Use Of Butylated Hydroxytoluene (Bht) As Antioxidant In Buffalo Semen Extender
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Publisher: 2011 Dissertation note: The Buffalo is the major milk producing animal in Pakistan. Nili-Ravi breed of buffalo is the one of the best breeds of the world. Buffalo contributes seventy percent of total milk production in Pakistan. The excellent buffalo bulls are few in number and their semen volume and sperm concentration is also low as compared to cattle. The animal has lower fertility particularly when inseminated with frozen semen. There are many reasons for poor fertility of buffalos and one of the most important reasons may reasons for poor fertility of buffalos and one of the most important reasons may be the production of Reactive Oxygen Species (POS) from the plasma membrane of spermatozoa causing damage to the membrane integrity due to lipid peroxidation. The addition of optimum level of antioxidants like BHT to buffalo semen extender before freezing may be answer to this problem. The main objective of the present study was to determine the level of total of antioxidants in the semen and to optimize the quantity of BHT in the semen extender of the Nili Rave Buffalo bulls.
In this study semen from Nili-Ravi buffalo bulls ( n=10) was collected twice per week and diluted with Tris egg yolk citrate extender. Antioxidant BHT was added to the extended semen at the rate of 1.0mM, 1.0 mM and 2 mM while Control was containing no BHT. The semen was visually analyzed under phase contrast microscope for motility, vitality (Eosin/Nigrosin staining), plasma membrane (HOS assay) and acrosomal integrity (NAR), at three stages i.e. after dilution, before freezing and after freezing. For the determination of total antioxidant capacity (TAC), semen samples were transported under ice to Spectophotometric Laboratary, GC. University Lahore. Data collected was presented as mean= SEM. Treatment groups were compared using one way ANOVA and Results were compared by using Duncan Multiple Range Test using SPSS (version 16.0) ; SPSS Chicago IL.
Result of this study show significant (P< 0.05) improvement in semen motility, vitality, plasma membrane and acrosomal integrity at 1.5 mM BHT concentration as compared to Control, however, insignificant difference was noticed with in treated groups and with in bulls. Maximum TEAC value (2.42) was observed in the extended semen containing two mM BHT and minimum TEAC values (0.58mM) in the post thaw semen sample with BHT.
It is concluded that addition of antioxidants in the buffalo semen improve the semen quality before and after freezing. Fortification of buffalo semen extender with BHT (@ of 1-1.5 mM) improved post thaw semen quality and hoped to increase the fertility of buffalo. However, field fertility trials need to be carried out for confirmation.
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Effect of Equine Chorionic Gonadotropin (eCG) on the Follicular Dynamics, Estrus Eepression, Ovulation and Pregnancy Rate in Control Internal Drug Release (CIDR) Based Estrus Synchronization Protocol in NIli-Ravi Buffalo.
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Publisher: 2013 Dissertation note: Buffalo is unique to livestock and dairy industry in Pakistan as its share is major (65%) in milk production major. Reproduction is crucial for profitable production in dairy sector. Buffalo reproduction is hampered due to small size ovaries, poor ovarian reserves, and less pronounced estrus intensity, ultimately leading to substandard fertility. Synchronization technology including CIDR based protocols are well established in cows and gaining popularity in buffaloes. However, this needs modifications based on estrous cycle physiology in buffaloes. It is hypothesized that addition of eCG at the time inducing luteolysis in CIDR based synchronization protocol will promote follicular growth, ovulation and pregnancy rates. The objective of this study is to determine the effect of addition of eCG to a CIDR based synchronization protocol on ovarian follicular dynamics, estrus rate, ovulation, and pregnancy rates in Nili-Ravi buffalo. Lactating multiparous suckled Nili-Ravi buffalo (n=63) milked twice daily were assigned in a CIDR (1.38 g progesterone Pfizer Co, USA) synchronization regimen. Animals were divided randomly to receive either saline (n=31) or 1000 IU eCG i.m. (n=32) (Chronogest PMSG, Intervet, Holland) concurrent with PGF2á (Dalmazine, cloprostenol, Fatro, Italy) treatment of the CIDR protocol (Day 6). Fixed time, two inseminations were performed at 48 and 60 hours after CIDR removal (Day 7). Number of follicles of variable size recruited at CIDR removal, dominant follicle size, its growth rate and ovulation time did not differ significantly between the groups. The estrus response was not higher significantly (P>0.14) in eCG group than control group but estrus intensity was significantly higher (P>0.001) in eCG group. Ovulation rate and 26 pregnancy per AI was higher (P>0.05 & 0.004) in buffaloes in eCG group. Therefore, It is concluded that eCG is helpful to improve estrus expression, ovulation rate and pregnancy per AI in FTAI program in Nili-Ravi buffaloes.
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Effect Of Alpha Lipoic Acid On Post Thaw Quality And In Vitro Incubation Of Nili Ravi Buffalo Bull Semen
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Publisher: 2015 Dissertation note: Cryopreservation is the freezing of cells or tissues to subzero temperatures, typically -196 º C. Many benefits have resulted from the process of cryopreservation. Damage induced by cryopreservation has been results cold shock, oxidative stress, osmotic changes, and formation of ice crystal and lipid–protein reorganizations within the cell membrane. Oxidative damage caused by reactive oxygen species (ROS) leads to impaired cell functions. Free radicals, includes ROS and RNS, are normal pro - oxidant molecules in aerobic metabolism. Alpha lipoic acid is a non-vitamin coenzyme that helps in significant metabolic and antioxidant functions in the body. Alpha lipoic acid has been reported to have extra functions by which they are able to synthesize vitamin C from its reduced form in the presence of glutathione. It is matchless among biological antioxidants, because it is equally lipid and water soluble. This allows it to nullify free radicals almost everywhere in the body, inside as well as outside the cells. Therefore, the objective of present study is to determine the effect alpha lipoic acid on post thaw quality and in vitro incubation of buffalo bull semen. Alpha lipoic acid scavenge on reactive oxygen species formed in semen during the process of cryopreservation, so it maintained good semen quality during post thaw and in vitro incubation. Three mature Nili-Ravi buffalo (Bubalis bubalis) bulls (4-8 year age) kept at SPU, Qadirabad Sahiwal Pakistan were used in the study. These bulls are being used as regular donors at SPU. There semen was collected with artificial vagina of temperature 42c; three ejaculates (one from each) was pooled and diluted (30 million sperms/ml) with extender of different inclusion levels (0.0, 0.5, 1, 1.5, 2, 2.5 mmol/ml) of alpha lipoic acid. Straws were filled and extended then semen was cooled for 2 hours and equilibrated for two hours. Semen was placed in Liquid nitrogen vapors for 10 minutes. Finally semen straws was put in liquid nitrogen, Total five replicates were performed. Now post thaw quality was checked in
which various tests were performed, like %age motility, Acridine orange assay for DNA integrity, HOST for plasma membrane integrity, Fitc-PNA/PI for viability and acrosomal integrity. Longevity test was performed by in vitro incubation of frozen thawed semen sample in SOF and evaluating it at 1.5, 3 and 4.5 hour interval in Carbon dioxide incubator. It was expected that Alpha lipoic acid shown positive effect on post thaw quality and in vitro incubation of buffalo bull semen, in the meaning of increased percentage motility, Less DNA damage during cryopreservation and incubation, Increased acrosomal and plasma membrane integrity. So alpha lipoic acid shown positive effect by counter acting on ROS during cryopreservation and in vitro incubation. Results acquired from this study shown that an increase in sperm motility, plasma membrane integrity, DNA integrity, Acrosomal integrity, viability and survival was caused by ALA competences in energy production and anti-oxidant properties, when used at the concentration of 0.5mM and 1mM. In summary, based on the results of our study, it can be concluded that an optimal concentration (0.5mM and 1mM) of ALA improved PMI, sperm motility and viability, minimize DNA damage and improved sperm survival.
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Development Of A Suitable Semen Extender For The Cryopreservation Of Nili Ravi Buffalo Bull (Bubalus Bubalis) Semen
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Publisher: 2015 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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