Abstract Circadian variation of serum concentrations of tylosin in broiler chickens after in-feed medication prompted a comparison study of the serum profiles of this drug after in-feed medication with standard tylosin phosphate (Tprf reference formulation group), and after in-feed medication with a sustained-release pellet formulation (Tpsr group), based on Patent No.MX/a/2012/013222 and PCT/MX2013/000137, in broiler chickens. Six hundred 4-week-old Ross broiler chickens were in-feed medicated with tylosin phosphate at an approximate dose of 25.2 mg/kg/d, based on daily feed consumption values and a final concentration of tylosin in feed of 200 mg/kg of feed. Approximately 2 to 3 mL of blood were obtained per 5 chickens every 2 h, avoiding the sampling of a bird more than once and during 72 h after making medicated feed available for the first time. Serum concentrations of tylosin were determined by HPLC. Gaussian multi-peak regressions were then fitted to serum concentration vs. time profiles. Day by d areas under the serum concentration vs. time profiles (AUC0–24), as well as overall AUC0–72, were statistically higher for the Tpsr group (P < 0.001). Also, maximum serum concentrations obtained and relative bioavailability for the Tpsr formulation were statistically higher (382.8%) as compared to the Tprf group (P < 0.01). Considering the referred improved values of AUC observed in the Tpsr formulation, as well as the fact that tylosin is a time-dependent antibacterial drug, better clinical responses are postulated with this pharmaceutical preparation intended for chickens. Tissue deposition studies for this new formulation of tylosin are required. DESCRIPTION OF PROBLEM Using a standard tylosin phosphate (Tp) premix as in-feed medication for chickens tends to yield circadian patterns of serum concentrations of the drug with troughs during the dark part of the daily dark: light cycle. The use of an experimental sustained-release formulation of Tt for in-feed medication, based on the gastro-retentive properties of its vehicle, maintains serum concentrations above minimum inhibitory concentration (MIC) values for the treatment of mycoplasmosis at night and increases overall bioavailability. This complies better with pharmacokinetic/pharmacodynamic (PK/PD) ratios required for tylosin and may prove useful in some resistant bacterial infections. Mycoplasma gallisepticum and M. synoviae are economically important pathogens in poultry production [1, 2]. In particular, M. gallisepticum has maintained a constant presence in commercial broiler and layer flocks around the world, causing problems mainly in the bird's respiratory apparatus. However, both also have been responsible for salpingitis, reduced egg production, and poor egg quality in commercial layer flocks [1, 2]. In spite of biosecurity practices in all stages of poultry production, eradication of Mycoplasma sp. is seldom successful . Hence, prophylaxis and treatment of mycoplasmosis are common courses of action to minimize economic losses caused by this disease. The macrolide class of antibacterial agents is often regarded as drugs of choice, particularly tylosin . PK/PD studies on tylosin have regarded this antibacterial drug as time dependent (t-d), i.e., ideally, serum concentration should be at or above the MIC level during the whole dose interval (T ≥ MIC 100% DI) [4, 5]. Although clinical results using tylosin are generally good, there is always room for better pharmaceutical design. For example, it has been shown that tylosin exhibits circadian-type serum concentration profiles in broiler chickens, with low serum concentrations of tylosin at night, due to the short half-life of the drug and the feeding and drinking habits of broilers . Such a biphasic serum profile of tylosin fails to optimize drug efficacy and favors the emergence of microbial resistance [7–9]. Hence, it appears reasonable to postulate that administering tylosin to chickens as a sustained-release pharmaceutical formulation, may optimize bioavailability and consequently its clinical efficacy, targeting serum concentrations of tylosin at or above minimum inhibitory concentrations as proposed for tylosin in this study (0.1 μg/mL)  during the whole dosing interval (T ≥ MIC 100% DI). Hence, the purpose of this study was to evaluate a novel dosage form of tylosin phosphate, named FOLA (F = bioavailability; O = optimal; LA = long acting) Patent No.MX/a/2012/01,3222 and PCT/MX2013/000137 (National Autonomous University of Mexico [UNAM]), aimed at complying better with optimal PK/PD ratios ascribed to this antibacterial drug. MATERIALS AND METHODS Experimental Birds This study was approved by the Institutional Committee of Research, Care and Use of Experimental Animals (CICUAL) of the UNAM, according to Mexican Official Regulation NOM-062-ZOO-2001. The trial was carried out in a commercial poultry house located near Mexico City (2,250 m above sea level; 19 240 N; 99 120 W). For the study, a total of 600, 4-week-old Ross broiler chickens, having a mean weight of 754 g ± 4.6 g SD, was separated from the flock by means of wire mesh and divided into 2 groups with equal numbers of birds and with 3 replicates of 110 birds each. Standard husbandry conditions for broiler chickens were observed . Group Tprf was medicated with tylosin (as tylosin phosphate) from the reference commercial preparation for in-feed medication (Tylan 40 premezcla®, Elanco Animal Health, Mexico City) at a rate of 2.27 kg (200 g tylosin phosphate) per ton of feed per label instructions. Group 2 (Tpsr) received tylosin phosphate formulated as a sustained-release preparation in pellet form, also at a rate of 200 g of tylosin phosphate per ton of feed . Pellets about the size of a grain of rice were prepared including one percent methocel  with butylhydroxytoluene as antioxidant in a base of 1:1 parts of wheat and corn flour. All elements were mixed, and a yellow to orange vegetable color was added. Finally, tylosine phosphate was incorporated at a rate of 2.27 g per kg of vehicle, and the mixture was extruded at temperatures no greater than 30°C, using ethyl-alcohol and cotton-seed oil as lubricants. Concentrations of tylosin in final medicated feed were assessed with HPLC in both groups, and concentration variations of five samples per group were never higher than 3.8% in either preparation. Both drug preparations were administered during 3 days. Actual dose achieved in terms of mg/kg, considering a 5% waste of food , was approximately 25.2 mg/kg/d based on mean daily feed consumption of 150 g/chicken. Composition of the basic diet used is presented in Table 1. Table 1. Diet composition for broilers. Ingredients Kg/ton Sorghum 565.11 Soybean meal 342.04 Vegetable oil 46.80 Calcium phosphate 15.16 Calcium carbonate 15.73 Salt 3.50 DL-Methionine 1.76 Vitamins1 2.50 Minerals2 1.00 Choline chloride 0.80 Coccidiostat 0.60 Fungicide 0.50 Antioxidant 0.50 Xanthophyls 4.00 Total 1000.00 Calculated analysis (per kg) MJ/kg 11.88 Crude protein 200 Total calcium 9.0 Inorganic phosphorus 4.5 Methioninepcystine 8.0 Lysine 10.2 Ingredients Kg/ton Sorghum 565.11 Soybean meal 342.04 Vegetable oil 46.80 Calcium phosphate 15.16 Calcium carbonate 15.73 Salt 3.50 DL-Methionine 1.76 Vitamins1 2.50 Minerals2 1.00 Choline chloride 0.80 Coccidiostat 0.60 Fungicide 0.50 Antioxidant 0.50 Xanthophyls 4.00 Total 1000.00 Calculated analysis (per kg) MJ/kg 11.88 Crude protein 200 Total calcium 9.0 Inorganic phosphorus 4.5 Methioninepcystine 8.0 Lysine 10.2 1Amount/kg: vitamin A 3000 000 IU, vitamin D375 0000 IU, vitamin E 6000 IU, vitamin K3 1.0 g, riboflavin 4 g, B12 0.060 g, pyridoxine 3.0 g, calcium pantothenate13.0 g, niacin 25 g, biotin 0.063 g, choline chloride 250 g. 2Amount/kg: selenium 0.2 g, cobalt 0.1 g, iodine 0.3 g, copper 10 g, zinc 50 g, iron 100 g, manganese 100 g, excipient cbp 1000 g. View Large Blood Samples Blood samples were obtained by direct radial or jugular puncture with 5 mL syringes and 22 gauge needles, obtaining approximately 2 to 3 mL per chicken. This procedure was repeated every 2 h during 48 h after making medicated feed available to chickens. Five broilers from each treatment and in each replicate were bled at each sampling time, and no bird was bled more than twice. Non-medicated, basal control samples were obtained at time zero. Blood samples were immediately centrifuged at 3,000 g, and serum was separated and stored frozen in liquid nitrogen until analyzed for concentrations of tylosin within 15 days. Tylosin Concentrations Serum concentrations of tylosin were determined by HPLC as proposed by Kowalski and Pomorska . The chromatographic method provided an acceptable degree of linearity, accuracy, and precision when applied to tylosin enriched serum samples. Linearity was demonstrated by means of the intra-assay coefficient of variance (< 1.9) and inter-assay error (< 1.5). The analytic assay was linear from 0.05 to 10 μg/mL, as linear concentration between the peak area and the tested concentration was confirmed by linear regression (y = 8,369,795.11x + 25,777.08, r2 = 0.999) and confidence interval from 8,248,465.47 to 8,490,929.75. Mean ± 1 SD recovery was 92 ± 2% (r = 0.96). Limit of detection was 0.01 μg/mL, and limit of quantification was 0.05 μg/mL. In this technique, 0.5 mL 0.2 M K2HPO4 (pH = 9) was added to 1 mL of serum, and the mixture was stirred and protein precipitated with 1 mL of 5% trichloroacetic acid. The mixture was centrifuged at 5,500 g for 15 minutes. The supernatant was diluted with 0.2 M phosphoric buffer (pH = 9) to 24 mL volume. The mixture was cleaned and thickened with solid-phase extraction (SPE) with C18 cartridges. The cartridges were activated with 10 mL of methanol and conditioned with 10 mL 0.1 M phosphoric buffer (pH = 8). Finally, the cartridges were cleaned with 5 mL phosphoric buffer (pH = 8) and dried. Dry residues with absorbed tylosin were eluted from cartridges with 0.5 mL acetonitrile and analyzed by HPLC. The mobile phase was composed of acetonitrile and 0.04 M KH2PO4 with pH adjusted to 2.6 (30:70 v/v/v), and was pumped isocratically at a flow rate of 1 mL/min. The variable wavelength UV detector was set at 280 nm. All analyses were performed at room temperature. Statistical Analysis Statistical analysis of serum concentrations of tylosin was carried out by means of Gaussian multi-peak regressions . Area under the serum concentration vs. time curve per d (AUC0–24) and overall AUC values (AUC0–72) were calculated by means of the trapezoidal method. CMAX and TMAX were obtained graphically. Relative bioavailability values (Fr%) were derived from comparing AUC0–24 and AUC0–72 from both groups. These values were compared by ANOVA and Bonferroni t test . RESULTS AND DISCUSSION Following oral administration of Tprf to broiler chickens, mean serum values approached maximum concentration (CMAX) of 0.2 μg/mL within approximately 8 h (TMAX). From this point onwards, serum concentrations of the drug declined constantly, reaching a trough below the limit of quantification (0.05 μg/mL) at 24 hours. This pattern also was observed during the following 2 days. In contrast CMAX for the Tpsr group approached 0.9 μg/mL, but with a TMAX of 26 hours. Other multiple peaks were observed. In this group, troughs were also multiple and ranged from 0.5 to 0.7 μg/mL. The mean plasma tylosin concentrations ± 1 SD obtained for each time point in both groups and their 3 replicates are presented in Figure 1 where Gaussian multi-peak regressions of best fit are superimposed (r2 > 0.9 in both cases). Table 2 shows day-by-day AUC values (AUC0–24), as well as the overall AUC (AUC0–72) for both serum profiles (Tpsr and Tprf groups). When AUC0–24 and AUC0–72 values were compared between groups, statistically higher values were obtained for Tpsr (P < 0.01). Also, Table 2 presents CMAX and TMAX values obtained for both groups. Again, statistically significant higher CMAX values were obtained for group Tpsr, as compared to Tprf in all cases (P < 0.01), with a much more prolonged TMAX value as well (P < 0.01). Serum concentrations were not statistically different until 18 h after in-feed medication; thereafter the Tpsr group had a steady superiority as far as concentrations of tylosin are concerned. Figure 1. View largeDownload slide Mean tylosin serum concentrations (± 1 SE) following oral administration of tylosin phosphate as the standard reference in-feed formulation (Tprf) and as an experimental sustained-release formulation (Tpsr), both at a dose of 200 ppm, equivalent to 25.2 mg/kg of tylosin/d, based on food consumption. Figure 1. View largeDownload slide Mean tylosin serum concentrations (± 1 SE) following oral administration of tylosin phosphate as the standard reference in-feed formulation (Tprf) and as an experimental sustained-release formulation (Tpsr), both at a dose of 200 ppm, equivalent to 25.2 mg/kg of tylosin/d, based on food consumption. Table 2. Basic pharmacokinetic parameters for 2 forms of oral administration of tylosin phosphate in poultry: as the standard reference in-feed formulation (Tprf) and as an experimental sustained-release formulation (Tpsr), both at a dose of 200 ppm, equivalent to 25.2 mg/kg of tylosin/d, based on food consumption. Parameters Tprf Tpsr AUC0–72 (μg/mL⋅h) 10.7± 1.1 41.0 ± 2.1 AUC0–24 (μg/mL⋅h) 3.6 ± 0.4 6.16 ± 0.8 AUC24–48 (μg/mL⋅h) 3.5 ± 0.5 15.8 ± 1.6 AUC48–72 (μg/mL⋅h) 3.5 ± 0.6 14.8 ± 1.4 AUMC0–72 (μg/mL⋅h2) 379 ± 10.4 1750 ± 98.7 CMAX1 (μg/mL) 0.21 ± 0.01 0.9 ± 0.1 CMAX2 (μg/mL) 0.2 ± 0.01 0.9 ± 0.1 CMAX3 (μg/mL) 0.22 ± 0.02 0.9 ± 0.1 CMAX4 (μg/mL) 0.22 ± 0.03 - TMAX1 (h) 8 26 TMAX2 (h) 12 38 TMAX3 (h) 22 50 TMAX4 (h) 52 - Overall Fr - 382.8 Parameters Tprf Tpsr AUC0–72 (μg/mL⋅h) 10.7± 1.1 41.0 ± 2.1 AUC0–24 (μg/mL⋅h) 3.6 ± 0.4 6.16 ± 0.8 AUC24–48 (μg/mL⋅h) 3.5 ± 0.5 15.8 ± 1.6 AUC48–72 (μg/mL⋅h) 3.5 ± 0.6 14.8 ± 1.4 AUMC0–72 (μg/mL⋅h2) 379 ± 10.4 1750 ± 98.7 CMAX1 (μg/mL) 0.21 ± 0.01 0.9 ± 0.1 CMAX2 (μg/mL) 0.2 ± 0.01 0.9 ± 0.1 CMAX3 (μg/mL) 0.22 ± 0.02 0.9 ± 0.1 CMAX4 (μg/mL) 0.22 ± 0.03 - TMAX1 (h) 8 26 TMAX2 (h) 12 38 TMAX3 (h) 22 50 TMAX4 (h) 52 - Overall Fr - 382.8 AUC = area under the curve; AUMC = area under the moment curve; MRT = mean residence time; CMAX: maximum serum concentration; TMAX = time to reach CMAX, Fr = overall relative bioavailability obtained with AUCTpsr0–72 (μg/mL⋅h)/AUCTprf0–72 x 100. View Large It is important to point out that the overall Fr (AUC0–72) of Tpsr as compared to Tprf was 382.8% in spite of administering the same dose. CONCLUSIONS AND APPLICATIONS The dosage form of antimicrobial agents is one of the key features in determining their efficacy [7, 18]. This is particularly relevant in veterinary medicine of productive species where physiological and behavioral differences may influence serum and tissue concentrations of the particular antibacterial agent . Hence, it appears reasonable to analyze whether novel pharmaceutical preparations allow a more rational use of antimicrobials in the commercial poultry industry . Consequently, this study was undertaken to assess the efficacy of a novel, sustained-release drug delivery formulation, intended for the oral administration of time-dependent agents, such as tylosin. Tylosin is an antibacterial drug that possesses wide distribution to most tissues and body fluids . However, studies have shown that oral administration leads to low absorption, and often higher doses may be needed to achieve a therapeutic effect . For example, the main manufacturer recommends 110 ppm as in-feed medication to control respiratory diseases in chickens, while Kowalski et al.  recommend 200 ppm of tylosin phosphate to obtain good clinical efficacy. Notwithstanding the above, it has been advanced that tylosin as a time-dependent antibacterial drug does not require high plasma and tissue concentrations to exert its antibacterial action . Ideally, serum concentrations should be at or above the MIC of the particular pathogen for as long as possible during dosing intervals (T > MIC). Hence, high dose rates should be administered when bacterial resistance is suspected. Also, the AUC/MIC ratio also has been indicated as relevant for clinical efficacy of macrolides . Considering T > MIC and AUC/MIC, it is likely to assume that clinical efficacy of tylosin may be compromised in case scenarios where the flock is affected by a disease caused by a less susceptible bacterial agent. Short half-life of tylosin combined with the diurnal rhythms for feeding, resting, drinking, walking, standing, foraging, and preening behaviors of broiler chickens, with little or no activity during the 7 h dark phase , tend to cause circadian serum concentrations of tylosin . The pellet preparation here studied may improve T > MIC and AUC/MIC ratios and, consequently, clinical efficacy. As shown by Gutierrez et al. , circadian serum concentrations also were found in this study in the Tprf, with a CMAX of 0.22 ± 0.03 μg/mL and a steady decline to reach a trough 23 h after medication was presented in the feeding pans. If a MIC value of a given pathogen is set as low as 0.02 μg/mL to 0.05 μg/mL [6, 22], a window for development of bacterial resistance is occurring overnight. In contrast, tylosin concentrations in the Tpsr group resulted in a CMAX of 0.9 ± 0.01 μg/mL, followed by discrete serum fluctuations in the concentrations of tylosin, as well as less pronounced troughs. This profile is likely to maintain serum concentrations of tylosin that are well above the referred MIC values during most of the time interval of this trial. Relative bioavailabilities of Tpsr assessed with AUC0–72 were also statistically higher for the Tpsr group (P < 0.01) with an overall Fr for AUC0–72 of 382% as compared to Tprf. Optimal ratios of AUC/MIC have not been established for tylosin in broilers, but based on the experience in humans for other macrolides , it is tempting to assume that it is preferable to obtain the highest possible values to treat a given infectious problem. Although this experiment aimed at defining the serum profiles of the reference formulation of tylosin phosphate in chickens and of an experimental formulation intended as a sustained-release formulation, it is tempting to propose a manner in which the Tpsr formulation achieves better AUC and serum concentrations of tylosin. The sustained release of tylosin is likely to be based on the properties of methocel (hypromellose) . This polymer has been used for decades in hydrophilic matrix systems in the human pharmaceutical industry. It is a nonionic substrate that minimizes interaction problems with both acidic or basic drugs. When mucoadherent polymers such as methocel come into contact with fluid of the gastrointestinal (GI) tract starting at the ingluvis, the polymer hydrates to form a gelatinous layer from which tylosin is released, mimicking zero-order kinetics . This can occur along the GI tract, but given transit time described for the proventriculus in commercial chickens (1 to 3 h), it is likely to occur in this organ. Then, the released tylosin will find a favorable proportion of duodenal loop absorption surface vs. amount of tylosin present at a given moment. In turn, this may explain an extended absorption phase, which will void the circadian cycle of tylosin concentrations previously described . To demonstrate that the PK/PD ratios obtained with the experimental formulation can result in superior clinical efficacy, comparative field studies must be carried out. Also, withdrawal times for this new formulation of tylosin will eventually be necessary before this experimental formulation could be considered for poultry medicine. Finally, considering that higher AUC was observed for the Tpsr formulation in this study, further research appears necessary to assess fecal loss of the drug. Footnotes Primary Audience: Growers, Producers, Veterinarians, Researchers REFERENCES AND NOTES 1. Kleven S. H. 2008. Control of avian mycoplasma infections in commercial poultry. Avian Dis. 52: 367– 374. Google Scholar CrossRef Search ADS PubMed 2. Peebles E. D., Branton S. L.. 2012. Mycoplasma gallisepticum in the commercial egg-laying hen: A historical perspective considering the effects of pathogen strain, age of the bird at inoculation, and diet on performance and physiology. J. Appl. Poult. Res. 21: 897– 914. Google Scholar CrossRef Search ADS 3. Gerchman I., Levisohn S., Mikula I., Manso-Silván L., Lysnyansky I.. 2011. Characterization of in vivo-acquiesistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry. Vet. Res . 42: 90. Google Scholar CrossRef Search ADS PubMed 4. Kowalski C., Rolinski Z., Zan R., Wawron W.. 2001. Pharmacokinetics of tylosin in broiler chickens. Polish. J. Vet. Sci . 5: 127– 130. 5. Ji L. W., Dong L. L., J H., Feng X. 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M., Kreizinger Z., Fekete L., Hrivnak V., Magyar T., Janosi S., Schweitzer N., Turcsanyi L., Erdelyi K., Gyuranecz M.. 2014. Antibiotic susceptibility profiles of Mycoplasma bovis isolated from cattle in Hungary, central Europe. BMC, Vet. Res , 10: 256. Google Scholar CrossRef Search ADS 10. Hannan P. 2000. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Vet. Res . 31: 373– 395. Google Scholar CrossRef Search ADS PubMed 11. Avigen. Ross 308 Manual cría pollo de engorde. Avigen editores. 2014. 12. Tylosin tartrate formulated as a sustained release preparation in pellet form (Patent No. MX/a/2012/013222 and PCT/MX2013/000137). 13. Standard methocel 400; Abaquim, Mexico City. 14. Xin H., Berry I. L., Arton B., Tabler G. T.. 1994. Feed and water consumption, growth, and mortality of male broilers. Poult. Sci . 73: 610– 616. Google Scholar CrossRef Search ADS PubMed 15. Kowalski C., Pomorska M.. 2006. 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Pharmacodynamic modeling of clarithromycin against macrolide-resistant [PCR-positive mef(A) or erm(B)] Streptococcus pneumoniae simulating clinically achievable serum and epithelial lining fluid free-drug concentrations. Antimicrob. Agents. Chemother . 46: 4029– 4034. Google Scholar CrossRef Search ADS PubMed 21. Gunnarsson S., Heikkila M., Valros A.. 2008. Effect of day length and natural versus incandescent light on perching and the diurnal rhythm of feeding behavior in layer chicks (Gallus g. domesticus). Acta. Agriculturae Scandinavica, Section A Animal Science. 58: 93– 99. Google Scholar CrossRef Search ADS 22. Jordan F. T. W., Knight D.. 1984. The minimum inhibitory concentration of kitasamycin, tylosin and tiamulin for Mycoplasma gallisepticum and their protective effect on infected chicks. Avian Pathol. 13: 151– 162. Google Scholar CrossRef Search ADS PubMed 23. Rajabi-Shiahboomi A. R., Jordan M. P.. 2000. Slow release HPMC matrix systems. Eur. Pharm. Rev. 5: 21– 23. 24. Patil S. A., Kuchekar B. S., Chabukswar A. R., Jagdale S. C.. 2010. Formulation and evaluation of extended-release solid dispersion of metformin hydrochloride. J. Young Pharm. 2: 121– 129. Google Scholar CrossRef Search ADS PubMed Acknowledgements This study was supported by a grant from the Department of Public Education (SEP) - National Council of Science and Technology (CONACyT), Mexico. © 2017 Poultry Science Association Inc.
Journal of Applied Poultry Research – Oxford University Press
Published: Mar 1, 2018
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