|Year : 2017 | Volume
| Issue : 2 | Page : 81-84
Impact of Silymarin enriched semen extender on bull sperm preservability
RI El-Sheshtawy, WS El-Nattat
Animal Reproduction and AI Dept., Veterinary Research Division, National Research Centre, Dokki, Egypt
|Date of Web Publication||25-Sep-2017|
R I El-Sheshtawy
Animal Reproduction and Artificial Insemination Department, 12622, National Research Centre, Dokki, Giza
Source of Support: None, Conflict of Interest: None
Objective: To explore the effect of silymarin on bull spermatozoa during cooling and cryopreservation. Methods: Pooled bull semen were diluted by Tris-Citrate-Fructose egg yolk diluents, purified silymarin powder (obtained from the milk thistle silybum marianum), purchased from Unipharma, Al Obour city, Egypt, was soaked in Tris-citric acid-fructose diluent for 48 h at 10 °C making a stock solution (70 mg/mL), from this stock solution we obtained concentrations of 0.18 mg/mL, 0.36 mg/mL, 0.54 mg/mL, 0.72 mg/mL, 0.90 mg/mL in addition to the control (0.00 mg/mL) reaching a final volume of 5 mL in each tube. Egg yolk was added to each tube to obtain silymarin enriched semen extender (SEE) with 20% egg yolk, cooled slowly up to 5 °C and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws (IMV, France). After equilibration periods, the straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen for 10 min and were then dipped in liquid nitrogen. Extended semen was subjected to evaluation (motility, alive%, abnormality%, intact sperm membrane (HOST)% and conception rate) in both chilled and frozen semen. Results: [Table 1] revealed that Sperm motility of the concentrations 2, 3 and 4 after 8 d of chilling were significantly (P<0.02) higher than control. Sperm motility of the concentration 2 (45.00%±2.89%) after 9 d of chilling was higher than control and the other concentrations. Addition of SEE in concentration 1 and 2 gave post thawing sperm motility as high as the control (47.50±2.81 and 45.00±2.58, respectively) while other concentration have lower effects on motility as compared to the control. Addition of silymarin improved post thawing alive% and was significantly higher (P<0.000 1) than the control. SEE decreased significantly (P<0.000 1) the % of post thawing abnormal sperm in concentration 3 and 4 (11.83±0.65 and 16.00±0.58, respectively). SEE improved significantly (P<0.018) the % of post thawing intact spermatozoa membranes (HOST%) in concentrations 2, 4 and 5 (71.17±0.83, 71.83±0.91 and 75.00±3.42, respectively) [Table 2]. Conclusion: It could be concluded that silymarin as a natural additive to semen extenders improved preservability in both chilled and frozen bull semen.
Keywords: Cattle, Semen, Preservation, Silymarin
|How to cite this article:|
El-Sheshtawy R I, El-Nattat W S. Impact of Silymarin enriched semen extender on bull sperm preservability. Asian Pac J Reprod 2017;6:81-4
|How to cite this URL:|
El-Sheshtawy R I, El-Nattat W S. Impact of Silymarin enriched semen extender on bull sperm preservability. Asian Pac J Reprod [serial online] 2017 [cited 2023 Feb 4];6:81-4. Available from: https://www.apjr.net/text.asp?2017/6/2/81/215608
| 1. Introduction|| |
Spermatozoa are the end point of male spermatogenesis and have particular anatomic and metabolic features. Nowadays, sperm cryopreservation and storage are of a great demand for conserving the supergenetic origins of the males, the development of artificial reproductive technologies such as artificial insemination (AI) and in vitro fertilization (IVF) are of a great interest. AI with frozen semen is essential in breeding and selection schedules contributing to increase production of domestic species. Nowadays, semen cryopreservation has many biotechnological applications. It can be used to solve infertility problems, life threatening diseases, preservation of semen and DNA from endangered species and conservation of biodiversity. The interaction of several factors (cooling rate, storage temperature, chemical ingredients of the extender, cryoprotectant concentration, over accumulation of oxygen free radicals, seminal plasma composition and hygienic control) are the key that affect the life-span of spermatozoa. Cryopreservation of bovine semen often induce an additional source for reactive oxygen species (ROS) attack on sperm due to decreased activities of antioxidant enzymes and the sperm membrane become more susceptible to lipid peroxidation which affect the membrane permeability. Natural antioxidants exert a protective effect preserving the metabolic activity and cellular viability of cryopreserved bovine spermatozoa. Nowadays, the use of herbal natural product has gained interest worldwide. Many of the herbs have been developed into herbal supplement which are claimed to assist in healthy life style. Among these herbs Silymarin is an extract from the seeds and fruits of the milk thistle silybum marianum that contains the flavonolignans silybin which is the major active component. Silymarin is a strong antioxidant used as a remedy for liver protection against oxidative stress and also as a protectant for the testicular tissue and improving semen quality through elevation of blood testosterone level,. Also, it prevents acetaminophen induced liver injury through restoration of glutathione level. No available literatures were found for interpreting the benefit for using Silymarin.
| 2. Materials and Methods|| |
2.1. Preparation of silymarin enriched semen extender (SEE)
Tris-citric acid-fructose diluent (TCF) was prepared according to Foote et al. purified silymarin powder (obtained from the milk thistle silybum marianum), purchased from Unipharma, Al Obour city, Egypt, was soaked in Tris-citric acid-fructose diluent (TCF), (silymarin: TCF) for 48 h at 10 °C making a stock solution (70 mg/mL), from this stock solution we obtained concentrations of 0.18 mg/mL, 0.36 mg/mL, 0.54 mg/mL, 0.72 mg/mL, 0.90 mg/mL in addition to the control (0.0 mg/mL) reaching a final volume of 5 mL in each tube. Egg yolk was added to each tube to obtain SEE with 20% egg yolk (SEEY).
2.2. Semen collection and initial evaluation
Three mature cattle bulls maintained at Semen Freezing Center, General Organization for Veterinary Services Ministry of Agriculture, Abbasia, Egypt, were used as semen donors. Ejaculates were collected using a bovine adapted artificial vagina at weekly intervals for 18 wk. Semen samples were initially evaluated for subjective sperm motility and sperm concentration. Ejaculates fulfilling minimum standard of sperm motility (70%) and sperm morphology were pooled in order to have sufficient semen for a replicate and to eliminate the bull effect. The semen was given a holding time for 10 min at 37 °C in the water bath before dilution.
2.3. Semen processing
Semen samples were diluted with TCF extender and used as control and other aliquots of pooled semen samples were diluted with TCF extenders containing the different concentrations of silymarin in order to provide concentration of 120 million sperm/mL. Extended semen was slowly cooled (approximately for 2 h) to 5 °C and equilibrated for 2 h. Semen was packed into 0.25 mL polyvinyl French straws. After equilibrium periods, the straws were horizontally placed on a rack and frozen in vapor 4 cm above liquid nitrogen for 10 min and were then dipped in liquid nitrogen. A fraction of extended semen from control and each concentration of the additive were kept at 5 °C for 7-10 d (chilling) and sperm motility was evaluated daily.
2.4. Assessment of semen quality parameters
The assessment was undertaken on after freeze thawing of bull spermatozoa. Also, sperm motility was evaluated for raw semen, 2 h after cooling and chilled semen daily up to 7-10 d. Frozen straws were thawed at 37 °C/1 min. The parameters studied were subjective semen characteristics (motility, alive, abnormality and hypoosmotic swelling test (HOST) %).
2.5. Statistical analysis
Statistical analysis data were analyzed using the SPSS (2005) computerized program v. 14.0 to calculate the analysis of variance (ANOVA) for the different parameters between control and additives replications. Significant difference between means was calculated using Duncan test at P<0.05.
| 3. Results|| |
3.1. Effect of SEE on cattle bull sperm motility during chilling
Sperm motility of the concentrations 2, 3 and 4 after 8 d of chilling were significantly (P<0.02) higher than control. Sperm motility of the concentration 2 (45.00%±2.89%) after 9 d of chilling was higher than control and the other concentrations [Table 1].
|Table 1: Effect of silymarin enriched extender on cattle bull sperm motility during chilling.|
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3.2. Effect of SEE on cattle post thawing sperm characteristics
Addition of SEE in concentration 1 and 2 gave post thawing sperm motility as high as the control (47.50±2.81 and 45.00±2.58, respectively) while other concentration have lower effects on motility as compared to the control.
Addition of silymarin improved post thawing alive% and was significantly higher (P<0.000 1) than the control. SEE decreased significantly (P<0.000 1) the % of post thawing abnormal sperm in concentration 3 and 4 (11.83±0.65 and 16.00±0.58, respectively). SEE improved significantly (P<0.018) the % of post thawing intact spermatozoa membranes (HOST%) in concentrations 2, 4 and 5 (71.17±0.83, 71.83±0.91 and 75.00±3.42, respectively) [Table 2].
|Table 2: Effect of silymarin enriched extender on cattle post thawing sperm characteristics.|
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| 4. Discussion|| |
Cryopreservation of sperm is of a great demand. According to Gadea et al., Uysal and Bucak and Bucak et al., minimizing the physical and chemical stresses of cooling, freezing and thawing of sperm cells and consequently improving viability and subsequent fertilizing capacity is achieved by including cryoprotectants in the semen extender. Cryopreservation causes wide-ranging physical, chemical and mechanical injures to sperm membranes of all mammalian species, which are attributed to temperature changes, alterations in the transition from the lipid phase, production of reactive oxygen species (ROS) and osmotic stress,. Moreover, the over accumulation of ROS causes a state of oxidative stress that involves structural damage of sperm membranes, fall of intracellular ATP levels causing decreasing in the viability and motility of cryopreserved sperm,. To decrease the harmful effects of ROS, seminal plasma possesses powerful source of ROS scavengers which offer protection for sperm, including enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and small molecular antioxidants such as ascorbic acid and a-tocopherol,. The herbal remedies contain antioxidants to counteract the deleterious action of reactive oxygen species (ROS). Sperm motility was kept high in chilled semen at the concentrations of silymarin 0.36, 0.54 and 0.72 mg/mL up to 8 d of chilling. This indicates that chilled extended semen with these concentrations could be used in AI up to 8 d. Sperm motility of the concentration 0.54 mg/mL was kept high up to 9 d of chilling. This means that semen extended with this concentration could be used in AI up to 9 d of chilling. Addition of silymarin improved post thawing alive%, sperm abnormalities, percent of post-thawing intact spermatozoa membranes and maintained the percent of sperm motility. The improved semen quality in both chilled and frozen semen is due to the strong antioxidant capacity of silymarin,. Wellington and Jarvis postulated that the mechanism of action of silymarin is through the stimulation of ribosomal RNA protecting the cell membrane from oxidative damage. They stated also that silymarin stimulates the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase. It could be concluded that silymarin as a natural additive to semen extenders improved preservability in both chilled and frozen bull semen.
Conflict of interest statement
The authors declare that they have no conflict of interest.
| References|| |
Medeiros CM, Forell F, Oliveira AT, Rodrigues JL. Current status of sperm cryopreservation: Why isn’t better. Theriogenology
Barbas JP, Mascarenhas RD. Cryopreservation of domestic animal sperm cells. Cell Tissue Bank
El-Sisy GA, El-Nattat WS, El-Sheshtawy RI. Buffalo semen quality, antioxidants and peroxidation during chilling and cryopreservation. Onl J Vet Res
Awda BJ, Mackenzie-Bell M, Buhr MM. Reactive oxygen species and boar sperm function. Biol Reprod
Câmara DR, Mello-Pinto MMC, Pinto IC, Brasil OO, Nunes JF, Guerr MMP. Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen. Small Ruminant Res
Fakurazi S, Nanthini U, Hairuszah I. Hepatoprotective and antioxidant action of Moringa oleifera
Lam. againsts acetaminophen induced hepatoxicity in rats. Int J Pharm
Luangpirom A, Junaimuang T, Kourchampa W, Somsapt P, Sritragool O. Protective effect of pomegranate (Punica granatum
Linn.) juice against hepatotoxicity and testicular toxicity induced by ethanol in mice. Anim Biol Anim Husb Int J Bioflux Soc
Foote RH, Brockett CC, Kaproth MT. Motility and fertility of bull sperm in whole milk extender containing antioxidants. Anim Reprod Sci
Salisbury GW, VanDemark NL, Lodge JR. Semen evaluation: In “Physiology of reproduction and artificial insemination of cattle
.” 2nd ed. USA: W.H. Freeman & Compagny, Sanfrancisco; 1978, p. 400-427.
SPSS. SPSS v.14.0 for Windows Evaluation Version Release 2005; 14.0.0.
Gadea J, Gumbo D, Novass C, Zquezf AZ, Grullol A, Gardo GC. Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro
fertilizing ability of frozen-thawed bull spermatozoa. Andrology
Uysal O, Bucak MN. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Vet Brno
Bucak MN, Atessahin A, Yuce A. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rum Res
El-Sheshtawy RI, El-Nattat WS, Sabra HA, Ali AH. Effect of honey solution on semen preservability of local breeds of cattle bulls. World Applied Sci J
Ortega Ferrusola C, González Fernández L, Morrell JM, Salazar Sandoval C, Macías García B, Rodríguez-Martinez H, et al. Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion dependent and is related to apoptotic-like changes. Reproduction
Baumber J, Ball BA, Gravence CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl
Agarwal A, Prahakaran SA, Said TM. Prevention of oxidative stress injury to sperm. J Androl
Aitken RJ, Baker MA. Oxidative stress and male reproductive biology. Reprod Fertil Dev
Sikka SC. Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl
Wellington K, Jarvis B. Silymarin: A review of its clinical properties in the management of hepatic disorders. Bio Drugs
[Table 1], [Table 2]