- Email: [email protected]
Effect of vitamin C supplementation on freezability of Barbari buck semen
Accepted Manuscript Title: Effect of Vitamin C Supplementation on Freezability of Barbari Buck Semen Author: Chetna Gangwar S.D. Kharche Ravi Ranjan Satish Kumar A.K. Goel S.K. Jindal S.K. Agrawal PII: DOI: Reference:
S0921-4488(15)00255-2 http://dx.doi.org/doi:10.1016/j.smallrumres.2015.06.002 RUMIN 4958
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
31-1-2015 3-6-2015 5-6-2015
Please cite this article as: Gangwar, C., Kharche, S.D., Ranjan, R., Kumar, S., Goel, A.K., Jindal, S.K., Agrawal, S.K.,Effect of Vitamin C Supplementation on Freezability of Barbari Buck Semen., Small Ruminant Research (2015), http://dx.doi.org/10.1016/j.smallrumres.2015.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights :
Ac ce p
te
d
M
an
us
cr
ip t
1. Supplementation of vitamin C improves the physico-morphological characteristics of buck spermatozoa. 2. Supplementation of vitamin C in buck semen dilutor improves the viability, motility as well as acrosomal integrity of the sperms 3. Vitamin C can be used as an effective antioxidant to improve the freeze ability of buck semen. 4. Very few literature is available on use of Vitamin C as an antioxidant in buck semen dilutor.
1 Page 1 of 11
Effect of Vitamin C Supplementation on Freezability of Barbari Buck Semen. Chetna Gangwar, S.D. Kharche*, Ravi Ranjan, Satish Kumar, A.K. Goel, S.K. Jindal, S.K. Agrawal
ICAR-CIRG, Makhdoom P.O. Farah-28112, Mathura (UP) INDIA
Corresponding
author. Email address: [email protected]
ip t
Mobile: +919897987074, Fax: +915652763246 Abstract
cr
Thirty six ejaculates from 6 adult Barbari bucks (2-4 years old) maintained at C.I.R.G under semi intensive management system were used to find out the freezability of buck semen at
us
different levels of vitamin C (0.0µM, 45.42 µM , 56.78 µM , 68.13 µM) by conventional method of freezing. The ejaculates were collected twice at weekly intervals by artificial vagina. The semen samples were diluted with Tris- Citric acid fructose diluents having 10% (v/v) egg yolk
an
and 6% (v/v) glycerol as cry protectant agent. The semen samples were extended to maintain sperm concentration approximately 100-120 million per dose. Filling and sealing of straws were
M
done at 50 C in cold handling cabinet after 4 hr of equilibration period, then straws were vapor frozen for 10 minutes above 2 cm of liquid nitrogen and finally stored into liquid nitrogen
d
container. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling test were conducted to check the freezability. Post thaw motility, live sperm intergrity
and hypo osmotic
te
count, acrosomal
swelling positive spermatozoa differed
significantly among groups and they were the highest in 1% group. The result indicated that
Ac ce p
vitamin C at the level of 56.78 µM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen. Keywords : Acrosomal integrity, caprine, freezing, semen, Vitamin C.
1. Introduction
An acceptable fertility of cryopreserved semen has always been a challenge and poor fertility of cryopreserved semen still remains a problem for the goat breeders and scientists engaged in cryopreservation of buck semen. Buck sperm cells contain a high proportion of polyunsaturated fatty acids, and therefore they are particularly susceptible to peroxidative damage (Asadpouret al.,2011).The production of Reactive Oxygen Species (ROS) is a normal physiological event in various organs and seminal plasma. However, the imbalance between ROS production and
2 Page 2 of 11
scavenging system can cause structural and functional damage to spermatozoa. To counteract the destructive effects of Reactive Oxygen Species (ROS), seminal plasma has an antioxidant system that seems to be very relevant to the protection of sperm (Alvarez and Storey, 1982). Their vulnerability increases especially following cryopreservation with a subsequent loss in
ip t
membrane integrity, impaired cell function, decreased motility and the fertilizing capability of the spermatozoa. Reactive oxygen species i.e., super oxide, hydroxyl radical and peroxide are
cr
produced by spermatozoa, immune cells in semen and also environmental factors (especially UV) during cryopreservation stages (Alvarez and Storey, 1982; Aitken and Fisher, 1994; Aitken
us
and Baker, 2004). The antioxidant capacity of spermatozoa is very limited to protect itself against ROS, compared with somatic cells. To control the level of ROS and promote motility and survival of sperm, numerous antioxidants have proven beneficial effect on improving buck
an
fertility (Azawi and Hussein, 2013). Vitamin C (ascorbic acid, ascorbate) is naturally present in seminal plasma to scavenge free radicals and its presence also assists various other mechanisms
M
in decreasing numerous disruptive free radical processes, including lipid peroxidation (Anane and Creppy, 2001).
d
Vitamin C represents the major water-soluble antioxidant in plasma. Ascorbic acid is required in vivo as a cofactor for at least eight enzymes and can also act as an antioxidant by reacting with
te
free radicals (Michael et al., 2008). The concentration of vitamin C in seminal plasma is 10 times greater than in blood plasma (364 vs. 40 μmol L-1).The addition of vitamin C in an extender
Ac ce p
could possibly improve sperm function by reducing cell damage through its continuous radicalscavenging action. However, a lot of studies were carried out on addition of different antioxidants in extenders to protect spermatozoa against detrimental effects of Reactive Oxygen Species (ROS) (Uysal and Bucak, 2007; Bucaket al., 2008) but there is limited information regarding the effects of the vitamin C in different extenders on buck semen quality. Therefore, the objective of this study was, to test the hypothesis that different levels of vitamin C might effectively protect buck semen from oxidative damage during cryopreservation in TRIS extender resulting in higher post thaw sperm viability, motility and fertility. 2. Materials and Methods 2.1 Source of animal and its management: The six adult Barbari bucks, of 2-4 years old, were selected for the study. The bucks were kept under semi-intensive system of management at
3 Page 3 of 11
Indian Council of Agricultural Research-Central Institutes for Research on Goats (ICAR-CIRG) Makhdoom. 2.2 Semen collection and evaluation: Thirty six ejaculates were collected from six adult Barbari bucks at twice a week interval using artificial vagina. The semen was collected from February,
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15th to March, 15th 2014. Immediately after collection, the volume, colour, consistency and mass motility of ejaculates were assessed. Semen samples having mass motility more than +4 were
cr
used for further study Ranjan et al. (2014).
us
2.3 Dilutor and dilution: Semen samples were extended with Tris-Citrate-Fructose yolk diluents (Tris-3.604g; Citric acid-1.902g; Fructose- 1g; Streptomycin- 100mg; Penicilline-100000 I.U.; Triple distilled water – 100 ml; pH- 6.8-6.9) having 10% (v/v) egg yolk and 6% (v/v) glycerol.
an
The samples were diluted to maintain the sperm concentration 100-120 million per dose or 400 million sperm/ml.
M
2.4 Vitamin C concentration in dilutor: Semen was diluted with Tris-Citrate-Fructose yolk at 50 C for 4 h before being frozen.
d
dilutor having 0%, 0.8%, 1% and 1.2 % vitamin C and diluted semen samples were equilibrated
te
2.5 Semen cryopreservation: After equilibration period of 4 hours semen was filled in 0.25 ml straws at 50 C and straws were sealed with polyvinyl alcohol powder. After filling and sealing,
Ac ce p
straws were vapour frozen in vapours of liquid nitrogen by keeping them 2-4 cm above the liquid nitrogen for 10 min. Finally straws were plunged into liquid nitrogen and stored into liquid nitrogen container.
2.6 Live and dead sperm: This was estimated as per standard staining procedure as described by Hancock (1951). The staining solution contains Eosin (0.67 g /100 mL) and Nigrosin (5 g /100 mL) and water to make the volume 100 ml. A drop of diluted semen mixed with eight drops of stain was incubated at 30°C for 2 min. Then smears made on pre-warmed slides were allowed to dry at 30°C. The excess stain was washed off in running tap water. The slide was then immersed briefly in ethanol to remove water. Then mounted smear was observed under 400× objective lens of the phase contrast microscope. Sperm abnormalities were also counted with same staining technique. Approximately 400 sperm were counted.
4 Page 4 of 11
2.7 Sperm staining and evaluation: Diluted semen (10 µl) was placed on a clean grease free pre warmed slide (370 C) with cover slip and observed under 40 X magnification of phase contrast microscogpe for assessing the progressive motility. The average values of two experts were considered for calculating the progressive motility. Giemsa stain was used to assess the swelling test was carried out as described by Gangwar et al. (2014).
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acrosomal integrity of frozen thawed buck spermatozoa as per Watson (1975). Hypo osmotic
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2.8 Statistical analysis: Data were analyzed by General Linear Model of SPSS 16 data analysis software. The factorial model included the effect of vitamin C as independent variables and
us
percent post thawed motility and live sperm count, abnormalities, acrosome intact sperm and hypo osmotic swelling positive sperm as dependent variables. Post Hoc Test Duncan LSD T3
an
was conducted to know the significant difference between different variables at P<0.05. Homogeneity test was also conducted to assign different superscript to variables based on
M
significant difference at P<0.05 between different variables. 3. Result and Discussion:
d
Results of the present experiment are shown in detail in table (1). In the present study the effects
te
of vitamin C as an antioxidant in buck semen diluter on post thaw semen quality was evaluated. The percentages of motile spermatozoa live and dead spermatozoa, hypo osmotic swelled
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spermatozoa and acrosome integrity for each vitamin C concentration were averaged. Results indicated that the progressive motility, live sperm count, abnormality, acrosomal integrity and hypo osmotic swelling positive spermatozoa (mean±SE) were 84.05±1.23, 89.71±0.94, 2.86±0.64, 91.57±0.91 and 75.43±1.03 respectively in fresh semen. Ravi Ranjan et al. (2009, 2014 and 2015) studied the above parameters at different egg yolk levels and equilibration period in Marwari and Jamunapari goats. The post thaw quality of semen frozen in dilutor having different percent of vitamin C as an antioxidant was found suitable for storage and further use in artificial insemination programme. In the present study 45.42 µM and 56.78 µM of vitamin C level were found suitable and have positive effect on post thaw motility and viability. Addition of vitamin C at the level 45.42 µM and 56.78 µM increases the post thaw motility 47.29±1.16% and 53.54±1.29% respectively and these values are significantly (p<0.05) higher than control group (41.04±1.08%). Similarly addition of vitamin C at the level of 45.42 µM and 56.78 µM also increases the live percentage count 61.87±1.43% and 68.21±1.37% respectively and again 5 Page 5 of 11
these values are significantly (p<0.05) higher than control group (56.92±1.49%). But 56.78 µM of vitamin C has significantly (p<0.05) improved the acrosomal integrity (83.62±1.15%) and hypo osmotic swelling positive spermatozoa (53.92±1.93%). In the present study 56.78 µM vitamin C level in diluter significantly (P<0.05) improves the post thaw motility, live percentage,
ip t
acrosomal intact spermatozoa and hypo osmotic swelling positive spermatozoa and the values were (mean ± SE) 53.54±1.29, 68.21±1.37, 83.62±1.15 and 53.92±1.93 respectively. Hence, the
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56.78 µM level of vitamin C is optimum as an antioxidant for buck semen diluter. However, 45.42 µM vitamin C also significantly (P<0.05) improves the post thaw motility (47.29 ±1.16)
us
and live sperm count (61.87 ±1.43). However there was no effect on acrosomal integrity and hypo osmotic swelling positive spermatozoa. While 68.13 µM level of vitamin C had no
an
significant effect on post thaw semen quality.
Caprine spermatozoa are highly sensitive to lipid peroxidation, which occurs as a result of the
M
oxidation of membrane lipids by superoxide, hydrogen peroxide and hydroxyl radicals. Spontaneous lipid peroxidation of the spermatozoa membranes destroys the structure of the lipid matrix. The attacks of the reactive oxygen species ultimately lead to the impairment of sperm
d
function, such as sperm motility, functional membrane integrity, leakage of intracellular enzymes
te
and damage to the sperm DNA through the oxidative stress (Alvarez and Storey, 1989). Ascorbic acid is water soluble natural antioxidant present in epididymal fluid and seminal plasma of many
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species Azawi et al. (2013).
Earlier Sonmez and Demirci (2003) found that the intramuscular injection of ascorbic acid for 30 days increased the semen quality of normal rams and increased its concentration in blood and seminal plasma. Fazeli et al. (2010) also reported that the subcutaneous vitamin C injection for 90 days increased sperm motility and the effect was evident up to 30 days after the cessation of injections. However, the percentage of live sperm and mass motility showed similar trends with control animals. In the present study 45.42 µM and 56.78 µM of vitamin C level were found suitable and have positive effect on post thaw motility and viability. Azawi and Hussein (2013) reported that the addition of vitamin C to semen preservation media could improve longevity and quality of cooled sperm in Awassi ram semen. Similar to our findings Azawi and Hussein (2013) reported that motility was improved from 35.9±1.9% to 54.7± 1.1% in vitamin C added diluter and live percentage was also improved from 40.9± 1.8% to 6 Page 6 of 11
59.7±1.3%. In addition, Asadpouret al. (2011) also reported positive effect of vitamin C (1 mM) in Citrate egg yolk (CEY) extender on lipid peroxidation but contrary to our findings supplementing Tris egg yolk (TEY) extender with different concentration of vitamin C produced no effect on the reduction of lipid peroxidation compared with the control group. These results
ip t
may be explained based on the fact that vitamin C protects the spermatozoa by preventing from endogenous oxidative DNA and membrane damages. It is also reported that vitamin C works by
cr
scavenging superoxide anions and singlet oxygen and can protect the plasma membrane from peroxidative damage. Findings of the present study suggested that ascorbic acid might be needed
us
to protect sperm against reactive oxygen species. In addition, the results of this study show that addition of vitamin C into TRIS extender suppressed deleterious effect of ROS on buck sperm. Similar results have been reported by other researchers (Sarloset al., 2002; Arabi and Seidaie,
an
2008). They have showed that vitamin C supplementation reduced ROS generation. The present findings are contrary with the observation made by Aurich et al. (1997) and Ball et al. (2001),
M
who did not detect any positive effect from the addition of vitamin C to cooled equine spermatozoa. Wittayaratet al. (2012) reported that supplementation of 0.5 mM vitamin C yielded
d
the highest percentages of sperm motility and viability in chilled semen; however, there was no beneficial effect on the plasma membrane and acrosomal integrity of the spermatozoa. Our
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Present findings are in agreement to those reported by Asghari et al. (1999), Maia et al. (2009) and (2010) in ram semen and Singh et al. (1996) on buffalo semen. The beneficial effect of (1999).
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adding vitamin C to diluted ram semen was in accordance with the results obtained by Asghari
Higher concentrations of vitamin C (2.5 mM) proved harmful to sperm motility in frozenthawed bull semen (Beconiet al., 1993). In the present study motility was declined at 1.2% level as compared to 1% level of vitamin C. Similarly Sonmez et al. (2005) also reported that addition of vitamin C in dose of more than 2mg per ml of diluted semen has adverse effect on ram semen quality and spermatic motility was reduced during their conservation in liquid state. This reduction in motility might be due to the sour nature (pH 2) of the vitamin C which reduces the pH of diluter due to its acidic property. 4. Conclusion: The result indicated that vitamin C at the level of 56.78 µM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen. 7 Page 7 of 11
5. Acknowledgement: The authors are thankful to the ICAR-CIRG for providing funding and lab facility to perform the experiments. 6. References balance of benefit and risk. Bioessays. 16, 259–267.
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Aitken, J. R., Fisher, H. 1994. Reactive oxygen species generation and human spermatozoa: the Aitken, R. J., Baker, M. A. 2004. Oxidative stress and male reproductive biology.Reprod. Fertil.
cr
and Dev. 16, 581-588.
Alvarez, J. G., Storey, B. T. 1982. Spontaneous lipid peroxidation in rabbit epididymal
us
spermatozoa: its effect on sperm motility. BiolReprod. 27, 1102–1108.
Alvarez, J. G., Storey, B. T. 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.Gamete
an
Res.23, 77-90.
Anane, R. and Creppy, E. E. 2001. Lipid peroxidation as pathway of aluminum cytotoxicity in
M
human skin fibroblast cultures: prevention by superoxide dismutase and catalase and vitamins E and C. Hum Exp Toxicol 20, 477–481.
d
Arabi, M., Seidaie, S. R. 2008.Assessment of motility and membrane peroxidation of bull Shahrekord. 2, 39-46.
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spermatozoa in the presence of different concentration of vitamin C. Vet Med J Asadpour, R., Jafari, R., Nasrabadi, H. T. 2011. Influence of added vitamin C and vitamin E on
Ac ce p
frozen-thawed bovine sperm cryopreserved in citrate and tris-based extenders.Vet. Res. Forum. 2 (1), 37 – 44.
Asghari, S. R. 1999. Reproductive characteristics of Ghezel and Mehraban rams and effect of vitamin C on semen parameters and fertility.MSc. Thesis. Shiraz University, Iran 155. Aurich, J. E., Schonherr, U., Hoppe, H. 1997. Effects of antioxidants on motility and membrane integrity of chilled stored stallion semen.Theriogenology. 48, 185–192. Azawi, O. I., Hussein, E. K. 2013. Effect of vitamins C or E supplementation to Tris diluent on the semen quality of Awassi rams preserved at 5 ˚C. Vet. Res. Forum. 4 (3), 157 – 160. Ball, B. A., Medina, V., Gravance, C. G. 2001. Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5°C.Theriogenology. 56, 577–589.
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Beconi, M. T., Francia, C. R., Mora, N. G.1993. Effect of natural antioxidants in frozen bovine semen preservation.Theriogenology. 40, 841-851. Bucak, M. N., Atessahin, A., Yuce, A. 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freez-thawing process.Small Rum.Res.75,128-134.
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Fazeli, P., Zamiri, M. J., Farshad, A., Khalili, B. 2010. Effects of vitamin C on testicular and seminal characteristics of Markhoz goats. Iran. J. of Vet. Res. Shiraz University. Vol.11,
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No. 3, Ser. No. 32, pp 267-372.
Gangwar, C., Ranjan R., Kumar, Satish., Kharche, S. D., Goel, A. K., Ramachandran, N., Jindal,
us
S. K. 2014. Use of chelating agent for optimum post thaw quality of buck semen. Ind. J. of Anim. Sci. 84(8), 839–841.
Hancock, J. L. 1951. A staining technique for the study of temperature shock in semen. Nature
an
(London). 167,323.
Maia, M. S., Bicudo, S. D., Azevedo, H. C. 2009. Motility and viability of ram sperm
M
cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants. Small Rumin. Res. 85, 85-90.
d
Maia, M. S., Bicudo, S. D., Sicherle, C. C. 2010. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with anti-oxidants.
te
Small Rumin. Res. 122, 118-123.
Michael, A., Alexopoulos, C., Pontiki, E. 2008. Quality and reactive oxygen species of extended
Ac ce p
canine semen after vitamin C supplementation.Theriogenology. 70, 827–835. Ranjan R,, Goel, A.K., Ramachandran, N., Kharche S.D. and Jindal, S.K. 2015. Effect of egg yolk levels and equilibration periods on freezability of jamunapari buck semen. The Indian Journal of Small Ruminants 21: 32-36. Ranjan R., Ramachandran N., Jindal S. K., Sinha N. K., Goel A. K., Kharche S. D. and Sikarwar A. K. S. 2009. Effect of egg yolk levels on keeping quality of Marawari buck semen at refrigeration temperature. Indian Journal of Animal Science. 79:10-13. Ranjan, R., Goel A. K., Kharche, S. D., Ramachandran, N., Gangwar, Chetna and Jindal, S. K., (2014). Comparison between normal and dual staining technique for evaluating acrosome status and viability in frozen thawed buck spermatozoa. The Ind. J. of Small Rumin. 20(2): 50-53.
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Sarlos, P., Molnar, A., Kokai, M. 2002. Comparative evaluation of the effect of antioxidants in the conservation of ram semen.Acta Vet Hun.50, 235-245. Singh, B., Chand, D., Singh, P. 1996. Effect of vitamin C addition in the diluent on the quality of deep frozen Murrah buffalo bull (Bubalus bubalis) semen.Int J Anim Sci.11, 131-132. quality in rams. J. Firat Univ. Health Vet.Sci.17, 195-201.
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Sonmez, M ., Demirci, E. 2003. The effect of intramuscular vitamin C administration on semen
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Sonmez, M., Turk, G., Yuce, A. 2005. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation, and testosterone levels in male Wistar rats. Theriogenology.
us
63, 2063-72.
Uysal, O., Bucak, M. N. 2007. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen.Acta vet. Brno.76, 383-390.
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Watson., P. F. 1975. Use of Giemsa stain to detect changes in acrosomes of frozen ram spermatozoa. Vet. Rec. 97, 12–15.
M
Wittayarat, M., Kimura, T., Kodama, R., Namula, Z., Chatdarong, K., Techakumphu, M., Sato, Y., Taniguchi, M., Otoi, T. 2012. Long-term preservation of chilled canine semen using
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te
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vitamin C in combination with green tea polyphenol. Cryo Letters. 33, 318-26.
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Table 1 Effect of different concentration of vitamin C (L-Ascorbic acid) in semen dilutor on post thaw quality of Barbari buck semen
Motility
Live
Dead
Abnormal
0.0 (control)
41.04±1.08c
56.92±1.49c
43.08±1.49a
3.58±0.61a
0.8
47.29±1.16b
61.87±1.43b
37.29±1.75b
3.12±0.58a
78.67±1.36b
46.58±1.41b
1.0
53.54±1.29a
68.21±1.37a
32.62±1.32c
2.83±0.59a
83.62±1.15a
53.92±1.93a
1.2
44.17±1.89bc
59.96±1.71bc
40.46±1.68ab
3.42±0.60a
76.75±1.76b
44.25±2.24b
Fresh semen
84.05±1.23
89.71±0.94
10.76±0.92
2.86±0.64
91.57±0.91
75.43±1.03
us
an
M
76.67±1.43b
cr
(Miligram %)
HOS
41.71±1.37b
Ac ce p
te
d
Acrosome
ip t
Concentration
11 Page 11 of 11
S0921-4488(15)00255-2 http://dx.doi.org/doi:10.1016/j.smallrumres.2015.06.002 RUMIN 4958
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
31-1-2015 3-6-2015 5-6-2015
Please cite this article as: Gangwar, C., Kharche, S.D., Ranjan, R., Kumar, S., Goel, A.K., Jindal, S.K., Agrawal, S.K.,Effect of Vitamin C Supplementation on Freezability of Barbari Buck Semen., Small Ruminant Research (2015), http://dx.doi.org/10.1016/j.smallrumres.2015.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights :
Ac ce p
te
d
M
an
us
cr
ip t
1. Supplementation of vitamin C improves the physico-morphological characteristics of buck spermatozoa. 2. Supplementation of vitamin C in buck semen dilutor improves the viability, motility as well as acrosomal integrity of the sperms 3. Vitamin C can be used as an effective antioxidant to improve the freeze ability of buck semen. 4. Very few literature is available on use of Vitamin C as an antioxidant in buck semen dilutor.
1 Page 1 of 11
Effect of Vitamin C Supplementation on Freezability of Barbari Buck Semen. Chetna Gangwar, S.D. Kharche*, Ravi Ranjan, Satish Kumar, A.K. Goel, S.K. Jindal, S.K. Agrawal
ICAR-CIRG, Makhdoom P.O. Farah-28112, Mathura (UP) INDIA
Corresponding
author. Email address: [email protected]
ip t
Mobile: +919897987074, Fax: +915652763246 Abstract
cr
Thirty six ejaculates from 6 adult Barbari bucks (2-4 years old) maintained at C.I.R.G under semi intensive management system were used to find out the freezability of buck semen at
us
different levels of vitamin C (0.0µM, 45.42 µM , 56.78 µM , 68.13 µM) by conventional method of freezing. The ejaculates were collected twice at weekly intervals by artificial vagina. The semen samples were diluted with Tris- Citric acid fructose diluents having 10% (v/v) egg yolk
an
and 6% (v/v) glycerol as cry protectant agent. The semen samples were extended to maintain sperm concentration approximately 100-120 million per dose. Filling and sealing of straws were
M
done at 50 C in cold handling cabinet after 4 hr of equilibration period, then straws were vapor frozen for 10 minutes above 2 cm of liquid nitrogen and finally stored into liquid nitrogen
d
container. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling test were conducted to check the freezability. Post thaw motility, live sperm intergrity
and hypo osmotic
te
count, acrosomal
swelling positive spermatozoa differed
significantly among groups and they were the highest in 1% group. The result indicated that
Ac ce p
vitamin C at the level of 56.78 µM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen. Keywords : Acrosomal integrity, caprine, freezing, semen, Vitamin C.
1. Introduction
An acceptable fertility of cryopreserved semen has always been a challenge and poor fertility of cryopreserved semen still remains a problem for the goat breeders and scientists engaged in cryopreservation of buck semen. Buck sperm cells contain a high proportion of polyunsaturated fatty acids, and therefore they are particularly susceptible to peroxidative damage (Asadpouret al.,2011).The production of Reactive Oxygen Species (ROS) is a normal physiological event in various organs and seminal plasma. However, the imbalance between ROS production and
2 Page 2 of 11
scavenging system can cause structural and functional damage to spermatozoa. To counteract the destructive effects of Reactive Oxygen Species (ROS), seminal plasma has an antioxidant system that seems to be very relevant to the protection of sperm (Alvarez and Storey, 1982). Their vulnerability increases especially following cryopreservation with a subsequent loss in
ip t
membrane integrity, impaired cell function, decreased motility and the fertilizing capability of the spermatozoa. Reactive oxygen species i.e., super oxide, hydroxyl radical and peroxide are
cr
produced by spermatozoa, immune cells in semen and also environmental factors (especially UV) during cryopreservation stages (Alvarez and Storey, 1982; Aitken and Fisher, 1994; Aitken
us
and Baker, 2004). The antioxidant capacity of spermatozoa is very limited to protect itself against ROS, compared with somatic cells. To control the level of ROS and promote motility and survival of sperm, numerous antioxidants have proven beneficial effect on improving buck
an
fertility (Azawi and Hussein, 2013). Vitamin C (ascorbic acid, ascorbate) is naturally present in seminal plasma to scavenge free radicals and its presence also assists various other mechanisms
M
in decreasing numerous disruptive free radical processes, including lipid peroxidation (Anane and Creppy, 2001).
d
Vitamin C represents the major water-soluble antioxidant in plasma. Ascorbic acid is required in vivo as a cofactor for at least eight enzymes and can also act as an antioxidant by reacting with
te
free radicals (Michael et al., 2008). The concentration of vitamin C in seminal plasma is 10 times greater than in blood plasma (364 vs. 40 μmol L-1).The addition of vitamin C in an extender
Ac ce p
could possibly improve sperm function by reducing cell damage through its continuous radicalscavenging action. However, a lot of studies were carried out on addition of different antioxidants in extenders to protect spermatozoa against detrimental effects of Reactive Oxygen Species (ROS) (Uysal and Bucak, 2007; Bucaket al., 2008) but there is limited information regarding the effects of the vitamin C in different extenders on buck semen quality. Therefore, the objective of this study was, to test the hypothesis that different levels of vitamin C might effectively protect buck semen from oxidative damage during cryopreservation in TRIS extender resulting in higher post thaw sperm viability, motility and fertility. 2. Materials and Methods 2.1 Source of animal and its management: The six adult Barbari bucks, of 2-4 years old, were selected for the study. The bucks were kept under semi-intensive system of management at
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Indian Council of Agricultural Research-Central Institutes for Research on Goats (ICAR-CIRG) Makhdoom. 2.2 Semen collection and evaluation: Thirty six ejaculates were collected from six adult Barbari bucks at twice a week interval using artificial vagina. The semen was collected from February,
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15th to March, 15th 2014. Immediately after collection, the volume, colour, consistency and mass motility of ejaculates were assessed. Semen samples having mass motility more than +4 were
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used for further study Ranjan et al. (2014).
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2.3 Dilutor and dilution: Semen samples were extended with Tris-Citrate-Fructose yolk diluents (Tris-3.604g; Citric acid-1.902g; Fructose- 1g; Streptomycin- 100mg; Penicilline-100000 I.U.; Triple distilled water – 100 ml; pH- 6.8-6.9) having 10% (v/v) egg yolk and 6% (v/v) glycerol.
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The samples were diluted to maintain the sperm concentration 100-120 million per dose or 400 million sperm/ml.
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2.4 Vitamin C concentration in dilutor: Semen was diluted with Tris-Citrate-Fructose yolk at 50 C for 4 h before being frozen.
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dilutor having 0%, 0.8%, 1% and 1.2 % vitamin C and diluted semen samples were equilibrated
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2.5 Semen cryopreservation: After equilibration period of 4 hours semen was filled in 0.25 ml straws at 50 C and straws were sealed with polyvinyl alcohol powder. After filling and sealing,
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straws were vapour frozen in vapours of liquid nitrogen by keeping them 2-4 cm above the liquid nitrogen for 10 min. Finally straws were plunged into liquid nitrogen and stored into liquid nitrogen container.
2.6 Live and dead sperm: This was estimated as per standard staining procedure as described by Hancock (1951). The staining solution contains Eosin (0.67 g /100 mL) and Nigrosin (5 g /100 mL) and water to make the volume 100 ml. A drop of diluted semen mixed with eight drops of stain was incubated at 30°C for 2 min. Then smears made on pre-warmed slides were allowed to dry at 30°C. The excess stain was washed off in running tap water. The slide was then immersed briefly in ethanol to remove water. Then mounted smear was observed under 400× objective lens of the phase contrast microscope. Sperm abnormalities were also counted with same staining technique. Approximately 400 sperm were counted.
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2.7 Sperm staining and evaluation: Diluted semen (10 µl) was placed on a clean grease free pre warmed slide (370 C) with cover slip and observed under 40 X magnification of phase contrast microscogpe for assessing the progressive motility. The average values of two experts were considered for calculating the progressive motility. Giemsa stain was used to assess the swelling test was carried out as described by Gangwar et al. (2014).
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acrosomal integrity of frozen thawed buck spermatozoa as per Watson (1975). Hypo osmotic
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2.8 Statistical analysis: Data were analyzed by General Linear Model of SPSS 16 data analysis software. The factorial model included the effect of vitamin C as independent variables and
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percent post thawed motility and live sperm count, abnormalities, acrosome intact sperm and hypo osmotic swelling positive sperm as dependent variables. Post Hoc Test Duncan LSD T3
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was conducted to know the significant difference between different variables at P<0.05. Homogeneity test was also conducted to assign different superscript to variables based on
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significant difference at P<0.05 between different variables. 3. Result and Discussion:
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Results of the present experiment are shown in detail in table (1). In the present study the effects
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of vitamin C as an antioxidant in buck semen diluter on post thaw semen quality was evaluated. The percentages of motile spermatozoa live and dead spermatozoa, hypo osmotic swelled
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spermatozoa and acrosome integrity for each vitamin C concentration were averaged. Results indicated that the progressive motility, live sperm count, abnormality, acrosomal integrity and hypo osmotic swelling positive spermatozoa (mean±SE) were 84.05±1.23, 89.71±0.94, 2.86±0.64, 91.57±0.91 and 75.43±1.03 respectively in fresh semen. Ravi Ranjan et al. (2009, 2014 and 2015) studied the above parameters at different egg yolk levels and equilibration period in Marwari and Jamunapari goats. The post thaw quality of semen frozen in dilutor having different percent of vitamin C as an antioxidant was found suitable for storage and further use in artificial insemination programme. In the present study 45.42 µM and 56.78 µM of vitamin C level were found suitable and have positive effect on post thaw motility and viability. Addition of vitamin C at the level 45.42 µM and 56.78 µM increases the post thaw motility 47.29±1.16% and 53.54±1.29% respectively and these values are significantly (p<0.05) higher than control group (41.04±1.08%). Similarly addition of vitamin C at the level of 45.42 µM and 56.78 µM also increases the live percentage count 61.87±1.43% and 68.21±1.37% respectively and again 5 Page 5 of 11
these values are significantly (p<0.05) higher than control group (56.92±1.49%). But 56.78 µM of vitamin C has significantly (p<0.05) improved the acrosomal integrity (83.62±1.15%) and hypo osmotic swelling positive spermatozoa (53.92±1.93%). In the present study 56.78 µM vitamin C level in diluter significantly (P<0.05) improves the post thaw motility, live percentage,
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acrosomal intact spermatozoa and hypo osmotic swelling positive spermatozoa and the values were (mean ± SE) 53.54±1.29, 68.21±1.37, 83.62±1.15 and 53.92±1.93 respectively. Hence, the
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56.78 µM level of vitamin C is optimum as an antioxidant for buck semen diluter. However, 45.42 µM vitamin C also significantly (P<0.05) improves the post thaw motility (47.29 ±1.16)
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and live sperm count (61.87 ±1.43). However there was no effect on acrosomal integrity and hypo osmotic swelling positive spermatozoa. While 68.13 µM level of vitamin C had no
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significant effect on post thaw semen quality.
Caprine spermatozoa are highly sensitive to lipid peroxidation, which occurs as a result of the
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oxidation of membrane lipids by superoxide, hydrogen peroxide and hydroxyl radicals. Spontaneous lipid peroxidation of the spermatozoa membranes destroys the structure of the lipid matrix. The attacks of the reactive oxygen species ultimately lead to the impairment of sperm
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function, such as sperm motility, functional membrane integrity, leakage of intracellular enzymes
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and damage to the sperm DNA through the oxidative stress (Alvarez and Storey, 1989). Ascorbic acid is water soluble natural antioxidant present in epididymal fluid and seminal plasma of many
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species Azawi et al. (2013).
Earlier Sonmez and Demirci (2003) found that the intramuscular injection of ascorbic acid for 30 days increased the semen quality of normal rams and increased its concentration in blood and seminal plasma. Fazeli et al. (2010) also reported that the subcutaneous vitamin C injection for 90 days increased sperm motility and the effect was evident up to 30 days after the cessation of injections. However, the percentage of live sperm and mass motility showed similar trends with control animals. In the present study 45.42 µM and 56.78 µM of vitamin C level were found suitable and have positive effect on post thaw motility and viability. Azawi and Hussein (2013) reported that the addition of vitamin C to semen preservation media could improve longevity and quality of cooled sperm in Awassi ram semen. Similar to our findings Azawi and Hussein (2013) reported that motility was improved from 35.9±1.9% to 54.7± 1.1% in vitamin C added diluter and live percentage was also improved from 40.9± 1.8% to 6 Page 6 of 11
59.7±1.3%. In addition, Asadpouret al. (2011) also reported positive effect of vitamin C (1 mM) in Citrate egg yolk (CEY) extender on lipid peroxidation but contrary to our findings supplementing Tris egg yolk (TEY) extender with different concentration of vitamin C produced no effect on the reduction of lipid peroxidation compared with the control group. These results
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may be explained based on the fact that vitamin C protects the spermatozoa by preventing from endogenous oxidative DNA and membrane damages. It is also reported that vitamin C works by
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scavenging superoxide anions and singlet oxygen and can protect the plasma membrane from peroxidative damage. Findings of the present study suggested that ascorbic acid might be needed
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to protect sperm against reactive oxygen species. In addition, the results of this study show that addition of vitamin C into TRIS extender suppressed deleterious effect of ROS on buck sperm. Similar results have been reported by other researchers (Sarloset al., 2002; Arabi and Seidaie,
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2008). They have showed that vitamin C supplementation reduced ROS generation. The present findings are contrary with the observation made by Aurich et al. (1997) and Ball et al. (2001),
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who did not detect any positive effect from the addition of vitamin C to cooled equine spermatozoa. Wittayaratet al. (2012) reported that supplementation of 0.5 mM vitamin C yielded
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the highest percentages of sperm motility and viability in chilled semen; however, there was no beneficial effect on the plasma membrane and acrosomal integrity of the spermatozoa. Our
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Present findings are in agreement to those reported by Asghari et al. (1999), Maia et al. (2009) and (2010) in ram semen and Singh et al. (1996) on buffalo semen. The beneficial effect of (1999).
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adding vitamin C to diluted ram semen was in accordance with the results obtained by Asghari
Higher concentrations of vitamin C (2.5 mM) proved harmful to sperm motility in frozenthawed bull semen (Beconiet al., 1993). In the present study motility was declined at 1.2% level as compared to 1% level of vitamin C. Similarly Sonmez et al. (2005) also reported that addition of vitamin C in dose of more than 2mg per ml of diluted semen has adverse effect on ram semen quality and spermatic motility was reduced during their conservation in liquid state. This reduction in motility might be due to the sour nature (pH 2) of the vitamin C which reduces the pH of diluter due to its acidic property. 4. Conclusion: The result indicated that vitamin C at the level of 56.78 µM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen. 7 Page 7 of 11
5. Acknowledgement: The authors are thankful to the ICAR-CIRG for providing funding and lab facility to perform the experiments. 6. References balance of benefit and risk. Bioessays. 16, 259–267.
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Aitken, J. R., Fisher, H. 1994. Reactive oxygen species generation and human spermatozoa: the Aitken, R. J., Baker, M. A. 2004. Oxidative stress and male reproductive biology.Reprod. Fertil.
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and Dev. 16, 581-588.
Alvarez, J. G., Storey, B. T. 1982. Spontaneous lipid peroxidation in rabbit epididymal
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spermatozoa: its effect on sperm motility. BiolReprod. 27, 1102–1108.
Alvarez, J. G., Storey, B. T. 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.Gamete
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Res.23, 77-90.
Anane, R. and Creppy, E. E. 2001. Lipid peroxidation as pathway of aluminum cytotoxicity in
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human skin fibroblast cultures: prevention by superoxide dismutase and catalase and vitamins E and C. Hum Exp Toxicol 20, 477–481.
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Arabi, M., Seidaie, S. R. 2008.Assessment of motility and membrane peroxidation of bull Shahrekord. 2, 39-46.
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spermatozoa in the presence of different concentration of vitamin C. Vet Med J Asadpour, R., Jafari, R., Nasrabadi, H. T. 2011. Influence of added vitamin C and vitamin E on
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frozen-thawed bovine sperm cryopreserved in citrate and tris-based extenders.Vet. Res. Forum. 2 (1), 37 – 44.
Asghari, S. R. 1999. Reproductive characteristics of Ghezel and Mehraban rams and effect of vitamin C on semen parameters and fertility.MSc. Thesis. Shiraz University, Iran 155. Aurich, J. E., Schonherr, U., Hoppe, H. 1997. Effects of antioxidants on motility and membrane integrity of chilled stored stallion semen.Theriogenology. 48, 185–192. Azawi, O. I., Hussein, E. K. 2013. Effect of vitamins C or E supplementation to Tris diluent on the semen quality of Awassi rams preserved at 5 ˚C. Vet. Res. Forum. 4 (3), 157 – 160. Ball, B. A., Medina, V., Gravance, C. G. 2001. Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5°C.Theriogenology. 56, 577–589.
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Beconi, M. T., Francia, C. R., Mora, N. G.1993. Effect of natural antioxidants in frozen bovine semen preservation.Theriogenology. 40, 841-851. Bucak, M. N., Atessahin, A., Yuce, A. 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freez-thawing process.Small Rum.Res.75,128-134.
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Fazeli, P., Zamiri, M. J., Farshad, A., Khalili, B. 2010. Effects of vitamin C on testicular and seminal characteristics of Markhoz goats. Iran. J. of Vet. Res. Shiraz University. Vol.11,
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No. 3, Ser. No. 32, pp 267-372.
Gangwar, C., Ranjan R., Kumar, Satish., Kharche, S. D., Goel, A. K., Ramachandran, N., Jindal,
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S. K. 2014. Use of chelating agent for optimum post thaw quality of buck semen. Ind. J. of Anim. Sci. 84(8), 839–841.
Hancock, J. L. 1951. A staining technique for the study of temperature shock in semen. Nature
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(London). 167,323.
Maia, M. S., Bicudo, S. D., Azevedo, H. C. 2009. Motility and viability of ram sperm
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cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants. Small Rumin. Res. 85, 85-90.
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Maia, M. S., Bicudo, S. D., Sicherle, C. C. 2010. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with anti-oxidants.
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Small Rumin. Res. 122, 118-123.
Michael, A., Alexopoulos, C., Pontiki, E. 2008. Quality and reactive oxygen species of extended
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canine semen after vitamin C supplementation.Theriogenology. 70, 827–835. Ranjan R,, Goel, A.K., Ramachandran, N., Kharche S.D. and Jindal, S.K. 2015. Effect of egg yolk levels and equilibration periods on freezability of jamunapari buck semen. The Indian Journal of Small Ruminants 21: 32-36. Ranjan R., Ramachandran N., Jindal S. K., Sinha N. K., Goel A. K., Kharche S. D. and Sikarwar A. K. S. 2009. Effect of egg yolk levels on keeping quality of Marawari buck semen at refrigeration temperature. Indian Journal of Animal Science. 79:10-13. Ranjan, R., Goel A. K., Kharche, S. D., Ramachandran, N., Gangwar, Chetna and Jindal, S. K., (2014). Comparison between normal and dual staining technique for evaluating acrosome status and viability in frozen thawed buck spermatozoa. The Ind. J. of Small Rumin. 20(2): 50-53.
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Sarlos, P., Molnar, A., Kokai, M. 2002. Comparative evaluation of the effect of antioxidants in the conservation of ram semen.Acta Vet Hun.50, 235-245. Singh, B., Chand, D., Singh, P. 1996. Effect of vitamin C addition in the diluent on the quality of deep frozen Murrah buffalo bull (Bubalus bubalis) semen.Int J Anim Sci.11, 131-132. quality in rams. J. Firat Univ. Health Vet.Sci.17, 195-201.
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Sonmez, M ., Demirci, E. 2003. The effect of intramuscular vitamin C administration on semen
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Sonmez, M., Turk, G., Yuce, A. 2005. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation, and testosterone levels in male Wistar rats. Theriogenology.
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Uysal, O., Bucak, M. N. 2007. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen.Acta vet. Brno.76, 383-390.
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Watson., P. F. 1975. Use of Giemsa stain to detect changes in acrosomes of frozen ram spermatozoa. Vet. Rec. 97, 12–15.
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Wittayarat, M., Kimura, T., Kodama, R., Namula, Z., Chatdarong, K., Techakumphu, M., Sato, Y., Taniguchi, M., Otoi, T. 2012. Long-term preservation of chilled canine semen using
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vitamin C in combination with green tea polyphenol. Cryo Letters. 33, 318-26.
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Table 1 Effect of different concentration of vitamin C (L-Ascorbic acid) in semen dilutor on post thaw quality of Barbari buck semen
Motility
Live
Dead
Abnormal
0.0 (control)
41.04±1.08c
56.92±1.49c
43.08±1.49a
3.58±0.61a
0.8
47.29±1.16b
61.87±1.43b
37.29±1.75b
3.12±0.58a
78.67±1.36b
46.58±1.41b
1.0
53.54±1.29a
68.21±1.37a
32.62±1.32c
2.83±0.59a
83.62±1.15a
53.92±1.93a
1.2
44.17±1.89bc
59.96±1.71bc
40.46±1.68ab
3.42±0.60a
76.75±1.76b
44.25±2.24b
Fresh semen
84.05±1.23
89.71±0.94
10.76±0.92
2.86±0.64
91.57±0.91
75.43±1.03
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76.67±1.43b
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(Miligram %)
HOS
41.71±1.37b
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Acrosome
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Concentration
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