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Department of Biological Sciences, National University of Singapore, 14, Science Drive 4, Singapore 117543
2To whom correspondence should be addressed. E-mail: dbsdjl{at}nus.edu.sg.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • yeast recombinant vitellogenin • Artemia enrichment • live yeast-feed for fish larvae • larval growth and survival
Larval nutrition is a key factor to successful larviculture. Sound nutrition promotes reproductive success and healthy larval development and is essential in the production of high-quality aquaculture products. In finfish, the yolk provides nutrition during embryonic development and early ontogenesis. The transition from endogenous to exogenous food supply at first feeding marks a critical phase during which high mortality may occur. Unfortunately, larval diets are difficult to evaluate, and current aquaculture practices are not always optimal for larval rearing. Suboptimal nourishment of the larvae due to improper or inadequate diets is a major cause of poor survival. Despite the clear importance of nutrition in influencing growth and survival, the nutrient requirements of the aquatic larvae remain poorly understood.
Presently, rotifer and Artemia are the quintessential live-food organisms for intensive culture of most first-feeding aquatic larvae before the larvae can be weaned onto artificially formulated diets. However, these live-preys are relatively costly but nutritionally incomplete (1). Variations of nutrient quality, primarily in the (n-3) highly unsaturated fatty acids (HUFA),3 which are essential for larval growth and survival, necessitate their enrichment before they can be offered to the larvae (2–4). To reduce dependence on live-food, countermeasures such as shortening of the preweaning period (5) and development of complete artificial diets for precocious cofeeding (6) were undertaken with some success. However, the exclusive use of artificial dry diets as first feed was deemed unsuitable until Cahu et al. (7) succeeded in developing a complete feed containing yeast and fish protein hydrolysate for the seabass larvae, totally replacing live-prey. Nevertheless, providing an acceptable first food with reliable availability, proper size and nutrient composition, remains a major challenge in aquaculture science.
Yeast single-cell proteins (SCPs) are playing a greater role in the evolution of aquaculture diets. With excellent nutrient profiles and capacity to be mass produced economically, SCPs have been added to aquaculture diets as partial replacement for fishmeal (8–10) and for HUFA-fortification of rotifer and Artemia (2). Some yeast strains with probiotic properties, such as Saccharomyces cerevisiae (SC) (11) and Debaryomyces hansenii (12), boost larval survival either by colonizing the gut of fish larvae, thus triggering the early maturation of the pancreas, or via the immuno-stimulating glucans derived from the yeast cell wall (13,14). However, many of these yeast supplements are deficient in sulfated amino acids, particularly methionine (11), which restricts their extensive use as the sole protein source.
Recently, we cloned a recombinant Pichia pastoris SMD1168H (SMD) capable of producing recombinant vitellogenin (rVtg), the precursor yolk protein of a tilapia fish (15). In the form of a large precursor protein, Vtg serves as a serum carrier that transports micronutrients into the oocytes. Within the oocytes, Vtg undergoes cleavage to form the storage yolk lipoproteins, which are the primary nutrient source of aquatic larvae. Interestingly, the rVtg clone has a high content of methionine and arginine, both of which are essential to fish (16). In addition to the high lipid content, it contains docosahexaenoic acid (DHA) [22:6(n-3)], which is absent in the host yeast.
With an aim of coalescing the broad spectrum of benefits that yeasts offer as SCP and the high nutrient value of the yolk proteins, we evaluated the prospects of using the yolk-enriched rVtg yeast as an alternative means of bioenriching Artemia and as a live-yeast feed for fish larvae. Considering that the endogenous yolk contains nutrients for sustaining the prefeeding larvae until first feeding, Heming and Buddington (17) logically proposed that the yolk content should reflect the nutritional and metabolic requirements of the prefeeding fish. We reasoned that an optimal first feed should simulate the yolk composition. As such, we aimed to use the rVtg-containing P. pastoris as a vehicle to deliver the rVtg protein either directly to the fish larvae or indirectly via Artemia. The exogenous rVtg protein provides an amino acid pool that mimics the natural yolk protein reserves. These supplements may buffer the abrupt switch of nutrient composition when the postlarvae start to feed on live-prey. This is the first report on the exclusive rearing of first-feeding fish larvae on a live yolk-producing P. pastoris. Contrary to the common notion that fish larvae are too large to ingest yeast directly, pure rVtg-based diets promote larval growth and survival.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Yeast cultures. P. pastoris strain SMD 1168H (SMD) and lyophilized baker’s yeast S. cerevisiae (SC), were obtained from Invitrogen and DCL, respectively. FL(-ss)pGAPzA in SMD1168H/500ZR #6, the high producer recombinant vitellogenin yeast clone (rVtg) previously selected for optimal intracellular expression of Vtg, was either cultured in BYPD medium or BYPD supplemented with 5% pure fish oil as described by Ding et al. (15). The latter culture, henceforth referred to as rVtg in 5% fish oil (rVtg-FO), was repeatedly washed to remove any unconsumed and adherent fish oil. The cells were freeze-dried using a FLEX1-DRYTM freeze drier, sealed, and kept at 4°C.
Artemia culture and enrichment. Artemia cysts were incubated at room temperature (26–30°C) with strong aeration, in 10 L of filtered seawater (30 g/L) held in a 15 L conical glass container. After 36 h, newly hatched nauplii were separated from the empty cysts and debris and transferred to clean filtered seawater with aeration. At instar II stage, the nauplii were sampled and counted for estimation of density. The nauplii were either starved or fed in 1 dose at 0.5 µg yeast/(Artemia · d) with 1 of the following yeasts: 1) SMD; 2) SC; 3) rVtg; or 4) rVtg-FO. The enrichment concentration for Artemia was established on the basis of earlier trials using various yeast dosages under laboratory culture conditions. Higher concentrations of yeasts caused the Artemia culture to crash. The enrichment was extended for 48 h because sampling of the enriched Artemia at 24 h showed no change in the PUFA contents. Before feeding, the freeze-dried yeasts were separately mixed to homogeneity in a small amount of water. A quantity sufficient for 3–5 d was prepared each time and kept at –30°C. Each dietary treatment was repeated 3 times using separate tanks. At 48 h, 50,000 unenriched or yeast-enriched Artemia were harvested on a nylon mesh. The Artemia were rinsed once with distilled water, blotted on absorbent tissue, transferred to a cryovial, and stored at –30°C for further analyses.
Lipid and fatty acid analyses. Total lipids were extracted from 1 g of each Artemia sample according to Folch et al. (18). Fatty acid analyses were performed as described by Ding et al. (15).
Fish larvae and experimental diets. Broodstock tilapia, Oreochromis mossambicus, were collected from the culture tanks. Fertilized eggs were retrieved from the mouth of the broodfish and were pooled from several fish to obtain the required quantity. They were then placed in a 3-L glass cylinder tank. The water was gently aerated and changed daily to facilitate hatching and to avoid fungal contamination. Hatched larvae were randomly distributed into separate 3-L flat-bottomed cylinder glass tanks at a density of 10 larvae/L; tanks were maintained between 26 and 30°C and were given gentle and constant aeration. Yolk-sac larvae at d 3 were used as the first-feeding larvae in all experiments. The procedures used in handling the broodfish and larvae complied with the guidelines stipulated by the National Advisory Committee for Laboratory Animal Research, Singapore.
Six diets were fed to separate groups of larvae, using a completely randomized design (Table 1). Treatment 1: between d 3 and 22, 4 groups of larvae were separately fed 1 type of live yeast, referred to as the "Single Yeast" diet: (i) SMD; (ii) SC; (iii) rVtg; or (iv) rVtg-FO. Treatment 2: 4 groups of larvae were each fed as in treatment 1 between d 3 and 12, followed by the freshly hatched unenriched Artemia between d 13 and 22. This regimen is termed the "Single Yeast >> unenriched Artemia" diet. Treatment 3: between d 3 and 22, 3 groups of larvae were each fed different combinations of yeasts (1:1) comprising either SC:SMD; SC:rVtg; or SC:rVtg-FO. This regimen is termed the "Combined Yeast" diet. Treatment 4: 3 groups of larvae were each fed from d 3 to 12 as in treatment 3, then weaned with unenriched Artemia from d 13 to 22. This diet is termed the "Combined Yeast >> unenriched Artemia" diet. Treatments 5 and 6: 4 groups of larvae were each fed either SC (treatment 5) or rVtg (treatment 6) from d 3 to 12 followed by Artemia that had been preenriched for 48 h with either SMD, SC, rVtg, or rVtg-FO from d 13 to 22. Treatment 5 is termed the "SC >> Yeast-enriched Artemia" diet and treatment 6 is termed the "rVtg >> Yeast-enriched Artemia" diet. The yeast and Artemia diets were both fed as 1 ration/d, at a density of 1 mg yeast/larva and 5 Artemia/mL, respectively.
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Statistical analysis. Differences in the total lipid and fatty acid composition among the experimental groups of Artemia were analyzed by 1-way ANOVA using WINKS Windows KWIKSTAT version 4.651 (TexaSoft Software). Differences between means were compared using Newman-Keuls multiple test. The data for tilapia larval length, weight, and survival were subjected to 1-way ANOVA with Tukey’s tests using Prism 4 for Windows, version 4.02 (GraphPad Software). The normality and variance homogeneity of the data were examined using D’Agostino and Pearson omnibus normality and Barlett’s tests, respectively. Percentage data, and data identified as nonhomogeneous were arcsine- and log-transformed, respectively, before ANOVA. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The increment of >90% of total lipids in the Artemia fed rVtg or rVtg-FO compared with the freshly hatched may be ascribed to the high lipid nature of the rVtg yeast. The rVtg was shown to contain 47% more total lipid than the host P. pastoris (SMD), in addition to being enriched with essential fatty acids (EFA) such as [18:2(n-6)], [20:5(n-3)], and [22:6(n-3)] (15). Overall, enrichment with rVtg or rVtg-FO improved the PUFA contents in the Artemia. However, the low DHA:EPA ratio of 0.1 and 0.2 attained by simple feeding of the Artemia with rVtg or rVtg-FO was far below the optimal dietary ratio of 2.0 as suggested for the seabass larvae (4). For many fish larvae, this low ratio would not sustain growth. Nevertheless, there are large variations in EFA requirements for different fish species and developmental stages. Previous findings on EFA requirements of the different tilapia species were variable. Although an earlier report documented that the Nile tilapia and Zilli’s tilapia did not need [20:5(n-3)] and [22:6(n-3)] (19), recent studies showed that the hybrid tilapia required these 2 HUFA in addition to [18:2(n-6)] (20,21). The quantitative requirements of the tilapia O. mossambicus, especially at the larval stage, have not yet been determined. Nonetheless, the high lipid content in the rVtg yeast and rVtg-enriched Artemia most likely favors larval development and survival because high dietary fat was shown to facilitate larval development in a study of the seabass (22).
This study demonstrated that feeding diets containing only rVtg-based yeasts rather than native SC or SMD between d 3 and 22, could sustain significantly greater growth and survival of the tilapia larvae. The growth parameters did not differ between the larvae fed rVtg or rVtg-FO, indicating that the inclusion of fish oil in the culture of rVtg is optional. The nutritional values of the rVtg yeasts appeared to surpass SC. This is reflected in the greater growth and survival of the larvae concurrently fed rVtg and SC than those fed only SC. The rVtg yeast contains low levels of [22:6(n-3)], which is lacking in the host yeasts, and higher contents of total amino acids and lipids, in particular methionine (98%) and arginine (80%), which are essential to fish (16). Because the 4 yeast diets were similarly reconstituted from lyophilized cultures, the growth difference attained with the rVtg and the SC or SMD diets likely arose from nutritional factors rather than variability in ingestion preference and digestibility of different yeasts. Although this study did not quantify the ingestion of the expressed rVtg by the tilapia larvae, immunocytochemical evidence (23) showed that the epithelial cells of the posterior intestine in fish larvae are capable of absorbing the lipovitellin or its resulting fragments from the Artemia. Nevertheless, the healthy growth manifested in the larvae fed the rVtg-based diets provides evidence for the importance of fortifying the first feed with yolk precursor proteins.
Prolonged feeding with pure yeast diets (d 3 to 22) delayed larval growth. This is in agreement with findings for seabass and striped bass larvae (7,10). Regardless of the yeast strains and fish species, inclusion of yeast in larval or juvenile diets typically lowers growth rates, although the survival rate is acceptable. On the basis of the results of our pilot-feeding trials, we deduce that to avoid compromising growth, the administration of pure live yeast diets in the form of yeast suspension is optimal if fed to d 3 larvae for a maximum of 10 d. The reduced growth experienced with 20 d of yeast feeding was likely due to inadequate ingestion because the yeast diets were supplied in the same form of suspension and at the same feeding ration throughout. Over time, the larvae increased rapidly in size and required diets of greater density and energy. Thus, the yeast suspension may not be sufficient to meet their nutrient requirements. Holt (6) reported that red drum larvae fed a yeast microdiet could achieve a growth rate comparable to those reared on live prey if they were fed progressively larger sizes of yeast microdiet. Thus, for future feeding trials, careful considerations must be given to the development of various sizes of rVtg micropellet.
We observed a consistent trend of growth acceleration by transferring the yeast-fed larvae to an Artemia diet, either in the form of newly hatched, or after yeast enrichment. Without HUFA enrichment, the newly hatched Artemia is DHA deficient and considered nutritionally inadequate to meet the demands of larval growth. Given the same initial diet of rVtg, a subsequent dietary switch to unenriched or yeast-enriched Artemia appeared to be both nutritionally adequate in promoting satisfactory larval catch-up growth. This implies that an initial rVtg-based diet (d 3–12) is adequate to overcome any nutritional deficiency in Artemia without any further enrichment. When unenriched Artemia was given as the weaning diet, the magnitude of growth acceleration appeared to be dependent on the prefeed. Comparing the use of rVtg-based diets alone with the combination of SC:rVtg or SC:rVtg-FO as the initial feeds, the length increment (
80% vs. 55%) and weight gain (>5.5-fold vs. 3-fold) were clearly better when rVtg was used as prefeed. This suggests that the rVtg yeast is an ideal starter diet that could improve the feed conversion efficiency in the subsequent diet.
This is the first time a diet comprising yolk-expressing yeast was used as a unifeed for tilapia larvae. Although tilapia larvae were shown to grow effectively when fed other raw SCP diets such as Spirulina platensis, the acceptability generally improved with larger larvae (24). The high survival within 22 d of larval life could be attributed to the nutritional "rVtg factors" and the P. pastoris "yeast factors." Although the present study did not address the mechanism by which the live rVtg yeast aided in sustaining survival, it is conceivable that the yolk-fortified yeast with probable probiotic properties provided an optimal balance of nutrients compared with the native yeasts. The rVtg protein, a phosphoglycolipoprotein by nature, may provide a pool of dietary phospholipids close to those found in the natural yolk. The growth-promoting effects of dietary phospholipids in larvae (25,26) and the immunostimulating properties of the P. pastoris cell wall (27) were verified.
Although the rVtg yeast diet cannot yet be considered as a complete substitute for the planktonic live-food, we observed that rearing of the first-feeding larvae on rVtg yeast, before weaning onto live-food, was feasible. The unique yolk-producing yeast has diverse applications as a partial substitute for live-food in larval feeding and for micro-algae in Artemia enrichment. We believe that Vtg, which carries the components of yolk proteins, should continue to be nutritionally appropriate for post-yolk sac larvae, particularly during the weaning period ("mixed-feeding phase"), when the larvae start to feed. This is especially so when appropriate or adequate food is not available as may be the case in an aquaculture practice. Furthermore, because rVtg yeast is nutritionally superior to the SC that is commonly incorporated into existing formulated diets for aquatic species at other developmental stages, it may be a potential replacement for SC on an isonitrogenous basis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DHA, docosahexaenoic acid; EFA, essential fatty acids; EPA, eicosapentaenoic acid; HUFA, highly unsaturated fatty acids; MUFA, monounsaturated fatty acids; rVtg, recombinant vitellogenin yeast clone; rVtg-FO, rVtg precultured in 5% fish oil; SC, Saccharomyces cerevisiae; SCP, single-cell proteins; SMD, Pichia pastoris SMD1168H.
Manuscript received 22 July 2004. Initial review completed 28 August 2004. Revision accepted 16 December 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Conceição, L.E.C., Grasdalen, H. & Rønnestad, I. (2003) Amino acid requirements of fish larvae and post-larvae: new tools and recent findings. Aquaculture 227:221-222.
2. McEvoy, L. A., Navarro, J. C., Hontoria, F., Amat, F. & Sargent, J. R. (1996) Two novel Artemia enrichment diets containing polar lipid. Aquaculture 134:339-352.
3. Rainuzzo, J. R., Reitan, K. I. & Olsen, Y. (1997) The significance of lipids at early stages of marine fish: a review. Aquaculture 155:103-115.
4. Sargent, J., McEvoy, L., Estevez, A., Bell, G., Bell, M., Henderson, J. & Tocher, D. (1999) Lipid nutrition of marine fish during early development: current status and future directions. Aquaculture 179:217-229.
5. Person Le Ruyet, J., Alexandre, J. C., Thébaud, L. & Mugnier, C. (1993) Marine fish larvae feeding: formulated diets or live prey?. J. World Aquacult. Soc. 24:211-224.
6. Holt, G. J. (1993) Feeding larval red drum on microparticulate diets in a closed recirculating water system. J. World Aquacult. Soc. 24:225-230.
7. Cahu, C., Zambonino Infante, J., Escaffre, A.-M., Bergot, P. & Kaushik, S. (1998) Preliminary results on sea bass (Dicentrarchus labrax) larvae rearing with compound diet from first feeding. Comparison with carp (Cyprinus carpio) larvae. Aquaculture 169:1-7.
8. Coutteau, P. & Lavens, P. (1989) The use of yeast as a single-cell protein in aquacultural diets. Artemia Reference Center, Med. Fac. Landbouww. Rijksuniv. Gent. 54:1583-1592.
9. Olvera-Novoa, M. A., Martínez-Palacios, C. A. & Olivera-Castillo, L. (2002) Utilization of torula yeast (Candida utilis) as a protein source in diets for tilapia (Oreochromis mossambicus Peters) fry. Aquacult. Nutr. 8:257-264.
10. Li, P. & Gatlin, D. M., III (2003) Evaluation of brewers yeast (Saccharomyces cerevisiae) as a feed supplement for hybrid striped bass (Morone chrysops x M. saxatilis). Aquaculture 219:681-692.
11. Oliva-Teles, A. & Gonçalves, P. (2001) Partial replacement of fishmeal by brewers yeast (Saccharomyces cerevisiae) in diets for sea bass (Dicentrarchus labrax) juveniles. Aquaculture 202:269-278.
12. Tovar, D., Zambonino, J., Cahu, C., Gatesoupe, F. J., Vázquez-Juárez, R. & Lésel, R. (2002) Effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae. Aquaculture 204:113-123.
13. Campa-Córdova, A. I., Hernández-Saavedra, N. Y., De Philippis, R. & Ascencio, F. (2002) Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to ß-glucan and sulphated polysaccharide. Fish Shellfish Immunol. 12:353-366.[Medline]
14. Burgents, J. E., Burnett, K. G. & Burnett, L. E. (2004) Disease resistance of Pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 231:1-8.
15. Ding, J. L., Lim, E. H., Li, H. F., Kumar, J. K., Lee, S. L. & Lam, T. J. (2004) Expression of recombinant vitellogenin in the yeast Pichia pastoris. Biotechnol. Bioeng. 85:330-339.[Medline]
16. Metabolism. De Silva, S. S. Anderson, T. A. eds. Fish Nutrition in Aquaculture (Aquaculture Series I) 1995:41-102 Chapman & Hall London, UK. .
17. Heming, T. A. & Buddington, R. K. (1988) Yolk absorption in embryonic and larval fishes. Hoar, W. S. Randall, D. J. eds. Fish Physiology 1988:407-446 Academic Press XI London, UK. .
18. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.
19. Sargent, J., Henderson, R. J. & Tocher, D. R. (1989) The lipids. Halver, J. E. eds. Fish Nutrition 2nd ed. 1989:153-218 Academic Press New York, NY. .
20. Chou, B.-S. & Shiau, S.-Y. (1999) Both n-6 and n-3 fatty acids are required for maximal growth of juvenile hybrid tilapia. N. Am. J. Aquac. 61:13-20.
21. Chou, B.-S., Shiau, S.-Y. & Hung, S.S.O. (2001) Effect of dietary cod liver oil on growth and fatty acids of juvenile hybrid tilapia. N. Am. J. Aquac. 63:277-284.
22. Zambonino Infante, J. L. & Cahu, C. L. (1999) High dietary lipid levels enhance digestive tract maturation and improve Dicentrarchus labrax larval development. J. Nutr. 129:1195-1200.
23. Kishida, M., Johanning, K. M., Bengtson, D. A. & Specker, J. L. (1998) Intestinal uptake of lipovitellin from brine shrimp (Artemia franciscana) by larval inland Silversides (Menidia beryllina) and striped bass (Morone saxatilis). Comp. Biochem. Physiol. 119A:415-421.
24. Lu, J., Yoshizaki, G., Sakai, K. & Takeuchi, T. (2002) Acceptability of raw Spirulina platensis by larval tilapia Oreochromis niloticus. Fish. Sci. 68:51-58.
25. Coutteau, P., Geurden, I., Camara, M. R., Bergot, P. & Sorgeloos, P. (1997) Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155:149-164.
26. Fontagné, S., Geurden, I., Escaffre, A.-M. & Bergot, P (1998) Histological changes induced by dietary phospholipids in intestine and liver of common carp (Cyprinus carpio L.) larvae. Aquaculture 161:213-223.
27. Rodríguez Valle, M., Montero, C., Machado, H. & Joglar, M. (2001) The evaluation of yeast derivatives as adjuvants for the immune response to the Bm86 antigen in cattle. BMC Biotechnol. 1:2.[Medline]
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