1.
Development Of A Suitable Semen Extender For The Cryopreservation Of Nili Ravi Buffalo Bull (Bubalus Bubalis) Semen
by Fazal Wadood (2007-VA-557) | Prof. Dr. Muhammad Aleem | Dr. Muhammad Younas | Prof. Dr. Nasim Ahmad | Prof. Dr. Ijaz Ahmad.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Presently, buffalo farmers are dissatisfied with fertility rates of the frozen semen used in the field and tend to use bulls. This study was designed to develop a suitable semen extender for cryopreservation of Nili Ravi buffalo semen that can improve conception rate in buffaloes.
Experiment-I, an attempt was made to develop semen extender with optimal osmotic pressure for buffalo semen using tris citric acid (TCAE), skim milk (SME) and coconut water (CWE) extenders (each extender have 260, 270, 280, 290 and 300 mOsm/kg osmotic pressure levels). In Experiment-II, best extender (TCAE: 300 mOsm/kg) of experiment-I was tried to improve post thaw spermatozoa characteristics by supplementing antioxidants [0.0, 1.75, 2.0 and 2.25 mM butylated hydroxy toluene (BHT) and 0.0, 2.0, 5.0 and 8.0 mM L-cysteine]. Post thaw spermatozoa motility, viability, plasma membrane integrity (PMI), DNA damage rate and lipid peroxidation were assessed in first two experiments. In Experiment-III, pregnancy rate assessment of extended semen was carried out by using Trial extender (best of experiment II) or Control extender of Semen Production Unit (SPU), Qadirabad, Pakistan (50 inseminations of each extender).
Higher spermatozoa motility at ≥ 270 mOsm/kg was noted in TCAE than both SME and CWE could be due to less intracellular ice formation in zwitterions extender. Higher spermatozoa viability in TCAE and CWE compared to SME may be attributed to extender effectiveness. Higher acrosomal integrity rate at 300 mOsm/kg in TCAE and SME may be because of less intracellular ice formation in isotonic extenders. At 290 mOsm/kg, higher spermatozoa PMI in SME and lesser DNA damage in three extenders might be due to lesser intracellular ice formation at cryopreservation. Decreased spermatozoa DNA damage in SME might be due to the presence of natural antioxidants i.e., casein. Higher lipid peroxidation in CWE than TCAE and SME may be due to presence of natural antioxidants (in SME) and higher cell dehydration potential of TCAE.
Higher spermatozoa motility recorded at 2.0 mM BHT compared to other BHT groups including DMSO might be due to fact that BHT protects spermatozoa mitochondria by reducing oxidative stress. Lower spermatozoa viability, PMI rates and higher DNA damage at 2.25 mM of BHT may be due to BHT toxic effects. Lower lipid peroxidation in BHT treated groups compared to DMSO and BHT control groups might be related to BHT strong antioxidant properties. L-cysteine caused higher spermatozoa DNA damage at highest level (i.e., 8 mM) that could also be due to antioxidant’s toxic effect.
Pregnancy rate 18 % higher was noted in Trial than Control semen extender; however no significant difference have been noted that might be due to less no of inseminations.
In conclusion, TCA extender (300 mOsm/kg) having BHT (2.0 mM) improved post thaw semen quality and yielded numerically better pregnancy rates. Results of study indicated that osmotic stress damaged the spermatozoa internal structures more severely than injury to plasma membrane.
Availability: Items available for loan: UVAS Library [Call number: 2360-T] (1).
2.
Effect Of Trehalose And L-Cysteine On Post Thaw Semen Quality, Antioxidant Enzyme Activity And Fertility In Nili Ravi Buffalo Bulls
by Sajid Iqbal | Dr. Amjad Riaz | Dr. Syed Murtaza Hassan Andrabi | Dr. Syed Murtaza Hassan Andrabi | Prof. Dr. Nasim Ahmad | Prof. Dr. Aneela Zameer Durrani.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Addition of various antioxidants in semen extender is one of the vital strategies being applied in reproductive biology for attaining functionally/structurally integral sperms and hence an appropriate conception rate. It is well established that chilling of buffalo semen results in decreased semen quality which is highly associated with decreased antioxidant activity and higher ROS production. Furthermore, buffalo bull spermatozoa are more susceptible to oxidative damage as compared to cattle bull spermatozoa. It is believed that this difference is due to higher contents of polyunsaturated phospholipids present in plasma membrane of buffalo bull spermatozoa. Freezing process accelerates the production of ROS molecules which may decrease the viability of buffalo bull spermatozoa during storage. Therefore, supplementation of antioxidants in semen extender is required to decrease the ROS-mediated damages to buffalo spermatozoa. The present study had, hence, been designed to monitor the effects of trehalose and L-Cysteine on the semen quality, antioxidant enzyme activity and fertility of Nili Ravi Buffalo bulls.
Semen samples (n= 20) from 4 buffalo bulls were diluted in Tris-citric acid based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30 and 45 mM of trehalose as compared to control. While profiles of superoxide dismutase (U/mL) and total glutathione (μM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At pre freezing, sperm catalase, superoxide dismutase and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione
were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45 and 60 mM of trehalose as compared to control and 15 mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30 mM trehalose as compared to control and 15 and 60 mM groups. While sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight linear velocity (μm/s), curvilinear velocity (μm/s), plasma membrane (structural and functional, %), acrosome (%) and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30 mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes.
Similarly Semen samples from 4 buffalo bulls were diluted in Tris-citric acid based extender having different concentrations of L-cysteine (0.0, 0.5, 1.0, 2.0, and 3.0 mM) and frozen into 0.5 ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase/reductase)] were significantly higher (P< 0.05) at pre freezing and post thawing in extender containing 2.0 mM L-Cysteine as compared to other groups. Post thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity(μm s-1), straight line velocity (μm s-1), curvilinear velocity (μm s-1), beat cross frequency (Hz), viable sperm with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with addition of 2.0 mM L-cysteine as compared to other groups (P < 0.05). The fertility rates (59 vs. 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mM of L-cysteine than the control. In conclusion, addition of 2.0 mM L-cysteine in extender improved the
SUMMARY
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antioxidant enzymes profile, post thaw quality and in vivo fertility of Nili Ravi buffalo bull spermatozoa.
Conclusion
It was concluded that addition of 30mM Trehalose and 2.0mM L-Cysteine in semen extender has significantly improved semen antioxidant enzymes activity, post thaw quality and fertility in Nili Ravi buffaloes. Availability: Items available for loan: UVAS Library [Call number: 2550-T] (1).
