Appropriate statistical methods to compare dose responses of methionine sources.

Two sources of methionine (Met) activity are frequently used in commercial feed formulation: DL-2-hydroxy-4-(methylthio) butanoic acid (HMTBA), most commonly available as an 88% solution with 12% water; and DL-methionine (DLM, 99% powder). Despite the fact that both compounds have been in commercial use for over 50 yr, controversy and confusion remain with respect to their relative bioefficacy (RBE). This paper presents a review of the use of a nonlinear common plateau asymptotic regression technique (NLCPAR) that has been used to compare the 2 Met sources with particular emphasis on the validity of the basic assumptions of that model. The thesis of this paper is that the controversy is due, at least in part, to the misapplication of this regression technique to estimate the RBE of HMTBA and DLM. The NLCPAR model is a bioassay with the key dependent assumptions that HMTBA is a dilution of DLM, and that each follows dose-response curves of the same form and approach a common plateau. Because both provide Met activity, it may be considered reasonable to accept these assumptions; however, specifically testing them demonstrated that the assumption of a common dose-response is not supported by data. The common plateau assumption was tested with an alternative approach of fitting nonlinear separate plateaus asymptotic regression (NLSPAR) to a set of 13 published broiler studies in which the NLCPAR model had been used to estimate RBE of HMTBA and DLM. The hypothesis of a common plateau was rejected (P < 0.01), meaning that the conclusion that HMTBA had lower bioefficacy than DLM based on the NLCPAR methodology was not valid. An example using published data demonstrated that the NLSPAR model was a significantly better fit than the NLCPAR model, and showed that HMTBA and DLM followed different dose responses. Consequently, there was no single value for RBE for the entire dose range; rather, the RBE of the 2 compounds varied with use level. The evidence presented here indicates that separate plateau models should be used when comparing these 2 products. These more valid models can then be used for predictions of differences between HMTBA and DLM at levels of expected use.

Production performance of dairy cattle administered recombinantly derived bovine somatotropin (USAN, Somavubove) daily: a dose range study.

Herds (n = 9) were used on which cattle (n = 598) received 0, 4.3, 8.6, 12.9 or 17.2 mg of recombinant bovine somatotropin (rbST; USAN, Somavubove) daily from about 75 d postpartum until lactation end. Minimum effective dose to increase (3.5% fat corrected milk) FCM of cows (multiparous) compared with controls is 2.0 mg rbST/d and maximum is 16.6 mg/d. Minimum effective dose to increase FCM of heifers (primiparous) is 5.0 mg rbST/d while the maximum is 16.7 mg/d. Dosing of 4.3 to 13.2 mg rbST/d enhances efficiency of FCM production in cows. Milk fat, protein, calcium and phosphorus were not effected by rbST except for instances of transient changes. Lactose was greatest in heifers given 12.9 mg rbST/d. Days of lost milk, incidents of mastitis, and somatic cell counts were not affected by rbST. Cows given 12.9 or 17.2 mg of rbST had reduced pregnancy and conception rates, whereas heifers did not. Total services, first service conception rate, services per conception, days open and days to first AI (artificial insemination) were not affected by rbST. Days between AI were greater in cows given 17.2 mg rbST/d. Restoration of body weight and condition occurred at slower rates in rbST-dosed cows. Heifers given 8.6 or 12.9 mg rbST/d gained more weight than controls. Net energy and protein balance was reduced (less so in heifers) near start of rbST but recovered as intakes increased. Cattle given rbST produce more milk than controls and should be managed accordingly.

LH and FSH response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin.

This study was conducted to describe the changes in serum LH and FSH concentrations in Holstein heifers following intramuscular (i.m.) injection of various dosages of fertirelin acetate and other commercially available GnRH products at their labeled dosages. The design was an incomplete Latin-square which gave nine replicates of each treatment. Treatments administered on Days 8 to 16 of the estrous cycle consisted of saline; 25, 50, 100 or 200 microg fertirelin acetate; 100, 250 or 500 microg gonadorelin; and 10 or 20 microg buserelin. Blood samples were collected via jugular catheters from 1 h before to 8 h after each injection. Log (Base 2) area under the LH and FSH curves (log AUC) were used to evaluate response to fertirelin acetate dosages and to determine difference (LSD: 0.05) and bioequivalence (alpha = 0.05) between the various dosages of GnRH products tested. Significant quadratic dose response relationships were detected for the LH and FSH log AUC data. Plots of the LH log AUC but not the FSH log AUC data suggested that a response plateau was being approached at the higher dosages of fertirelin acetate. Bioequivalence (alpha = 0.05) was based on the assumption that two means are equivalent if they differ by no more than 20% of the reference log AUC mean. Using these criteria fertirelin acetate is 2.5 to 10 times more potent than gonadorelin, whereas buserelin is approximately 10 to 20 times more potent than fertirelin acetate in the bovine for LH and FSH release.

Efficacy of ceftiofur hydrochloride for treatment of experimentally induced colibacillosis in neonatal swine.

Ceftiofur hydrochloride was tested for effectiveness against induced colibacillosis in neonatal swine. In this model, pigs less than 12 hours old were inoculated via stomach tube with a virulent, K99+, nalidixic acid-resistant strain of Escherichia coli. Six hours after challenge exposure, 1 dose of ceftiofur was administered either IM or orally in experiment 1 and orally only in experiment 2. Mortality, shedding of bacteria, fecal consistency scores, and body weight changes were monitored for 10 days. In experiment 1 (n = 383 pigs), all treatments at dosage that ranged between 0.5 and 64.0 mg of ceftiofur/kg of body weight significantly (P less than 0.001) reduced mortality, bacterial shedding, and diarrhea and increased weight gain, compared with findings in untreated controls. There were no detectable differences between oral and IM routes, except that there was greater reduction in bacteria shedding associated with the oral route of administration. In experiment 2 (n = 505 pigs), ceftiofur was administered orally either once at 6 hours after challenge exposure or twice at 6 and at 48 hours after the first dose. Dosage of ceftiofur was 0, 5, 10, 20, 30, or 60 mg/kg administered once, or half the same dose was administered at each of 2 times. At the optimal dosage (10 mg/kg), a single dose was as effective as 2 doses. The single administration at all dosages reduced mortality, bacterial shedding, and diarrhea scores and increased body weight gain, compared with findings in untreated pigs (P less than 0.01). In this induced infection model, the optimal treatment dosage was determined to be 10 mg/kg administered once.

Microbiological determination of neomycin in feeds: collaborative study.

A modification of the AOAC microbiological determination of neomycin in feeds was collaboratively studied by 12 laboratories. The official method was modified by substituting a constant salt concentration diluent for the feed extract diluent, preparing the agar medium in tris buffer, and performing the test with a monolayer plating system. Each laboratory performed single assays on 8 samples in a randomized sequence. The samples included duplicates of a cattle and swine feed at 2 different marketed concentrations. The mean recovery across all laboratories was 110.7% of theory with a range of means of 69.4-128.6 across the 12 laboratories. The results of one laboratory and 2 additional values from different laboratories were deemed outliers and excluded from statistical analysis. The statistical analysis gave a confidence interval of +/- 26% for individual assays.

Effects of PGE1 or PGE2 and/or acetazolamide on expulsion of Nippostrongylus brasiliensis from rats.

PGE1 and PGE2 have been reported to enhance natural expulsion of Nippostrongylus brasiliensis, a nematode parasite, from the intestine of the rat. Mucus production may also be a key element of worm rejection. Our study attempts to determine if 1) PGE1 or PGE2 alter the normal course of infection with N. brasiliensis in rats, 2) a known mucous enhancing drug, acetazolamide, can augment the rate of worm expulsion, and 3) combinations of prostaglandins and acetazolamide affect N. brasiliensis in the rat. Rats were inoculated with approximately 1,000 infective larvae of N. brasiliensis. Animals were administered, intraduodenally, one of the following: 0.2 ml 0.9% NaCl; 0.2 ml 100% ethanol; 250 micrograms PGE1/0.2 ml 100% ethanol; 250 micrograms PGE2/0.2 ml 100% ethanol; 250 micrograms acetazolamide/0.2 ml 100% ethanol; 250 micrograms PGE1 or PGE2 + 250 micrograms acetazolamide/0.2 ml 100% ethanol. These solutions were given in a single bolus on day 6 postinoculation (PI) or twice daily on days 6-9 PI. Following these treatments the number of parasite ova per gram feces per day for days 6-10 PI and numbers of worms present at necropsy on day 10 PI were determined. Treatment with prostaglandins or acetazolamide or both failed to adversely affect egg deposition by adult female worms or the number of worms in the small intestine. These results do not support the involvement of prostaglandins in the expulsion of N. brasiliensis from the host intestine.

Determining animal drug combinations based on efficacy and safety.

A procedure for deriving drug combinations for animal health is used to derive an optimal combination of 200 mg of novobiocin and 650,000 IU of penicillin for nonlactating cow mastitis treatment. The procedure starts with an estimated second order polynomial response surface equation. That surface is translated into a probability surface with contours called isoprobs. The isoprobs show drug amounts that have equal probability to produce maximal efficacy. Safety factors are incorporated into the probability surface via a noncentrality parameter that causes the isoprobs to expand as safety decreases, resulting in lower amounts of drug being used.

Evaluation of a topical treatment, alone and in combination with a detergent, for generalized demodicosis.

Thirty dogs (20 treated, 10 controls) with naturally-acquired generalized demodicosis were utilized to evaluate the bio-activity and safety of a liquid concentrate formulation of amitraz, with or without the addition of a nonionic detergent. The detergent was added to the treatment mixture to enhance wetting and thereby reduce the number of treatments required to return diseased animals to a normal state. Three--six miticide treatments were topically applied to dogs at 14-day intervals, at a concentration of 250 parts per million active drug. The liquid concentrate with or without detergent, was equally effective and safe as a dermatotherapy for demodicosis; addition of the nonionic detergent grossly improved the wetting characteristics of the treatment mixture; however, it did not alter the biological activity or the safety of the therapy.

Microbiological determination of neomycin in feeds and formulated products.

An AOAC modified method is described for the microbiological assay of neomycin, which has been adapted to include complete feeds, supplements, premixes, liquids, oil suspensions, boluses, and antibiotic-impregnated paper. The method features a more sensitive standard response line with a monolayer plating system. The use of a buffered plating medium in place of the water-prepared medium results in a curve with less degree of slope, which allows for more accurate interpretation of the standard response. The feed extract diluent used for standard response line dilution, which is prepared from exposure of the feed extract fluid to pH changes, heat, and sodium hypochlorite, has been eliminated. The constant salt concentration diluent used for the preparation of standards is the same as the salt concentration of the sample extract solution to be tested. Results for 50 commercial complete feeds and 50 commercial premixes received over the last 5 years produced an overall mean recovery of 101% with a mean percent recovery range of 80-112%. A statistical analysis of these 100 commercial, complete feeds and premixes, ranging in concentration from 47 g/ton to 70 g/lb, indicates the assay has little, if any, concentration-related bias. Precision and accuracy of the method was supported by laboratory studies of 20 assays that produced a mean recovery of 101% and standard deviation of 3.

Evaluation of a sponge-on therapy for canine scabies.

Forty dogs (20 treated, 20 controls) were utilized to evaluate a new treatment for naturally acquired canine scabies. A liquid concentrate formulation of amitraz was diluted and applied as a sponge-on therapy. Ninety-four percent of the dogs treated with the scabicide were cleared of mites and returned to clinical normality with a single topical treatment; one dog was retreated, cleared of mites and was also returned to normality. All dogs treated with the miticide responded clinically, therefore the treatment also may be useful when trial therapy is necessary to differentially diagnose the disease. The miticide was well tolerated by all dogs, and there was no evidence of dermal or ocular irritation. Topical treatment with the liquid concentrate was efficacious and safe as a therapy for naturally acquired canine scabies. Placebo controls did not improve clinically and these animals retained their mite populations.

Chemotherapeutic treatment of naturally acquired generalized demodicosis.

Fifty-two dogs naturally parasitized with Demodex canis and having the generalized form of the disease were utilized to evaluate the efficacy and safety of single or multiple topical treatments with a liquid concentrate formulation of amitraz. Ten dogs (5 treated, 5 controls) were utilized to evaluate a single treatment. A single topical treatment with the miticide did not significantly reduce the incidence of dogs with mites, however, significant clinical improvement resulted. Side-effects were not observed after treatment. Forty-two dogs (26 treated, 16 controls) were utilized to evaluate multiple topical treatments with the liquid concentrate. A series of 3-6 treatments was applied topically at 14-day intervals. The dogs treated with the miticide received an average of 4.5 topical treatments. All (100%) of the dogs responded clinically, and the mean rate of improvement at four weeks post-treatment was 99.1%. Most dogs (96.2%) were cleared of mites after 3-6 treatments, and Mitaban did not cause any dermatologic, ocular, or other clinical side-effects. Multiple treatments with the liquid concentrate were highly efficacious and safe for treatment of generalized demodicosis. Control dogs did not improve clinically and retained mite populations.

Microbiological determination of lincomycin in feeds and supplements containing high concentrations of bentonite.

A microbiological method is described for determining lincomycin in complete feeds, supplements, and pre-mixes greater than 100 g/ton and containing high levels of bentonite. The AOAC method currently used is unsatisfactory for analyzing feeds with concentrations of bentonite greater than 5.5%. Study indicates that low lincomycin recoveries from high level bentonite feeds are a function of aqueous contact and subsequent binding. The present method involves an alternative extraction technique using formamide as the primary extractant. A binary solvent system of ethanol and phosphate buffer aids in the extraction, miscibility, and conversion to water solubility for subsequent testing. Forty-one feeds containing as high as 60% bentonite were assayed by the reported method and gave a mean recovery of 106% and a range of 94-114%. An inter-laboratory confirmation study produced a mean recovery of 103%, with a range of 93-114%. A factorial analysis of variance of the interactive effects between lincomycin level and bentonite level within the AOAC method and within the bentonite method showed no interactions which would influence percent recovery in either assay method.

Lincomycin dose response for treatment of necrotic enteritis in broilers.

A dose response study was conducted to determine the appropriate (optimal) lincomycin concentration in drinking water for the treatment of necrotic enteritis (NE) in broilers. The study was replicated twice over time using a total of 2,895 broilers. Birds were raised in a facility containing a built-up litter from a source that experienced NE. They were commingled from 1 day of age until NE was observed. Lincomycin was mixed in drinking water at a rate of 0, .528, 2.114, 8.454, or 33.818 mg/liter for a period of 7 days. The study was terminated 3 weeks after initiation of therapy. Necrotic enteritis was diagnosed by mortality and pathological findings. The data on mortality were analyzed statistically using analysis of variance procedures. The Walker-Carmer technique was applied to estimate the appropriate lincomycin concentration. Lincomycin was effective for the treatment of NE in broilers at concentrations of greater than or equal to 2.114 mg/liter of drinking water. The minimal effective dose that would achieve maximal treatment response (optimal dose) against NE in broilers was estimated to be 16.9 mg lincomycin/liter of drinking water.

Therapeutic effects of parenteral administration of lincomycin on experimentally transmitted swine dysentery.

The therapeutic effects of 2 dose levels of lincomycin and a reference drug (tylosin) were compared in 80 growing pigs with experimentally transmitted swine dysentery (SD). The pigs were allotted equally to 4 groups. Treatment was initiated 5 days after pigs were exposed to SD. Lincomycin was administered IM at doses of 11.0 or 4.4 mg/kg of body weight once daily for 3 to 7 days. Tylosin was injected IM at a dose of 8.8 mg/kg once daily for 3 days (highest recommended dose). The control (nontreated) infected pigs were not given the drug. The effects was terminated 22 days after exposure to SD. The effects were measured in terms of mortality, survival, physical activity, performance, and necroscopy findings. The 2 drugs reduced the clinical signs of SD. Pigs treated with either dose of lincomycin had a better treatment response than did pigs treated with tylosin, as evidenced by less mortality, longer survival time, and greater feed intake (P = 0.05). In addition, pigs treated with the larger dose of lincomycin, 11.0 mg/kg, had better treatment responses in 12 of the 14 measured criteria than did pigs treated with tylosin. Also, these pigs treated with the larger lincomycin dose had better treatment responses tha did the pigs treated wih the smaller dose of lincomycin, 4.4 mg/kg, as evidenced by dysentery, fecal consistency, physically active and intermediately active pig days, body weight gain, and feed intake.

Gas-liquid chromatographic determination of melengestrol acetate in cattle feed supplements: collaborative study.

The assay for melengestrol acetate (MGA) in cattle feed supplements was the subject of a collaborative study using 6 laboratories. Two feed formulations were each fortified with 0, 0.0625, 0.125, 0.500, 1.00, and 1.50 mg MGA/lb of supplement, and each laboratory assayed 4 samples of each level of each formulation by electron capture gas chromatography after liquid-liquid extraction of each sample followed by solvent partition and column chromatography cleanup procedures. Overall recovery was 83.2% with a pooled within-laboratory sample-to-sample standard deviation of 9.5%. This includes the variability due to formulation, batches, days, levels of MGA, samples, and their interactions. Including the collaborators as a source of variance results in an overall standard deviation of 15.0%. The major sources of variability were associated with collaborator, formulation, and level of MGA. Lower than expected recoveries were attributed to difficulty in recovering MGA from samples containing concentrations of MGA below the range of the method and the inadvertent substitution by some laboratories of ethanol-free chloroform for the ethanol-containing reagent grade chloroform specified in the methodology. On the basis of the collaborative results, the method has been adopted as official first action for MGA in cattle feed supplements at concentrations from 0.125 to 1.00 mg MGA/lb.

Efficacy of Linco-Spectin medication on mycoplasma meleagridis airsacculitis in turkey poults.

The efficacy of Linco-Spectin (LS) water medication was determined against Mycoplasma meleagridis (MM) airsacculitis in turkey poults under controlled conditions. The poults were obtained from commercial flocks naturally infected with MM. Water medication was given for the first five days of life. In Georgia and Ohio, LS was given at 0, 1, 2, and 4 g per gallon of drinking water. In California and Minnesota, LS was administered at 0 and 2 g per gallon of drinking water. At three weeks old, the birds were weighed individually and examined serologically, culturally, and grossly for MM airsacculitis. LS at 2 g activity per gallon of drinking water was beneficial in controlling MM airsacculitis in all four trials.

Effect of a single injection of lincomycin, spectinomycin, and linco-spectin on early chick mortality caused by Escherichia coli and Staphylococcus aureus.

The efficacies of lincomycin (L) and spectinomycin (S), alone and in various combinations (L/S), were determined against Escherichia coli (EC) and Staphylococcus aureus (SA) of avian origin, both in vitro and in vivo. L contributed significantly to L/S activity against SA, while S contributed significantly to L/S activity against EC, and L/S (2.5 mg L + 5.0 mg S) was more effective than either L or S against SA and EC. The suggested optimum dose for controlling early chick mortality caused by SA and EC is 2.5/5.0 mg of L/S per chick.

Social status and sex ration effects on mating frequency of cockerels.

Cockerels of 3 early- and 3 late-maturing strains of White Leghorns were observed for agonistic and mating behavior while housed with pullets in floor pens. All male strains were equally represented in each of 6 flocks; replicated flocks contained 6, 12, or 18 males with male:female rations of 1:24, 1:12, or 1:8, respectively. Differences associated with selection for early or late sexual maturity were not detected for either social status or mating frequency. Contrary to expectation, social status of cockerels had little effect on frequency of mating. Flocks with fewer males had lower fertility, but sex ratios (in the range used) had no effect on frequency of mating of individual males. Implications of these results are discussed.