurement according to the following model:

Y_ijk = μ+T_i+P_j+S (P)_ijk+(T*P)_ij+E_ijk

The least square mean was calculated by modulating the data higher than 70% and less than 30%. Differences with p<0.05 were considered significant.

RESULTS

Progressive motility and viability of Egyptian buffalo bull spermatozoa were significantly higher (p<0.05) in extenders containing 20, 40 and 60 mM glutamine than those with 0, 80 and 100 mM at all stages of cryopreservation (Fig. 1 and 2).

The farther increase in glutamine to 100 mM caused a significant reduction in the progressive motility and viability of buffalo spermatozoa as compared with control and other glutamine extenders at all stages of cryopreservation. The addition of glutamine improved sperm abnormalities at all concentrations tested (from 20-100 mM) at any stage of cryopreservation (Fig. 3).

The addition of 20, 40 and 60 mM glutamine to freezing extender was more effective than the other glutamine levels on decreasing of sperm buffalo abnormalities after cooling and freezing-thawing process.

Fig. 1:	Progressive motility of buffalo spermatozoa (%) at different stages of cryopreservation. The values with different letters are significantly different from one another (p<0.05) between treatments (a, b, c,...) and between cryopreservation stages (A, B, C,...), respectively

Fig. 2:	Live buffalo spermatozoa (%) at different stages of cryopreservation. The values with different letters are significantly different from one another (p<0.05) between treatments (a, b, c,....) and between cryopreservation stages (A, B, C,...), respectively

Fig. 3:	Abnormal buffalo sperm (%) at different stages of cryopreservation. The values with different letters are significantly different from one another (p<0.05) between treatments (a, b, c,....) and between cryopreservation stages (A, B, C,...), respectively

Fig. 4:	Plasma membrane integrity (%) of buffalo sperm at different stages of cryopreservation. The values with different letters are significantly different from one another (p<0.05) between treatments (a, b, c,...) and between cryopreservation stages (A, B, C,...), respectively

The percentages of acrosome and plasma membrane integrity showed higher (p<0.05) in extender containing 60 mM after dilution and equilibration while, adding 20 and 40 mM glutamine significantly (p<0.05) increased sperm acrosome and membrane integrity at post-thawing process (Fig. 4 and 5). Whereas, the increase of glutamine to 100 mM led to a significant reduction in both acrosome and membrane integrity of buffalo spermatozoa in compared with that extended in other glutamine concentrations and control extenders.

The fertility rate was statistically higher (p<0.05) for spermatozoa diluted in extender containing 20 mM glutamine (72.7%) followed by that diluted in 40 mM (64.3%) and 60 mM (60%) extenders in compared with those diluted in 80 (50%), 100 mM (30%) and 0 mM control (40%) (Fig. 6).

DISCUSSION

The increase in sperm sensibility to oxidative stress and cryodamage affect spermatozoa quality, shortening their life span resulting in a reduction in sperm motility, viability, antioxidant enzyme activity and fertilizing capacity during extending, freezing and thawing process¹⁶. Frozen-thawed bull semen is more easily peroxidized than fresh semen. Additionally, intracellular antioxidant capacity in sperm decreases following freezing-thawing process¹⁷.

In the present study, progressive motility of buffalo bull spermatozoa was high when the extender was supplemented with 20-60 mM glutamine than that in control one. The further increase in glutamine concentration to 100 mM in extender significantly decreased sperm motility percentage.

Fig. 5:	Acrosome integrity (%) of buffalo sperm at different stages of cryopreservation. The values with different letters are significantly different from one another (p<0.05) between treatments (a, b, c,...) and between cryopreservation stages (A, B, C,...), respectively

Fig. 6:	Conception rate (%) of buffalo sperm. The values with different letters are significantly different from one another (p<0.05) between treatments (A, B, C,...)

These results are inline with that in a previous study on cryopreserved stallion semen which indicated that glutamine addition to the INRA 82 extender significantly increased the motility parameters of stallion spermatozoa after freezing and thawing for the concentration of 30-80 mM and become ineffective when added at concentration of 120 and 160 mM resulted in a significant decrease in sperm motility¹⁸. Also, El-Sheshtawy et al.¹⁹ noticed that 25 mM glutamine offered protection for frozen-thawed buffalo spermatozoa but the higher concentration of glutamine (50 mM) in extender did not improve the results whereas, the 100 mM concentration significantly decreased sperm motility. It is believed that glutamine improves the progressive motility of buffalo bull spermatozoa by reducing the Reactive Oxygen Species (ROS) in the semen extender which are responsible for reducing lipid peroxidation of bio-membrane system that associated with sperm motility during the freeze-thawing process^20-22. Sperm viability and intact acrosome are responsible to achieve capacitation, acrosome reaction and final fertilization^14,23. In the present study, viability of buffalo bull spermatozoa was higher in samples cryopreserved in tris-egg yolk extender containing 20-60 mM glutamine in compared to the control extender, whereas, the presence of 100 mM significantly decreased sperm viability. In addition, higher intact acrosome percentages were observed in cryopreserved buffalo bull spermatozoa due to addition of glutamine from 20-60 mM. These results were in agreement with that of Bucak et al.²⁴ in Ovine semen and Ansari²⁰ in buffalo semen. Bucak et al.²⁵ suggested that the addition of amino acid to the semen extender maintain the viability of buffalo spermatozoa by coping with ROS levels through increasing the intracellular activity of antioxidants. Glutamine has an extracellular mechanism of action. An increase in the concentration glutamine in extender leads to increase in osmotic pressure²⁶. When the concentration gets too high, spermatozoal motility was compromised and this was confirmed in the present study. Watson and Martin²⁷ suggested that a dual glutamine action was seen: The first is extracellular and cryoprotective, the second is intracellular and metabolic with the intervention of glutamine in the Krebs cycle via glutamine and α-cetoglutate. In the present study, all glutamine concentrations in extenders causes a significant decrease in abnormal and sperm plasma membrane damage at all stages of cryopreservation process compared to the control. Current results with respect to sperm parameters are in agreement with that of Trimeche¹¹ on Poitou jackass, Li et al.²⁸ on cynomonolgus monkey, Khlifaoui et al.²⁹ on stallion, El-Sheshtawy¹⁹ and Topraggaleh et al.³⁰ on buffaloes and Sariozkan³¹ on rabbit. With respect to fertility results based on 45-60 day rectal palpation after AI, the addition of glutamine at doses ranging from 20-60 mM in semen extender led to have a higher fertility rates compared to the control. While, adding 100 mM had adversely effect on fertility rate. These findings disagree with that of previous studies where there was no improvement in fertility rate when certain additives were added in semen extender^32,33,20,34.

CONCLUSION

The present results concluded that the addition of glutamine at doses ranging from 20-60 mM in tris freezing extender not only improved post-thaw motility of buffalo bull spermatozoa but also maintained sperm viability, abnormalities, acrosome and plasma membrane integrity during freezing-thawing process and enhanced the fertilizing ability of frozen-thawed buffalo spermatozoa.