Printer Friendly

Antifungal phytochemical compounds of Cynodon dactylon and their effects on Ganoderma boninense.

INTRODUCTION

Antifungal activity from medicinal plants had been studied intensively by previous researchers [2, 25, 23]. Not only important in medicine, crude extracts of some well-known medicinal plants are also used in controlling some of the plants pathogens [24]. Despite the remarkable anti-microbial activity from medicinal plants against human fungal pathogens, it had been well acknowledged by scientific community and the agricultural practitioners that some medicinal plants also possess great potential in combating fungal plant pathogens. The uses of plant-derived products as disease control agents have been studied, since they tend to have low mammalian toxicity, less environmental effects and wide public acceptance [20]. The devastating Basal Stem Rot (BSR) disease in oil palm which caused by Ganoderma boninense is a fatal disease and considered the most serious disease affecting oil palm in South East Asia [4]. The losses caused by BSR can up to 80% after repeated planting cycles and it is also estimated about 90% of the estates in West Malaysia were reported with the presence of G. boninense [18]. Cynodon dactylon is a type of perennial grass that possesses great medicinal values. It is traditionally used as a rejuvenator, wound healers and was believed to be able to cure many diseases and infections [15]. Scientifically it has been reported to possess many pharmacological activity including antidiabetic, cardioprotective, antidiarrheal and antibacterial properties [19, 3, 1]. However, the role of C. dactylon in combating plant fungal pathogen was scantly reported. Therefore, the present study has been designed to screen the potential anti-fungal activity from some phytochemical compounds of C. dactylonagainst G. boninense.

Objectives:

This study is aimed to investigate the antifungal activity of C. dactylonSolid Phase Extraction (SPE) extract against the most devastating pathogen in oil palm, G. boninense and to identify the possible antifungal compounds via Liquid Chromatography-Mass Spectrometry (LCMS) analysis.

Materials and Methods

3.1. Plant Collection:

Wild ecotype of the plant was collected in area of Kota Kinabalu (Lat: 6.034826, Long: 116.12316), Sabah, Malaysia. Voucher (jgobilik 1090/2011) was kept in School of Sustainable Agriculture (SPL), Universiti Malaysia Sabah (UMS) and a duplicate was submitted to BORH Herbarium, Institute of Tropical Biology and Conservation (ITBC), UMS for future reference.

3.2. Cynodon dactylon Ethanol Crude Extraction:

The whole plant of C. dactylon was thoroughly cleaned using distilled water to remove soil and dirt and then dried for 24-72 hours in a drying chamber at 40-50[degrees]C to remove water content from the plant. To optimize and enhance the extraction yield, the dried plant was homogenized using a mechanical blender (Waring[R] Commercial Blender). Approximately 100g of the plant powder later was soaked into 200mL of ethanol and shaken on a platform shaker (LabCompanion[TM]) at 150 rpm at temperature of 25[degrees]C to obtain the plant extracts. The soaking process was repeated three times for each extraction to obtain a complete extraction. The extracts obtained were then evaporated and concentrated under reduced pressure (768mmhg to 7mmhg) using Rota Vapor[TM] (BUCHI) to achieve final concentration of 1g of extract per mL of solvent. The Aliquot was then kept in -20[degrees]C until further use.

3.3. Preparation of Cynodon dactylon Solid Phase Extraction (SPE) Extract:

Strata[TM]-X 33um Polymeric Sorbent reverse phase (200mg/6mL) (Phenomenex) cartridges with 12-cartridges manifold system was used. Methanol absolute (1 mL) was used to activate the sorbent and further equilibrated with sterile deionized distilled water (1 mL). Ethanol extract of C. dactylon was then loaded into the cartridges and left inside the SPE sorbent matrix for few seconds up to a minute. The loaded sample was then washed with 1% methanol (1 mL). The resulted fraction yielded from wash procedure was collected and labelled as 'flush fraction'. Finally, the remaining samples inside the SPE sorbent matrix were eluted with 2mL of methanol:acetonitrile (1:1; v/v), collected and labelled as 'elute fraction'. The aliquots were taken to dryness using purified nitrogen gas. Dried aliquots were stored in -20[degrees]C for further bioassays. Both flush and elute fractions were collected and tested for their respective antifungal activity.

3.4. Ganoderma boninense Culture:

Ganoderma boninense was isolated from

infected oil palms in Kota Marudu Sabah and their identity was molecular identified [5]. The fungal cultures were maintained on Potato Dextrose Agar (PDA) for further use.

3.5. Agar diffusion bioassay:

Agar diffusion bioassay for elute and flush fractions of C. dactylon ethanol SPE extract was conducted onG. boninense. In this bioassay, 1mL from each fraction was incorporated into their respective media (PDA) to make a final concentration of 10mg[mL.sup.-1]prior the hardening of media. The dried C. dactylon ethanol extact was first dissolved in acetone before incorporated into the media. Approximately 7-8 days old culture of G. boninense was plucked from the edge of the culture using sterile cork-boarer with 0.8cm diameter and placed at the middle of the media containing C. dactylon SPE extracts. For determination of minimum inhibitory concentrations (MICs), a series of concentrations of C. dactylon SPE extract (0, 5, 10, 20 mg[mL.sup.-1]) was incorporated into PDA. Agar without plant extract, but containing an identical concentration of acetone, served as controls. The growth of the pathogen was expressed as radial growth (cm).

3.6. Mass Analysis and Compound Identification:

The Liquid Chromatography-Mass Spectrometry (LCMS) analysis was carried out using an Agilent 1200 series coupled with Agilent 6200 series Quadrapole Time of Flight (Q-ToF) Mass Spectrometry (MS) Dual Electrospray Ionization (ESI) detector. Mass spectra analysis on elute fraction of C. dactylon SPE extract was done using the Agilent MassHunter Work station Qualitative analysis Software. In this software, few mass to charge ratio (m/z) peaks from a respective chromatogram were generated and the most abundant m/z was selected for generating the most probable mass for particular compound through Find by Molecular Feature algorithm. Each identified mass representing particular compounds was subjected to compound identification using online metabolites spectral database, METLIN (http://metlin.scripps.edu). The identity of compounds from SPE fractions was identified by matching their true molecular mass with existing chemical compound databases in METLIN. Besides, METLIN, other online databases including PubChem, KEGG and HMDB were also utilized to enhance the compounds identification.

Results:

4.1. Antifungal effects of C. dactylon against G. boninense:

Both elute and flush fractions of ethanol SPE extract are subjected to agar diffusion bioassay to determine the antifungal activity against oil palm pathogen, G. boninense. After 14 days of incubation along with continuous observation, the elute fraction of ethanol SPE extract showed greater antifungal activity via inhibition of radial growth of G. boninense in contrast to the flush fraction (Table 1). The elute fraction of C. dactylon ethanol SPE extract was able to suppress the fungal growth with more than 70% of radial growth (final growth of G. boninense = 2.34[+ or -]0.15cm) after 14 days of incubation in contrast to the control plate (final growth of G. boninense = 8.00[+ or -]0.00cm). Meanwhile, the flush fraction of the plant ethanol SPE extract was only able to suppress G. boninense growth up to approximately 38% of radial growth (final growth of G. boninense = 4.92[+ or -]0.11cm). Due to stronger antifungal activity exerts by the elute fraction, subsequent bioassay was done to determine the minimum inhibitory concentration (MIC) of the extract to fully suppress G. boninense growth. Figure 1 shows different concentration of the plant elute fraction of ethanol SPE extracts along with controls and their effect on G. boninense growth. Among four of the concentrations tested, approximately 20.00mg[mL.sup.-1] of the plant extract was able to fully suppress the fungal growth up to 14 days of incubation (final growth of G. boninense = 0.74[+ or -]0.05), although at days 14, little growth of G. boninense was observed from the plates, which might probably suggesting the overcome effect due to the degradation active compounds proposed by Gonzalez-Lamothe et al. [13]. No significant different (P < 0.05) in growth of G. boninense on control media compared to media with no SPE-elute fraction extract showed no adverse effect exert by the solvent used while no growth of G. boninense on Nystatin-containing media after the 14 days of incubation showed the fungi is not antifungal resistant.

4.2. Identification of Phytochemical compounds:

Some potential antifungal compounds were identified from the elute fraction through mass spectral analysis (Figure 2). Saponin is one of the antifungal compounds that presence in the elute fraction. Three types of saponin (Tokoronin, Ophiopogonin C and Cyclopassiflosides) were identified based on their mass spectral analysis. Meanwhile, Elemicin, one of the phenolic compounds that possess antifungal properties [29] was identified. Fatty acid and its derivatives were known to possess great antifungal activity [16, 7]. In the present study, numbers of fatty acid compounds were identified. However, only some of them were reported to possess antifungal properties such as 5-oxo-7-octenoic acid [12], Stearidonic acid [30] and 17-Hydroxylinolenic acid [31]. Neocnidilide, a carboxylic acid was also the antifungal compound [27] that identified from the elute fraction. Other compounds that reported to exhibit antifungal properties such as Gingerglycolipid B [28] and Apiole [8] were also identified from the elute fraction.

Discussion:

The present study revealed the potential of C. dactylon as an alternative source for biocontrol agent against G. boninense. The use of plants as a natural source for developing fungicide and pesticide are now getting attention due to the pitfall possess by chemicals and pesticides towards human health and environment. The finding was remarkable as the elute fraction from the extract was able to suppress the fungal growth compare to the flush fraction. Phytochemical compounds identificationbased on mass spectra analysis revealed the presence of possible antifungal compounds in elute fractions. Saponins, which are believed to form the main constituents of many plant drugs and folk medicines, and are considered responsible for numerous pharmacological properties [11]. Although to date, no report on the antifungal activity from the identified saponin compounds, numerous studies have been done previously which prove the saponins' role as antifungal agent [10, 6]. Sindambiwe et al. [26] had demonstrated the role of maesasaponin mixture which contains same class of saponins with Tokoronin, a steroidal saponin against Epidermophyton floccosum, Microides interdigitalis and Trichophyton rubrum. Li et al. [21] have shown a triterpenoidal saponin, jujubogenin saponin which also in the same class with Ophiopogonin C which found in the present study to possess antifungal activity against Candida albicans, Crytococcus neoformans and Aspergillus fumigatus. Meanwhile, triterpenoid saponins from the seeds of

Chenopodium quinoa (Chenopodiaceae) have been reported to have antifungal activity against Candida albican [32]. In this study, two triterpenoidal saponins, Cyclopassifloside V and Cyclopassifloside VII were identified from the elute fraction, which might possess same antifungal activity as reported by the previous authors. In the present work, saponins were only detected in the elute fraction, which might possibly contribute to the antifungal activity of this fraction against G. boninense. Fatty acidis one of the most interesting compound that can exhibit great biological activities. In this study, several fatty acids were identified based on mass spectral analysis. Kabarra et al. [16] have intensively discussed the role of fatty acid as antimicrobial agents. In the present study, three fatty acids (5-oxo-7-octenoic acid, Stearidonic acid and 17-hydroxylinolenic acid) were identified and might responsible for the inhibition on Ganoderma boninense growth. The finding in this study was in aggreement with the study done by Jananie et al. [14] and Kanimozhi et al. [17] which showed C. dactylon to contain fatty acid compounds such as Stearidonic acid and Hydroxylinolenic acid. From the previous works, Gershon and Shanks [12] had reported the role of 5-oxo-7-octenoic acid against Aspergillus niger, Myrotheciumverrucaria and Trichoderma viride. Thibane et al. [30] have demonstrated the antifungal activity of Stearidonic acid against C. albicans and C. dubliniensis. Meanwhile, 17-hydroxylinolenic acid had been studied for its antifungal effect against C. albicans [16], Crinipellis pernicosa, Pyrenophora avanae, Pythium ultimum, Rhizoctonia solani [31], Alternaria solani and Fusarium oxysporum [22]. It is suggested that these compounds might contribute to the antifungal activity against G. boninense and might be a significant component responsible for antifungal activity in elute fraction. In our study, the only antifungal carboxylic acid identified was Neocnidilide. Suzuki et al. [27] have demonstrated the role of Neocnidilide against mycotoxin-producing fungi. Neocnidilide might contribute to the antifungal effect in the elute fraction against G. boninense due to its antifungal effect. Other identified carboxylic acids were not reported to possess antifungal effect and they are most probably yielded from metabolic activity in the plant. Apart from the bioactive constituents discussed above, two compounds; Apiole and Gingerglycolipid B were identified from other classes of metabolites which might responsible for antifungal effect. Diego [9] have demonstrated the role of Apiole as an antifungal agent extracted from Piper auritum and Piper holtonii against Colletotrichum acutatum and Botryodiplodia theobromae. In another work, da Silva et al. [8] have found the abundance of Apiole in Piper krukoffii which might contribute to larvicidal and antifungal activity inthe plant methanol extract. Meanwhile, a glycolipid, Gingerglycolipid B was also reported by Tarawneh et al. [28] to exert exert significant antifungal effect against Cryptococcus neoformans. The present study suggests the potential of C. dactylon to be developed as a biocontrol agent against G. boninense.

Acknowledgment

The authors acknowledge their profound gratitude to the Sustainable Palm Oil Research Unit (SPOR)and School of Science and Technology, Universiti Malaysia Sabahor providing the facilities for research work.

Financial Disclosure:

There is no conflict of interest.

Funding/Support:

The study was financially supported by Fundamental Research Grant Scheme (FRGS), Ministry of Education Malaysia.

Received: 25 June 2014; Received: 8July 2014; Accepted: 10 August May 2014; Available online: 30 August 2014

References

[1.] Abdullah, S., J. Gobilik, K.P. Chong 2012. Preliminary phytochemical study and antimicrobial activity from various extract of cynodon dactylon (L.) pers.(bermuda) against selectedpathogens. International Journal of Pharmacy & Pharmaceutical Sciences, 4(5): 227-230.

[2.] Arif, T., J.D. Bhosale, N. Kumar, T.K. Mandal, R.S. Bendre, G.S. Lavekar, R. Dabur, 2009. Natural products-antifungal agents derived from plants. Journal of Asian Natural Products Research, 11(7): 621-638.

[3.] Babu, D.S.R., V. Neeharika, V. Pallavi, M.B. Reddy, 2009. Antidiarrheal activity of Cynodon dactylon. Pers, 5(19): 23-27.

[4.] Chong, K.P., M. Atong, S. Rossall, 2012. The roles of syringic, caffeic and 4- hydroxybenzoic acids in Ganoderma-oil palm interaction. Asian Journal of Microbiology, Biotechnology and Environmental Sciences, 14(2):157-166.

[5.] Chong, K.P., M.S. Lum, C.P. Foong, C.M.V.L. Wong, M. Atong, S. Rossall, 2013. First identification of Ganoderma boninense isolated from Sabah based on PCR and sequence homology. African Journal of Biotechnology, 10(66): 14718-14723.

[6.] Coleman, J.J., I. Okoli, G.P. Tegos, E.B. Holson, F.F. Wagner, M.R. Hamblin, E. Mylonakis, 2010. Characterization of plant-derived saponin natural products against Candida albicans. ACS Chemical Biology, 5(3): 321-332.

[7.] Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12(4): 564-582.

[8.] da Silva, J.K., E.H. Andrade, M.J. Kato, L.M. Carreira, E.F. Guimaraes, J.G. Maia, 2011. Antioxidant capacity and larvicidal and antifungal activities of essential oils and extracts from Piper krukoffii. Natural Product Communications, 6(9): 1361-1366.

[9.] Diego, L., 2012. Chemical composition and antifungal activity of Piper auritum Kunth and Piper holtonii C. DC. against phytopathogenic fungi. Chilean Journal of Agricultural Research, 72(4): 507-515.

[10.] Escalante, A.M., C.B. Santecchia, S.N. Lopez, M.A. Gattuso, A. Gutierrez Ravelo, F. Delle Monache, M.G. Sierra, S.A. Zacchino, 2002. Isolation of antifungal saponins from Phytolacca tetramera, an Argentinean species in critic risk. Journal of Ethnopharmacology, 82(1): 29-34.

[11.] Estrada, A., G.S. Katselis, B. Laarveld, B. Barl, 2000. Isolation and evaluation of immunological adjuvant activities of saponins from Polygala senega L. Comparative Immunology, Microbiology and Infectious Diseases, 23(1): 27-43.

[12.] Gershon, H. and L. Shanks, 1978. Antifungal activity of fatty acids and derivatives: structure-activity relationships. In: Kabara, J.J. (eds). The Pharmacological Effect of Lipids. Champaign, IL: American Oil Chemists' Society, pp: 51-62.

[13.] Gonzalez-Lamothe, R., G. Mitchell, M. Gattuso, M.S. Diarra, F. Malouin, K. Bouarab, 2009. Plant antimicrobial agents and their effects on plant and human pathogens. International Journal of Molecular Sciences, 10(8): 3400-3419.

[14.] Jananie, R.K., V. Priya, K. Vijayalakshmi, 2011. Determination of bioactive components of Cynodon dactylon by GCMS analysis. New York Science Journal, 4: 16-20.

[15.] Joy, P.P., J. Thomas, S. Mathew, B.P. Skaria, 1998. Medicinal plants. Tropical horticulture, 2: 449-632.

[16.] Kabara, J.J., D.M. Swieczkowski, A.J. Conley, J.P. Truant, 1972. Fatty acids and derivatives as antimicrobial agents. Antimicrobial Agents and Chemotherapy, 2 (1): 23-28.

[17.] Kanimozhi, D., V.R. Bai, 2012. Evaluation of Phytochemical Antioxidant Antimicrobial Activity Determination of Bioactive Components of Ethanolic Extract of Aerial And Underground Parts of Cynodon dactylon L. International Journal of Scientific Research and Reviews, 1: 33-48.

[18.] Khairuddin, H., T.C. Chong, 2008. An overview of the current status ofGanodermabasal stem rot and its management in a large plantation group in Malaysia. The Planter, 84: 469-482.

[19.] Kumar, A., P. Kashyap, H. Sawarkar, B. Muley, A. Pandey, 2011. Evaluation of Antibacterial Activity of Cynodon dactylon (L.) Pers. International Journal of Herbal Drugs Research, 1(2): 31-35.

[20.] Lee, S.O., K.S. Jang, K.Y. Cho, 2007. Antifungal activity of five plant essential oils as fumigant against postharvest and soilborne plant pathogenic fungi. The Plant Pathology Journal, 23(2): 97-102.

[21.] Li, X.C., H.N. ElSohly, A.C. Nimrod, A.M. Clark, 1999. Antifungal Jujubogenin Saponins from Colubrina retusa. Journal of Natural Products, 62(5): 674-677.

[22.] Liu, S., R. Weibin, L. Jing, X. Hua, W. Jingan, G. Yubao, W. Jingguo, 2008. Biological control of phytopathogenic fungi by fatty acidsMycopathologia, 166: 93-102.

[23.] Parveen, S., A.H. Wani, A.A. Ganie, S.A. Pala, R.A. Mir, 2014. Antifungal activity of some plant extracts on some pathogenic fungi. Archives of Phytopathology and Plant Protection, 14(3): 1-6.

[24.] Quiroga, E.N., A.R. Sampietro, M.A. Vattuone, 2001. Screening antifungal activities of selected medicinal plants. Journal of Ethnopharmacology, 74(1): 89-96.

[25.] Ravikumar, M.C., R.H. Garampalli, 2013. Antifungal activity of plants extracts against Alternaria solani, the causal agent of early blight of tomato. Archives of Phytopathology and Plant Protection, 46(16): 1897-1903.

[26.] Sindambiwe, J.B., M. Calomme, S. Geerts, L. Pieters, A.J. Vlietinck, D.A. Vanden Berghe, 1998. Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata. Journal of Natural Products, 61(5): 585-590.

[27.] Suzuki, H., A. Tanaka, K. Yamashita, 1987. Synthesis and Absolute Configuration of Neocnidilide (Organic Chemistry). Agricultural and Biological Chemistry, 51(12): 3369-3373.

[28.] Tarawneh, A.H., F. Leon, M.M. Radwan, L.H. Rosa, S.J. Cutler, 2013. Secondary metabolites from the fungus Emericella nidulans. Natural product communications, 8(9): 1285-1288.

[29.] Tavares, A.C., M.J. Gonsalves, C. Cavaleiro, M.T. Cruz, M.C. Lopes, J. Canhoto, L.R. Salgueiro, 2008. Essential oil of Daucus carota subsp. halophilus: Composition, antifungal activity and cytotoxicity. Journal of Ethnopharmacology, 119(1): 129-134.

[30.] Thibane, V.S., J.L.F. Kock, S.R. Ell, P.W.J. Van Wyk, C.H. Pohl, 2010. Effect of marine polyunsaturated fatty acids on biofilm formation of C. albicans and C. dubliniensis. Marine Drugs, 8: 2597-2604.

[31.] Walters, D., L. Raynor, A. Mitchell, R. Walker, K. Walker, 2004. Antifungal activities of four fatty acids against plant pathogenic fungi. Mycopathologia, 157:87-90.

[32.] Woldemichael, G.M., M. Wink, 2001. Identification and biological activities of triterpenoid saponins from Chenopodium quinoa. Journal of Agricultural and Food Chemistry, 49(5): 2327-2332.

(1) Syahriel Abdullah, (2) Januarius Gobilik, (1) Khim-PhinChong

(1) Sustainable Palm OH Research unit (SPOR), School of Science and Technology, Universiti Malaysia Sabah

(2) School of Sustainable Agriculture, Universiti Malaysia Sabah

Corresponding Author: Khim-PhinChong, Sustainable Palm Oil Research unit (SPOR), School of Science and Technology, Universiti Malaysia Sabah.

Tel: 60-88320000; ext: 5571; Fax: 60-88435324; E-mail: chongkp@ums.edu.my

Table 1: Agar diffusion bioassay of C. dactylon ethanol SPE elute
and flush fractions against G. boninense. The efficacy of the
extracts was measured by the ability of the extracts to inhibit
the fungal radial growth.

Day     Growth * of G.borunense in different         Control
             ethanol SPE extract fractions

              A                    B

1     0.70 [+ or -] 0.00   0.70 [+ or -] 0.00   0.7 [+ or -] 0.00
2     0.70 [+ or -] 0.00   0.70 [+ or -] 0.00   0.7 [+ or -] 0.00
3     0.70 [+ or -] 0.00   0.70 [+ or -] 0.00   0.7 [+ or -] 0.00
4     0.84 [+ or -] 0.05   0.70 [+ or -] 0.00   1.00 [+ or -] 0.11
5     1.18 [+ or -] 0.04   0.70 [+ or -] 0.00   1.80 [+ or -] 0.12
6     1.56 [+ or -] 0.05   0.70 [+ or -] 0.00   3.50 [+ or -] 0.16
7     1.96 [+ or -] 0.04   0.70 [+ or -] 0.00   5.10 [+ or -] 0.23
8     2.68 [+ or -] 0.08   0.70 [+ or -] 0.00   7.30 [+ or -] 0.05
9     3.77 [+ or -] 0.09   0.70 [+ or -] 0.00   7.90 [+ or -] 0.04
10    4.61 [+ or -] 0.09   0.76 [+ or -] 0.09   8.00 [+ or -] 0.00
11    4.78 [+ or -] 0.09   1.00 [+ or -] 0.07   8.00 [+ or -] 0.00
12    4.83 [+ or -] 0.11   1.48 [+ or -] 0.08   8.00 [+ or -] 0.00
13    4.90 [+ or -] 0.11   1.96 [+ or -] 0.11   8.00 [+ or -] 0.00
14    4.92 [+ or -] 0.11   2.34 [+ or -] 0.15   8.00 [+ or -] 0.00

* After 14 days of incubation, elute fraction of C. dactylon
ethanol SPE extract showed stronger antifungal activity against
G. boninense. Growth of G.bormensevias measured in cm base on
radial growth; the concentration of both elute and flush
fractions of EtOH SPE extracts are 10mg[mL.sup.-1]; Values
presented are means of five replicates, [+ or -] stand, dev.

A=Ethanol SPE flush fraction; B= Ethanol SPE elute fraction
COPYRIGHT 2014 American-Eurasian Network for Scientific Information
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Research Article
Author:Abdullah, Syahriel; Gobilik, Januarius; Khim-PhinChong
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9MALA
Date:Aug 30, 2014
Words:3687
Previous Article:The potential of local trees for phytostabilization of heavy metals in gold cyanidation tailing contaminated soils of West Lombok, Indonesia.
Next Article:Effect of different centrifugation duration on Simmental bull sperm quality and membrane status after sexing, cooling and freezing processes.
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters |