- Email: [email protected]
In Vitro Supplements Improves Motility and Progressive Score of Spermatozoa in Jermasia Goats
Available online at www.sciencedirect.com
ScienceDirect APCBEE Procedia 8 (2014) 329 – 333
2013 4th International Conference on Agriculture and Animal Science (CAAS 2013) 2013 3rd International Conference on Asia Agriculture and Animal (ICAAA 2013)
In Vitro Supplements Improves Motility and Progressive Score of Spermatozoa in Jermasia Goats M. Shikh Maidina , N.F.Adanana, M.T. Aminudina, A. Tawangb b
a Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia.
Abstract Several supplement intakes exert a marked effect on sperm quality, and this is useful in Artificial Insemination practice which is widely used in goat farming. The aim of this study was to determine the effect of in vitro supplementations in different concentrations (selenium, L-Arginine and Vitamin E) on sperm qualities; motility and progressive score of Jermasia goats. Results shown from observation at time interval; 2, 2.5, 3, 4 and 6 hours that the percentage of motility and progressive score of spermatozoa treated with 0.01 mM L-Arginine and 1 mg/ml vitamin E were significantly higher between 2 and 3 hours 0.01 mM L-Arginine and 1 mg/ml vitamin E compared to Control and other supplementation groups (p<0.05). Meanwhile, after 2 hours, the motility and progressive score of 0.6 ppm selenium were declined drastically compared to other groups (p < 0.05). The combination of L-Arginine and vitamin E supplements leads to increase productions of Nitric Oxide, hence stimulates the metabolism of glucose and triggering the ATP production in the sperm. In conclusion, both L-Arginine and vitamin E supplements are significantly stimulated and the motility of fresh semen of goats in a concentration-dependent manner is improved. © 2013 2014 The Authors. by Published by Elsevier B.V. Thisand/or is an open access article underresponsibility the CC BY-NC-ND license © Published Elsevier B.V. Selection peer review under of Asia-Pacific (http://creativecommons.org/licenses/by-nc-nd/3.0/). Chemical, Biological & Environmental Engineering Society Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Keywords: SELENIUM, L-ARGININE, ANTIOXIDANT
Corresponding author. Tel.: +603 8946 6614; fax: +603 8656 7454. E-mail address: [email protected].
2212-6708 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2014.03.049
330
M. Shikh Maidin et al. / APCBEE Procedia 8 (2014) 329 – 333
1. Introduction Poor sperm quality would affect the success rate of artificial insemination practise, hence reducing e goat productivity. Intensive farming for goat would assist in increasing the goat productivity, which is anticipated to increase demand in livestock industry. A well-organized reproductive system is needed to produce fertile animals; hence increasing the livestock production rate. Therefore, the fertility of both male and female goats is important to improve their reproductive performance. In addition, the importance of male fertility is related with reproductive success in female reproduction. In male, nutrition has a direct impact on the potency of sperm performance such as motility and progressive score. Literature in ruminant stated that component of trace elements such as selenium as supplementations are required in small amounts and it beneficially improves their reproductive success. In addition, deficiency of selenium reduces sperm quality in rats, mice, chickens, pigs, sheep, cattle, buffalo, boars and goats as reported in [1]. Beside Selenium, L-Arginine is also found to affect the reproductive process. [2] have determined that L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation and enhances the cell metabolism. Moreover, low concentrations of L-Arginine increase the sperm motility whereas high L-Arginine concentration decreases sperm motility [3] and [4]. In addition, nitric oxide synthesized from L-Arginine also helps to induce acrosome reaction, sperm chemotaxis, and sperm-egg interactions [4] and [5]. It also has been suggested that conjunction works of both supplement and vitamin E would improve semen quality especially on sperm motility by preventing free radical damage on sperm body, thus, increasing the rate of pregnancy to produce good quality of viable offspring. Therefore, the aim of this study was to determine the effectiveness of in vitro supplements; selenium, LArginine and vitamin E within different concentration and combination on sperm parameters (motility and progressive score) on male Jermasia goats. 2. Materials and Methods 2.1. Animal Management This study was conducted at Mini Farm, Institute of Science Biology, University of Malaya, Malaysia (3°7’25’’N&101°39’11’’E) from January to May 2013. Six sexually mature and healthy bucks of Jermasia male goats age ranges from 6 to 8 years and weighing 44.8 ± 0.57 kg were subjected to routine semen collection using artificial vagina. The extended pooled samples were divided into 4 groups (Table 1). Two replicates are prepared for each group. Table 1. Details of pooled semen’s groups (percentage of motility and progressive score of spermatozoa) using sodium selenate for selenium and α-tocopherol for vitamin E. Group of semen analysis Control; C
Supplement No supplementation
Group 1; G1
0.3 ppm selenium
Group 2; G2
0.6 ppm selenium
Group 3; G3
0.6 ppm selenium and 1.0 mg/mL Vitamin E
Group 4; G4
0.01mM L-Arginine
Group 5; G5
0.1 mM L-Arginine
Group 6; G6
0.01 mM L-Arginine and 1 mg/ml vitamin E
M. Shikh Maidin et al. / APCBEE Procedia 8 (2014) 329 – 333
2.2. Semen Analysis 2.2.1. Semen collection The process of semen collection was performed using artificial vagina (AV). The ejaculated semen was incubated in a water bath with temperature maintained at 37°C to preserve the quality of the semen and sperm. Pooled fresh semen was used and semen with normal pH, color, volume and motility >80% was selected for the experiment. 2.2.2. Motility (percentage of motile spermatozoa) and progressive score analyses Sperm motility (% of motile) and progressive analysis was assessed by subjective measurement after the semen is diluted with tri-citric egg yolk extender; with a ratio of 1:9 (semen : dilution). Observations for the semen analysis (% of motility and progressive score were carried out for 5 times at different intervals; 2, 2.5, 3, 4 and 6 hours. Sperm progressive score was subjectively estimated by observing the sperm motility and the movement is scored on 0-5 scale while viewing under the Microscope Olympus CX41.Two slides were used for each sample and 3 fields were observed for every slide. The score of 0-5 was based on the following criteria: 0 = no motility, 1 = non-progressive motility, 2 = slow progressive motility, 3 = side-to-side movement accompanied by slow progressive motility, 4 = faster progressive motility and 5 = very fast progressive motility [6]. 2.3. Statistical Analyses All the data were anaylsed using OneWay analysis (ANOVA) SPSS version 20.0; statistical software. The evaluation and assessments of the quality of spermatozoa was analysed by Duncan’s test by comparison between groups and time interval; 2, 2.5, 3, 4 and 6 hours. Data are presented by means ± SE; differences were regarded as significant at p < 0.05. 3. Results 3.1. Percentage of Motility The motility of spermatozoa a is significantly higher between 2 and 3 hours in Group 6 (0.01 mM LArginine and 1 mg/ml vitamin E) compared to Control and other groups (Table 2: p<0.05). Meanwhile, within selenium groups (Groups 1 to 3), semen treated with 0.3 ppm selenium (Group 1) showed a higher percentage of motility compared to others groups. However, the percentage motility of L-Arginine groups are generally higher than 0.3 ppm selenium (Group 1) at time interval of 6 hours. It is clearly shown that the percentage of motility is immediately decreased in Group 2 (0.6 ppm selenium) after 2 hours and onwards compared to other groups (p < 0.05). Hence, the supplement is found to be toxic to Jermasia sperm. 3.2. Progressive Score In Table 3, progressive score in semen treated with 0.01 mM L-Arginine and 1 mg/ml vitamin E (Group 6) is higher than other groups throughout a time interval of 2 to 6 hours. In addition, progressive score of LArginine groups (Groups 4 to 6) are numerically higher than selenium groups and Control group (Table 3). In addition, it is clearly shown that the percentage of motility immediately decreased in Group 2 (0.6 ppm selenium) after 2 hours onwards compared to other groups (p < 0.05).
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Table 2. The analysis of motility percentage between groups (Control=non-supplemented group; G1=0.3 ppm selenium; G2 =0.6 ppm selenium; G3=0.6 ppm selenium and 1.0 mg/mL Vitamin E; G4=0.01 mM of L-Arginine; G5=0.1 mM L-Arginine and G6=0.01 mM LArginine and 1 mg/ml vitamin E) in pooled semen (n=6; mean ± SE). Time; hour
Control
G1 b
2
81.50 ± 0.29
2.5
81.38 ± 0.24 b bc
a, b, c, d, e
G2
84.50 ± 0.50
c
G3 a
G4 bc
G5
G6 84.25 ±0.85c
83.00 ± 0.41
84.25 ± 0.48d
21.50 ± 1.55a
81.25 ± 0.75 b
83.75 ± 0.25cd
82.00 ± 0.00bc
84.50 ± 0.29 d
de
a
b
e
cd
84.00 ± 0.58e
3
79.63 ± 0.24
4
79.75 ± 0.25 cd
78.75 ± 1.49cd
4.75 ± 0.85a
73.25 ± 2.69 b
82.50 ± 0.65 d
78.00 ± 1.22c
82.50 ± 0.96 d
6
d
c
a
b
d
d
79.50 ± 0.96 d
76.13 ± 0.43
53.25 ±2.14
8.00 ± 0.82 1.55 ± 0.25
78.50 ± 0.96 10.50 ± 1.85
83.25 ± 0.48
82.00 ± 0.82
bc
74.75 ± 1.70
83.00 ± 0.41
83.75 ± 0.25
bc
79.00 ± 0.58
81.25 ± 0.48 77.00 ± 0.58
Significantly different within times (p <0.05)
Table 3. The analysis of progressive score between groups (Control=non-supplemented group; G1=0.3 ppm selenium; G2 =0.6 ppm selenium; G3=0.6 ppm selenium and 1.0 mg/mL Vitamin E; G4=0.01 mM of L-Arginine; G5=0.1 mM L-Arginine and G6=0.01 mM LArginine and 1 mg/ml vitamin E) in pooled semen (n=6; mean ± SE) Time; hour
Control
2
4.18 ± 0.03ab
G2
G3
4.52 ± 0.05c
3.97 ± 0.09a
d
3.28 ± 0.09
a
2.30 ± 0.15
a
G4
4.43 ± 0.02c
4.40 ± 0.09b
4.18 ± 0.10ab
4.43 ± 0.09c
4.20 ± 0.07
bc
4.40 ± 0.07cd
4.23 ± 0.02
bc
4.35 ± 0.09bc
3
4.10 ±0.02
b
4
4.10 ±0.04c
4.13 ± 0.14c
1.88 ± 0.18a
3.73 ± 0.11b
4.33 ± 0.03c
4.18 ± 0.03c
4.30 ± 0.06c
6
d
c
a
b
d
d
4.00 ± 0.08d
3.64 ±0.07
4.43 ± 0.05 2.85 ± 0.21
c
1.13 ± 0.13
4.28 ± 0.02
4.13 ± 0.07 1.75 ± 0.14
b
4.33 ± 0.07
bcd
G6
4.18 ±0.06
4.48 ± 0.02
bcd
G5
b
2.5
a, b, c, d
G1
4.35 ± 0.06
bc
3.93 ± 0.08
3.80 ± 0.07
Significantly different within times (p <0.05)
4. Discussion and Conclusion The combination of L-Arginine and Vitamin E (Group 6) is able to sustain the percentage of motility above 80%, up to 6 hours after semen collection. This mixture of supplement could possibly be implemented in AI process. L-Arginine is able to increase the production of nitric oxide (NO) in cells, while Vitamin E acts as antioxidants also able to increase synthase activity of NO [7] and [11]. Nitric oxide is well known in improving male reproductive performance by improving the sperm motility. The present of NO in cells by mitochondrial nitric oxide synthase helps to regulate the oxygen level uptake for cellular metabolism [8]. Therefore, the combination of L-Arginine and Vitamin E is able to sustain the motility of spermatozoa for a period of few hours. Sustainability of motility shown in Group 6 is also related to a high rate of glucose metabolism in spermatozoa, whereas the production rate of ATP is enhanced [9]. However, the enhancement contributes to the damage of sperm membrane as the L-Arginine concentration is not sufficient to produce NO for reducing the lipid peroxidation [9] and [10]. Semen treated with L-Arginine (Group 4) shows similar results. Selenium is also able to prolong the sperm motility, however it lasted for a mere 3 hours. The possible reason of this is due to the level of selenium concentration. Moreover, the content of egg yolk in tricitric extender could interfere the motility of sperm during microscopic observation. The difficulties to observe the movement of impaired sperm is possibly due to yolk globules in the tri-extender used [12].
M. Shikh Maidin et al. / APCBEE Procedia 8 (2014) 329 – 333
As a conclusion, in vitro supplementations of L-Arginine and vitamin E promotes better semen parameters and able to sustain the motility of sperm Jermasia goat. However, these results are based on in vitro evaluations, further studies are requires as to ensure the mechanism of how and where specifically the supplements react with the sperm. This is important to identify the factors affecting the results more accurately. Acknowledgements We thank Mr. Razali Jonit and Dr. Shafishuhaza Sahlan for their assistance with the experiment and Universiti Putra Malaysia (Vote: 9374600) for financial support. References [1] Dorostkar K, Alavi-Shoushtari SA, Mokarizadeh A. Effects of in vitro selenium addition to the semen extenders on the spermatozoa characteristics before and after freezing in water buffaloes (Bubalus bubalis).VetResForum.2012;3(4): 263-268. [2] O'Flaherty C, Rodriguez P, Srivastava S. L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa. Bioch EtBiophyA(BBA)-General Subjects.2004;1674(2):215-221 [3] Hassanpour H, Mirshokrai P, Shirazi A, Aminian A. Effect of nitric oxide on ram sperm motility in vitro. Pakistan J BiolSc: PJBS.2007;10(14):2374-2378. [4] Hassanpour H, Teshfam M, Karimi Goodarzi A, Tajik P, Mirshokraei P. In vitro effects of l-arginine on motion parameters in ram epididymal sperm. Comparative Clinical Pathology.2010;19(4):351-355. [5] Revelli A, Costamagna C, Moffa F, Aldieri E, Ochetti S, Bosia A, Massobrio M, Lindblom Bo, Ghigo D. Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa. BiolReproduc.2001;64(6):1708-1712. [6] Sontakke SD, Umapathy G, Sivaram V, Kholkute SD, Shivaji S. Semen characteristics, cryopreservation, and successful artificial insemination in the blue rock pigeon (Columba livia). Theriogenology.2004;62:139–153. [7] Husein RH, Ahmed MO, Muhammed SM. Effects of L-arginine, vitamin E and their combinations on sperms morphology in albino male mice. J Al-Nahrain University.2010;14(2):137-143. [8] Finocchietto PV, Franco MC, Holod S, Gonzalez AS, Converso DP, Antico Arciuch VG, Serra MP, Poderoso JJ, Carreras MC. Mitochondrial nitric oxide synthase: A masterpiece of metabolic adaptation, cell growth, transformation, and death. ExpBiol Med.2009;234(9):1020-1028 [9] Srivastava S, Desai P, Coutinho E, Govil G. Mechanism of action of L-arginine on the vitality of spermatozoa is primarily through increased biosynthesis of nitric oxide. BiolReproduc.2006;74(5): 954-958. [10] Munne-Bosc S. The role of alpha-tocopherol in plant stress tolerance. (Special issue: Vitamin E in plant, man, and animals). J PlantPhy.2005;162(7):743-748. [11] Newaz MA, Nawal NNA, Rohaizan CH, Muslim N, Gapor A. α-Tocopherol increased nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats. Am J Hypertens.1999;12(8): 839-844. [12] Thun R, Hurtado M, and Janett F. Comparison of Biociphos-Plus and TRIS-egg yolk extender for cryopreservation of bull semen.Therio.2002; 57(3):1087-1094.
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ScienceDirect APCBEE Procedia 8 (2014) 329 – 333
2013 4th International Conference on Agriculture and Animal Science (CAAS 2013) 2013 3rd International Conference on Asia Agriculture and Animal (ICAAA 2013)
In Vitro Supplements Improves Motility and Progressive Score of Spermatozoa in Jermasia Goats M. Shikh Maidina , N.F.Adanana, M.T. Aminudina, A. Tawangb b
a Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia.
Abstract Several supplement intakes exert a marked effect on sperm quality, and this is useful in Artificial Insemination practice which is widely used in goat farming. The aim of this study was to determine the effect of in vitro supplementations in different concentrations (selenium, L-Arginine and Vitamin E) on sperm qualities; motility and progressive score of Jermasia goats. Results shown from observation at time interval; 2, 2.5, 3, 4 and 6 hours that the percentage of motility and progressive score of spermatozoa treated with 0.01 mM L-Arginine and 1 mg/ml vitamin E were significantly higher between 2 and 3 hours 0.01 mM L-Arginine and 1 mg/ml vitamin E compared to Control and other supplementation groups (p<0.05). Meanwhile, after 2 hours, the motility and progressive score of 0.6 ppm selenium were declined drastically compared to other groups (p < 0.05). The combination of L-Arginine and vitamin E supplements leads to increase productions of Nitric Oxide, hence stimulates the metabolism of glucose and triggering the ATP production in the sperm. In conclusion, both L-Arginine and vitamin E supplements are significantly stimulated and the motility of fresh semen of goats in a concentration-dependent manner is improved. © 2013 2014 The Authors. by Published by Elsevier B.V. Thisand/or is an open access article underresponsibility the CC BY-NC-ND license © Published Elsevier B.V. Selection peer review under of Asia-Pacific (http://creativecommons.org/licenses/by-nc-nd/3.0/). Chemical, Biological & Environmental Engineering Society Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Keywords: SELENIUM, L-ARGININE, ANTIOXIDANT
Corresponding author. Tel.: +603 8946 6614; fax: +603 8656 7454. E-mail address: [email protected].
2212-6708 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2014.03.049
330
M. Shikh Maidin et al. / APCBEE Procedia 8 (2014) 329 – 333
1. Introduction Poor sperm quality would affect the success rate of artificial insemination practise, hence reducing e goat productivity. Intensive farming for goat would assist in increasing the goat productivity, which is anticipated to increase demand in livestock industry. A well-organized reproductive system is needed to produce fertile animals; hence increasing the livestock production rate. Therefore, the fertility of both male and female goats is important to improve their reproductive performance. In addition, the importance of male fertility is related with reproductive success in female reproduction. In male, nutrition has a direct impact on the potency of sperm performance such as motility and progressive score. Literature in ruminant stated that component of trace elements such as selenium as supplementations are required in small amounts and it beneficially improves their reproductive success. In addition, deficiency of selenium reduces sperm quality in rats, mice, chickens, pigs, sheep, cattle, buffalo, boars and goats as reported in [1]. Beside Selenium, L-Arginine is also found to affect the reproductive process. [2] have determined that L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation and enhances the cell metabolism. Moreover, low concentrations of L-Arginine increase the sperm motility whereas high L-Arginine concentration decreases sperm motility [3] and [4]. In addition, nitric oxide synthesized from L-Arginine also helps to induce acrosome reaction, sperm chemotaxis, and sperm-egg interactions [4] and [5]. It also has been suggested that conjunction works of both supplement and vitamin E would improve semen quality especially on sperm motility by preventing free radical damage on sperm body, thus, increasing the rate of pregnancy to produce good quality of viable offspring. Therefore, the aim of this study was to determine the effectiveness of in vitro supplements; selenium, LArginine and vitamin E within different concentration and combination on sperm parameters (motility and progressive score) on male Jermasia goats. 2. Materials and Methods 2.1. Animal Management This study was conducted at Mini Farm, Institute of Science Biology, University of Malaya, Malaysia (3°7’25’’N&101°39’11’’E) from January to May 2013. Six sexually mature and healthy bucks of Jermasia male goats age ranges from 6 to 8 years and weighing 44.8 ± 0.57 kg were subjected to routine semen collection using artificial vagina. The extended pooled samples were divided into 4 groups (Table 1). Two replicates are prepared for each group. Table 1. Details of pooled semen’s groups (percentage of motility and progressive score of spermatozoa) using sodium selenate for selenium and α-tocopherol for vitamin E. Group of semen analysis Control; C
Supplement No supplementation
Group 1; G1
0.3 ppm selenium
Group 2; G2
0.6 ppm selenium
Group 3; G3
0.6 ppm selenium and 1.0 mg/mL Vitamin E
Group 4; G4
0.01mM L-Arginine
Group 5; G5
0.1 mM L-Arginine
Group 6; G6
0.01 mM L-Arginine and 1 mg/ml vitamin E
M. Shikh Maidin et al. / APCBEE Procedia 8 (2014) 329 – 333
2.2. Semen Analysis 2.2.1. Semen collection The process of semen collection was performed using artificial vagina (AV). The ejaculated semen was incubated in a water bath with temperature maintained at 37°C to preserve the quality of the semen and sperm. Pooled fresh semen was used and semen with normal pH, color, volume and motility >80% was selected for the experiment. 2.2.2. Motility (percentage of motile spermatozoa) and progressive score analyses Sperm motility (% of motile) and progressive analysis was assessed by subjective measurement after the semen is diluted with tri-citric egg yolk extender; with a ratio of 1:9 (semen : dilution). Observations for the semen analysis (% of motility and progressive score were carried out for 5 times at different intervals; 2, 2.5, 3, 4 and 6 hours. Sperm progressive score was subjectively estimated by observing the sperm motility and the movement is scored on 0-5 scale while viewing under the Microscope Olympus CX41.Two slides were used for each sample and 3 fields were observed for every slide. The score of 0-5 was based on the following criteria: 0 = no motility, 1 = non-progressive motility, 2 = slow progressive motility, 3 = side-to-side movement accompanied by slow progressive motility, 4 = faster progressive motility and 5 = very fast progressive motility [6]. 2.3. Statistical Analyses All the data were anaylsed using OneWay analysis (ANOVA) SPSS version 20.0; statistical software. The evaluation and assessments of the quality of spermatozoa was analysed by Duncan’s test by comparison between groups and time interval; 2, 2.5, 3, 4 and 6 hours. Data are presented by means ± SE; differences were regarded as significant at p < 0.05. 3. Results 3.1. Percentage of Motility The motility of spermatozoa a is significantly higher between 2 and 3 hours in Group 6 (0.01 mM LArginine and 1 mg/ml vitamin E) compared to Control and other groups (Table 2: p<0.05). Meanwhile, within selenium groups (Groups 1 to 3), semen treated with 0.3 ppm selenium (Group 1) showed a higher percentage of motility compared to others groups. However, the percentage motility of L-Arginine groups are generally higher than 0.3 ppm selenium (Group 1) at time interval of 6 hours. It is clearly shown that the percentage of motility is immediately decreased in Group 2 (0.6 ppm selenium) after 2 hours and onwards compared to other groups (p < 0.05). Hence, the supplement is found to be toxic to Jermasia sperm. 3.2. Progressive Score In Table 3, progressive score in semen treated with 0.01 mM L-Arginine and 1 mg/ml vitamin E (Group 6) is higher than other groups throughout a time interval of 2 to 6 hours. In addition, progressive score of LArginine groups (Groups 4 to 6) are numerically higher than selenium groups and Control group (Table 3). In addition, it is clearly shown that the percentage of motility immediately decreased in Group 2 (0.6 ppm selenium) after 2 hours onwards compared to other groups (p < 0.05).
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Table 2. The analysis of motility percentage between groups (Control=non-supplemented group; G1=0.3 ppm selenium; G2 =0.6 ppm selenium; G3=0.6 ppm selenium and 1.0 mg/mL Vitamin E; G4=0.01 mM of L-Arginine; G5=0.1 mM L-Arginine and G6=0.01 mM LArginine and 1 mg/ml vitamin E) in pooled semen (n=6; mean ± SE). Time; hour
Control
G1 b
2
81.50 ± 0.29
2.5
81.38 ± 0.24 b bc
a, b, c, d, e
G2
84.50 ± 0.50
c
G3 a
G4 bc
G5
G6 84.25 ±0.85c
83.00 ± 0.41
84.25 ± 0.48d
21.50 ± 1.55a
81.25 ± 0.75 b
83.75 ± 0.25cd
82.00 ± 0.00bc
84.50 ± 0.29 d
de
a
b
e
cd
84.00 ± 0.58e
3
79.63 ± 0.24
4
79.75 ± 0.25 cd
78.75 ± 1.49cd
4.75 ± 0.85a
73.25 ± 2.69 b
82.50 ± 0.65 d
78.00 ± 1.22c
82.50 ± 0.96 d
6
d
c
a
b
d
d
79.50 ± 0.96 d
76.13 ± 0.43
53.25 ±2.14
8.00 ± 0.82 1.55 ± 0.25
78.50 ± 0.96 10.50 ± 1.85
83.25 ± 0.48
82.00 ± 0.82
bc
74.75 ± 1.70
83.00 ± 0.41
83.75 ± 0.25
bc
79.00 ± 0.58
81.25 ± 0.48 77.00 ± 0.58
Significantly different within times (p <0.05)
Table 3. The analysis of progressive score between groups (Control=non-supplemented group; G1=0.3 ppm selenium; G2 =0.6 ppm selenium; G3=0.6 ppm selenium and 1.0 mg/mL Vitamin E; G4=0.01 mM of L-Arginine; G5=0.1 mM L-Arginine and G6=0.01 mM LArginine and 1 mg/ml vitamin E) in pooled semen (n=6; mean ± SE) Time; hour
Control
2
4.18 ± 0.03ab
G2
G3
4.52 ± 0.05c
3.97 ± 0.09a
d
3.28 ± 0.09
a
2.30 ± 0.15
a
G4
4.43 ± 0.02c
4.40 ± 0.09b
4.18 ± 0.10ab
4.43 ± 0.09c
4.20 ± 0.07
bc
4.40 ± 0.07cd
4.23 ± 0.02
bc
4.35 ± 0.09bc
3
4.10 ±0.02
b
4
4.10 ±0.04c
4.13 ± 0.14c
1.88 ± 0.18a
3.73 ± 0.11b
4.33 ± 0.03c
4.18 ± 0.03c
4.30 ± 0.06c
6
d
c
a
b
d
d
4.00 ± 0.08d
3.64 ±0.07
4.43 ± 0.05 2.85 ± 0.21
c
1.13 ± 0.13
4.28 ± 0.02
4.13 ± 0.07 1.75 ± 0.14
b
4.33 ± 0.07
bcd
G6
4.18 ±0.06
4.48 ± 0.02
bcd
G5
b
2.5
a, b, c, d
G1
4.35 ± 0.06
bc
3.93 ± 0.08
3.80 ± 0.07
Significantly different within times (p <0.05)
4. Discussion and Conclusion The combination of L-Arginine and Vitamin E (Group 6) is able to sustain the percentage of motility above 80%, up to 6 hours after semen collection. This mixture of supplement could possibly be implemented in AI process. L-Arginine is able to increase the production of nitric oxide (NO) in cells, while Vitamin E acts as antioxidants also able to increase synthase activity of NO [7] and [11]. Nitric oxide is well known in improving male reproductive performance by improving the sperm motility. The present of NO in cells by mitochondrial nitric oxide synthase helps to regulate the oxygen level uptake for cellular metabolism [8]. Therefore, the combination of L-Arginine and Vitamin E is able to sustain the motility of spermatozoa for a period of few hours. Sustainability of motility shown in Group 6 is also related to a high rate of glucose metabolism in spermatozoa, whereas the production rate of ATP is enhanced [9]. However, the enhancement contributes to the damage of sperm membrane as the L-Arginine concentration is not sufficient to produce NO for reducing the lipid peroxidation [9] and [10]. Semen treated with L-Arginine (Group 4) shows similar results. Selenium is also able to prolong the sperm motility, however it lasted for a mere 3 hours. The possible reason of this is due to the level of selenium concentration. Moreover, the content of egg yolk in tricitric extender could interfere the motility of sperm during microscopic observation. The difficulties to observe the movement of impaired sperm is possibly due to yolk globules in the tri-extender used [12].
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As a conclusion, in vitro supplementations of L-Arginine and vitamin E promotes better semen parameters and able to sustain the motility of sperm Jermasia goat. However, these results are based on in vitro evaluations, further studies are requires as to ensure the mechanism of how and where specifically the supplements react with the sperm. This is important to identify the factors affecting the results more accurately. Acknowledgements We thank Mr. Razali Jonit and Dr. Shafishuhaza Sahlan for their assistance with the experiment and Universiti Putra Malaysia (Vote: 9374600) for financial support. References [1] Dorostkar K, Alavi-Shoushtari SA, Mokarizadeh A. Effects of in vitro selenium addition to the semen extenders on the spermatozoa characteristics before and after freezing in water buffaloes (Bubalus bubalis).VetResForum.2012;3(4): 263-268. [2] O'Flaherty C, Rodriguez P, Srivastava S. L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa. Bioch EtBiophyA(BBA)-General Subjects.2004;1674(2):215-221 [3] Hassanpour H, Mirshokrai P, Shirazi A, Aminian A. Effect of nitric oxide on ram sperm motility in vitro. Pakistan J BiolSc: PJBS.2007;10(14):2374-2378. [4] Hassanpour H, Teshfam M, Karimi Goodarzi A, Tajik P, Mirshokraei P. In vitro effects of l-arginine on motion parameters in ram epididymal sperm. Comparative Clinical Pathology.2010;19(4):351-355. [5] Revelli A, Costamagna C, Moffa F, Aldieri E, Ochetti S, Bosia A, Massobrio M, Lindblom Bo, Ghigo D. Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa. BiolReproduc.2001;64(6):1708-1712. [6] Sontakke SD, Umapathy G, Sivaram V, Kholkute SD, Shivaji S. Semen characteristics, cryopreservation, and successful artificial insemination in the blue rock pigeon (Columba livia). Theriogenology.2004;62:139–153. [7] Husein RH, Ahmed MO, Muhammed SM. Effects of L-arginine, vitamin E and their combinations on sperms morphology in albino male mice. J Al-Nahrain University.2010;14(2):137-143. [8] Finocchietto PV, Franco MC, Holod S, Gonzalez AS, Converso DP, Antico Arciuch VG, Serra MP, Poderoso JJ, Carreras MC. Mitochondrial nitric oxide synthase: A masterpiece of metabolic adaptation, cell growth, transformation, and death. ExpBiol Med.2009;234(9):1020-1028 [9] Srivastava S, Desai P, Coutinho E, Govil G. Mechanism of action of L-arginine on the vitality of spermatozoa is primarily through increased biosynthesis of nitric oxide. BiolReproduc.2006;74(5): 954-958. [10] Munne-Bosc S. The role of alpha-tocopherol in plant stress tolerance. (Special issue: Vitamin E in plant, man, and animals). J PlantPhy.2005;162(7):743-748. [11] Newaz MA, Nawal NNA, Rohaizan CH, Muslim N, Gapor A. α-Tocopherol increased nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats. Am J Hypertens.1999;12(8): 839-844. [12] Thun R, Hurtado M, and Janett F. Comparison of Biociphos-Plus and TRIS-egg yolk extender for cryopreservation of bull semen.Therio.2002; 57(3):1087-1094.
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