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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.
Contributor(s): Dr. Muhammad Younas | Prof. Dr. Nasim Ahmad | Prof. Dr. Ijaz Ahmad.
Material type: materialTypeLabelBookPublisher: 2015Description: 88p.Subject(s): Department of Theriogenology | Phd. thesisDDC classification: 2360-T Dissertation 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.
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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.

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