Effect Of L-Cysteine And Glutathione On Post Thaw Quality Of Sahiwal Bull Spermatozoa
Material type: Book ; Format:
; Literary form:
Publisher: 2015 Dissertation note: Freezing and thawing of semen leads to production of reactive oxygen species (ROS)
due to plasma membrane lipid peroxidation. Because of this semen quality can be
compromised. To overcome this problem, antioxidants have been used in cryopreservation
medium. Glutathione and cysteine have thiol groups which penetrate into the cell and protect
it from oxidative stress. In this study, effect of different concentrations of cysteine and
glutathione on post thaw quality of Sahiwal bull spermatozoa was determined.
Semen was collected with artificial vagina from five mature regular donor Sahiwal
bulls kept at the Semen Production Unit Qadirabad, Sahiwal. Semen samples possessing
>60% motility and >500x10
sperm/ml were included in study. After collection, semen
samples from five bulls were pooled, divided into seven equal aliquots and kept at 37 ºC in
water bath. After that dilution was done with Tris citric egg yolk extender having different
concentrations of cysteine and glutathione as Con (0.0 mM), C1 (1.0 mM cystein), G1 (1.0
mM glutathione), CG0.5/1(0.5 mM Cysteine+1.0 mM glutathione), CG1/0.5 (1.0 mM
cysteine+0.5 mM Glutathione), CG0.5/0.5 (0.5 mM cysteine+0.5 mM glutathione) and
CG1/1 (1.0 mM cysteine+1.0 mM glutathione). Diluted samples were cooled to 4ºC in two
hours and equilibrated for 4 hours at 4
C. After that they were packaged into 0.5 ml French
semen straws (20x10
sperm/straw). All semen straws were placed 4cm above liquid nitrogen
surface in vapors for 10 minutes. Then, semen straws were plunged into liquid nitrogen for
freezing and stored until post thaw analysis. The experiment was repeated for five times
(replicates = 5). Four semen straws/treatment were thawed for 30 seconds in water bath at
37ºC and evaluated for visual motility, plasma membrane integrity (PMI), acrosome integrity,
mitochondrial trans membrane potential and CASA motility parameters and kinematics.
PMI in group CG0.5/0.5 was significantly higher (40.00±1.42 %) as compared to Con
26.67±0.80 (P<0.5). Plasma membrane integrity in groups CG1/1, CG0.5/1, G1 and C1 was
significantly higher (36.00±1.88 %, 36.20±1.07 %, 33.60±1.21 % and 32.80±0.80 %
respectively) as compared to Con (26.67±0.80 %) (P<0.05). There was no significant
difference in C1 (32.80±0.80 %) and G1 (33.60±1.21 %) (P>0.05). In case of acrosome
integrity, NAR value of group CG0.5/0.5 was significantly higher (71.40±1.08 %) as
compared to Con (59.67±0.37 %) (P<0.05). All other groups also showed significant
differences as compared to Con (P<0.05). CG0.5/0.5 also showed significantly higher NAR
value (71.40±1.08 %) as compared to C1 (64.40±1.40 %) and G1 (67.60±2.07 %) (P<0.05).
CG0.5/0.5 had significantly higher value (71.40±1.08 %) as compared to CG1/0.5 and CG1/1
(65.60±0.81 % and 68.80±0.97 % respectively) (P<0.05). CG0.5/0.5 had significantly higher
subjective motility (54.00±1.88) as compared to Con (36.66±0.92)
Mitochondrial transmembrane potential of CG0.5/0.5 was significantly higher
(37.00±0.71 %) as compared to Con (25.33±1.28 %) (P<0.05). All the other treatment groups
also had higher mitochondrial transmembrane potential as compared to Con (P<0.05). In
groups of combination of cysteine and glutathione, CG0.5/0.5 showed significant difference
(37.00±0.71 %) as compared to CG1/1 and CG1/0.5 (29.00±1.00 % and 33.80±0.86 %)
CASA results showed that CG1/1 had significantly higher motility as compared to the
control. But the percentage of progressive spermatozoa was significantly higher in
CG0.5/0.5. VSL of group CG0.5/0.5 was significantly higher (53.33±2.90 %) as compared to
Con (45.10±0.50 %). However, VSL, VCL, ALH and BCF did not vary significantly among
groups. STR and LIN of group CG0.5/0.5 were significantly higher as compared to the
In conclusion, addition of cysteine and glutathione in tris citric egg yolk extender
improved the post thaw quality of Sahiwal bull spermatozoa. In case of additive effect of
cysteine and glutathione, CG0.5/0.5 showed higher plasma membrane integrity, acrosome
integrity, mitochondrial transmembrane potential, progressive and rapid spermatozoa as
compared to CG0.5/1, CG1/0.5 and CG1/1.
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Comparison Of Commercial Triladyl Extender With A Tris-Citric-Egg-Yolk (TCEY) Extender On Post-Thaw Semen Quality Of Nili Ravi Buffalo
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Cryopreservation of semen is the most important step for its usage in artificial insemination. Freezing of semen leads to a remarkable reduction in post-thaw semen quality. Therefore, selection of a better semen extender has always been considered priority that could serve as a good cryoprotectant.. Our semen production units (SPUs) have been using Tris based egg yolk semen extender since long time. Some modern SPUs like CEBG are using commercially available semen extenders for better post-thaw semen quality.
After collection pooled semen divided into two equal aliquots in separate sterilized test tubes and kept in water bath at 37 ºC. Semen was diluted with each of extender (TCEY and Triladyl) on the basis of sperm concentration (40x106sperm/ ml). Diluted semen was placed bottles and placed in safety cabinet cooled to 4 ºC over and equilibrated for 4 hrs. After equilibration semen was filled in 0.5 ml French straws (20x106sperm/ 0.5 ml). All semen straws placed in automatic freezer 4cm above liquid nitrogen surface in vapors for 10 minutes. Liquid Nitrogen vapors used in automatic programmable freezer to reduce temperature from 4 ºC to -180 ºC and then plunged into liquid nitrogen -196 ºC for freezing and was stored until analyzed. The experiment was repeated for seven times (replicates = 07)
CASA sperm motility parameter and kinematics were analyzed at Center of Excellence for Bovine Genetics (CEBG) Renala khurd District Okara. For further analysis frozen semen straws were brought to the Department of Theriogenology UVAS, Lahore. Effects of Triladyl and TCEY on post-thaw semen quality of the Nili Ravi buffalo semen were compared.
In Triladyl group, significantly (P<0.05) higher post-thaw motility (PTM %), Plasma membrane integrity (PMI, %),) DNA integrity (%), Live percentage was found. However, no significant (P<0.05) difference was found regarding NAR results between both groups. Sperm abnormalities were found significantly lower in Triladyl group as compared to TCEY group.
In overall assessment regarding and post-thaw CASA motility parameters, CASA motility, (PROG %), rapid (RAP %), medium (MED%), and slow (Slow, %) and sperm motility kinematics (VAP μm/sec), (VSL μm/sec), (VCL μm/sec), (ALH μm), (BCF HZ), (STR%) and (LIN%) Triladyl was found better than TCEY.
This was concluded that use of commercial semen extender Triladyl resulted in significantly better post-thaw semen quality as compared to Tris citric egg yolk (TCEY) extender.
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Comparison Of The Cryoprotective Effect Ofquailand Chicken Egg Yolk On The Freezability Of Nili-Ravi Buffalo Bull Spermatozoa
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Pakistan is one of the most important agricultural country. Livestock as a subsector of agriculture contributes 11.4% in the national economy and 56.3% in agriculture. Buffalo is honored as the black gold of Pakistan and Nili-Ravi is the most important animal in this regard. Approximately 60% of the total milk produced within a country comes from buffalo. Artificial insemination is considered to be the most important tool for the prompt genetic improvement of livestock. Mammalian spermatozoa undergo many structural and biochemical changes during cryopreservation which may leads to an impaired fertility. Buffalo bull spermatozoa are sensitive and more prone to damage than cattle. Various experiments have been conducted in order to improve the post thaw semen quality which includes supplementation of egg yolk from different bird species that tends to work as a cryoprotectent against cold shock. In the present study it is assumed that replacement of chicken egg yolk with quail egg yolk in cryodiluents improves the freezability of Nili-Ravi buffalo bull spermatozoa.
Three adult Nili-Ravi buffalo bulls without any clinical reproductive anomaly and kept at Semen Production Unit (SPU), Qadirabad District Sahiwal were used in this study. Semen from these three experimental bulls was collected twice a day twice a week by using an artificial vagina already maintained at 42 ºC. Each bull was subjected to a minimum of seven replicas. Both semen samples were collected on each collection day from each of the three experimental bulls with an interval of about ten minutes between the ejaculates. After initial evaluation for sperm motility and concentration, both ejaculates collected from the same bull at the same collection day were pooled and again evaluated for sperm cell concentration. Every pooled semen sample was then divided into five aliquots and extended with one of the five experimental extenders namely A, B, C, D, and E in order to maintain a final concentration of 40 million spermatozoa per 0.54 ml of diluted semen. Instantly after dilution motility percentile for each semen sample was recorded. After filling, open ends of straws were sealed with polyvinyl pyrolidine powder and allowed to cool and then it was stored at 4-5 ºC for equilibration. Finally the samples were frozen and stored in liquid nitrogen. On post thaw evaluation, motility, live/dead count, plasma membrane integrity, acrosomal integrity, and DNA integrity along with CASA evaluation parameters for motility characteristics were performed. Data were analyzed by means of two way ANOVAand comparison between means was done by using Duncan’s multiple range test. Results showed that post-extension motility and DNA integrity did not differ significantly between extender A and E. While post-thaw motility, plasmolemma intactness, acrosomal integrity, and viable sperm count was found to be higher in extender E compared to extender A. Similarly, CASA evaluation factors like progressive sperm cells motility, straight line velocity, straightness, linearity index, and the percentile of rapidly moving spermatozoa were also significantly different among extender E and A. While curvilinear velocity, and amplitude of lateral head displacement was higher in extender B that contained 5% QEY. So it was concluded that substitution of 20% CEY with 20% QEY in cryodiluents resulted in an improved post-thaw motility, viable sperm count, plasmolemma intactness, and sperm kinematics. Furthermore, it was seen that reduction in the concentration of QEY from 20% to 5% resulted in decreased sperm motility characteristics.
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Effect Of Β-Carotene And Tocopherol On Pregnancy Rate In Cidr Synchronized Nili-Ravi Buffaloes
Material type: Book ; Literary form:
Publisher: 2017 Dissertation note: Buffalo is of unique importance in livestock and dairy industry of Pakistan due to its high milk production and shares 65% of total milk production. Reproduction is important to get good production and profit in dairy sector. Reproduction in buffalo is compromised due to its small size ovaries, poor ovarian reserves and less pronounced estrus intensity, resulting as low fertility. Synchronization techniques including CIDR based protocols are well established in cows and getting popularity in buffaloes but with low results, comparatively. Therefore, some modifications are required based on physiology of estrus cycle in buffalo. This was hypothesized that additional injection of Dalmavital in CIDR base protocol will enhance the pregnancy rates and embryonic liability by minimizing the oxidative stress. Therefore, present study is conducted to evaluate the effect Dalmavital on estrus response, Estrus intensity, Pregnancy rates and embryonic losses in CIDR synchronized Nili-Ravi buffalo. For this, buffaloes were scanned ultrasonically for the reproductive tract evaluation. Reproductively sound buffaloes were selected and randomly allocated to one of the two treatment group. 86 buffaloes with normal reproductive tract were assigned in two groups 1; CIDR group (n = 43) and 2; CIDR+D (n = 43). AI was performed twice at 48 and 60 hours after CIDR removal. Estrus response (ER) did not differ significantly (P >0.05) in groups, CIDR and CIDR-D but estrus intensity (EI) was statistically significant (P<0.05) in treatment group. Pregnancy rates were also non-significant (P>0.05) in treatment and control group but improved comparatively in CIDR-D group (63% in CIDR-D group and 56% in CIDR group). Embryonic and fetal losses were also non-significant (P>0.05) between the control and treatment group. Results were also compared in cyclic and non-cyclic, Milking and dry, BCS and parity. Results were non-significant in milking and dry, BCS and Parity. Pregnancy rates were found different significantly (P<0.05) in cyclic and non-cyclic animals, when treatment
is ignored. From the present study it can be concluded that Dalmavital may have good effect on estrus intensity in CIDR synchronized Nili-Ravi buffalo.
Nili-Ravi buffalo is known as black gold of Pakistan. They produce about 2500 liters of milk with 6.5% butter fat. Despite of benefits, this breed is highly influenced with low reproductive activity that include prolonged pubertal period, poor exhibition of estrus, inadequate ovarian activity, long calving interval, high embryonic mortality and low fertility rate with artificial insemination. These factors reduce buffalo’s reproduction which leads to great economic losses. Therefore, there is dire need to address these problems and to orchestrate novel approaches to enhance the reproductive efficiency of buffalo. In cows, during last six to decades, researchers have considerably devised certain strategies to synchronize estrus with the help of prostaglandins, progestagens and estrogens. The advent of these hormones or synchronization protocols helped significantly in improving reproductive management. Moreover, the use of fixed time artificial insemination protocols resulted in acceptable fertility. However, these tools provide inconsistent results to manage reproduction in buffalo. Therefore, unprecedented approaches are required to facilitate and improve the buffalo reproduction.
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