Patent Application: US-96814401-A

Abstract:
a method to improve the health and growth of suckling dairy calves on dairy farms includes supplementing calves &# 39 ; diets with a ruminal fluid preparation obtained from the rumen of a cow . this supplement provides the calves with the protection needed to develop faster and healthier . it also leads to a decreased incidence of scours in the treated calves .

Description:
the present invention utilizes a preparation of ruminal fluid to improve the health and growth of suckling dairy calves . this improvement in calf rearing allows for the extraction of a naturally occurring ruminal fluid from an adult cow . this fluid is readily available , and therefore , inexpensive . our results indicate that ruminal fluid preparations improve the calves &# 39 ; average daily weight gain by as much as 100 %, and the number of days that the calves had diarrhea was 5 - fold lower . in addition , the ruminal fluid can be sterilized to negate the risks of pathogenic organisms populating the fluid . because sterilized ruminal fluid preparations have as much activity as those containing live bacteria , disease transmission through administration of ruminal fluid should not be a problem . this research has thus elucidated a novel , safe mechanism for fighting disease , and provides an alternative to antibiotics . the following experiments sought to examine the effect of ruminal fluid ( rf ) on the growth and health of new - born , milk - fed dairy calves . ruminal fluid was fractionated by centrifugation and autoclaved so the mechanism of action could be more precisely defined . a series of five ( 5 ) experiments were conducted over a period of 4 - years ( 1998 to 2001 ) to examine the effects of rf extracts on calf health and growth . research was implemented at the cornell university research center in dryden , n . y . pregnant , non - lactating holstein dairy cattle were fed a total mixed ration ( 3 . 8 kg corn silage , 1 . 7 kg haylage , 2 kg grass hay , 1 . 2 kg high moisture shelled corn , 0 . 2 kg whole cottonseed , and 1 . 1 kg soybean meal / d ) to meet nrc recommendations ( nrc , 1989 ). several hours before parturition , the pregnant cows were placed in a clean pen that was bedded with sawdust . soon after parturition ( less than 15 minutes ), the cow was milked , and 2 kg of colostrum was given to the new - born calf via a stomach tube . at approximately 1 hours after birth , the calf was given another 2 kg of colostrum orally . calves were housed outside in individual hutches ( approximately 1 . 7 × 1 . 2 m , cal - tel delux , hampel corp ., germantown , wis .). the hutches were located on clean , coarse gravel that was changed each year to prevent the build up of disease - causing microorganisms . calves were given clean bedding ( straw ) each day , and manure was removed 3 times per week . calves were raised under husbandry conditions that are typical of the cornell research center , subject to the recommendations of the cornell center for research animal resources ( iacuc # 91 - 32 - 00 ). calves were fed either milk or milk replacer and the amounts varied with the experiment . rf preparations and dosage time also varied with the experiments . calves consumed all of the milk or milk replacer that was provided . all calves were provided with water and a commercial grain mix ( agway calf prestarter , agway , inc ., syracuse , n . y ., 22 % crude protein , 81 % tdn , 2 . 0 mcal / 1 kg , 1 . 3 mcal / 1 kg neg ) ad libitum starting at 3 days of age . supplemental hay was not provided . body weight gains were assessed according to the guidelines of larson et al . ( 1977 ). approximately 4 hours after birth , the calves were placed in a cart that restrained movement , the cart was placed on a platform scale , and initial body weight was determined as the difference between the weight of the cart and the total . calves that had an initial birth weight greater than 53 kg or less than 34 kg were not included in the experiments . thereafter , the calves were weighed in similar fashion approximately 6 hours after the morning feeding . the calves were restrained , and the weight measurements were relatively constant during the procedure (& lt ; 0 . 25 kg variation ). the calves were inspected five times per day by the dairy farm workers ( approximately 5 different individuals ), and the appearance of feces on the hair surrounding the rectum and in the bedding was scored . in most cases , the calves defecated immediately after they were aroused . in other cases , fecal matter that had not yet dried or crusted over was used as an index of recent defecation . scours were defined as fresh fecal material that had a runny or watery texture and either a white or gray color . according to larsen et al . ( 1977 ), this definition corresponds to code 4 stools . if calves with scours appeared to be dehydrated ( e . g ., sunken eyes , sluggish body movements , and loss of skin elasticity ), they were supplemented with an aqueous mixture of electrolytes ( 83 % corn sugar , 4 . 3 % sodium chloride , 4 . 8 % potassium chloride , 4 . 9 % sodium bicarbonate , 4 % potassium phosphate , w / v ). fresh ruminal fluid ( frf ) for experiment 1 was removed from the rumen of a lactating dairy cow via a fistula . the fistulated cow was fed the total mixed ration ( see above ) ad libitum . the fluid was withdrawn from the rumen with a suction device via a pipe that had holes ( as described in example 1 ). the holes ( 6 mm diameter ) in the pipe filtered the frf so that it would not contain large feed particles . in experiments 2 , 3 and 4 , frf was taken from a non - lactating dairy cow that was fed timothy hay ad libitum . the frf was brought to the laboratory and placed in a 39 ° c . water bath . after gas production from the fermentation had buoyed feed particles to the top of the flask , and protozoa had sedimented to the bottom , frf containing mixed ruminal bacteria was withdrawn from the center of the flask . the frf was centrifuged ( 10 , 000 × g , 30 min , 5 ° c .). the supernatant ( rfs ) was removed , and the cell pellet ( rfc ) was re - suspended in an equal volume of 0 . 9 % nacl . frf that had been autoclaved ( 4 l , 121 ° c ., 40 min ) is referred to as autoclaved ruminal fluid ( arf ). the ruminal fluid cell pellet ( rfc ), ruminal fluid supernatant ( rfs ) and arf were dispensed into plastic vials ( 8 ml ) and frozen (− 15 ° c .) until use . bacterial protein in rfc and rfs was analyzed by the method of bradford ( 1967 ) ( using bovine serum albumin as a standard ) after the cells had been heated to 100 ° c . in 0 . 2 n naoh for 15 min . the rna and dna were measured using an orcinol - fecl3 reaction ( schneider , 1957 ) and ribose was used as the standard . the frf that had been centrifuged to remove feed particles ( 100 × g , 5 min , 5 ° c .) was assayed by the phenol sulfuric acid method ( 0 . 3 ml phenol , 2 . 1 ml 70 % ( v / v ) sulfuric acid , 100 ° c ., 10 min , 485 nm ; ashwell , 1966 ) to estimate total frf - bps . the frf was then centrifuged at higher speed to remove the bacteria ( 10 , 000 × g , 15 min , 5 ° c . ), and the resulting rfs was again assayed by the phenol sulfuric acid method to estimate rfs - bps . the rfs was then treated with 1 % ( w / v ) cetyltrimetylammonium bromide ( ctab ) to precipitate bacterial polysaccharides ( ausubel et al , 1997 ), and the polysaccharide - free supernatant ( pf - rfs ) was also assayed by the phenol sulfuric acid method to estimate non - specific rf interference . preliminary results indicated ctab ( 200 μl of a 1 % solution ) did not interfere with the phenol sulfuric acid assay . true rfs - bps was defined as rfs - bps - pf - rfs . true rfc - bps was defined as ( frf - bps - pf - rfs )- true rfs - bps . glucose was used as a standard . experiment 1 : twenty four heifer calves were randomly allotted to control ( no addition ) or the frf treatment ( 12 calves / group ). treated - calves received 8 ml frf / day in the colostrum or morning milk feeding for 6 weeks . both groups of calves were fed equal amounts of whole milk two times / day ( approximately 12 hour interval between feedings , 4 . 5 kg / day ). the calves were weaned at 6 weeks of age . calves were weighed at birth and at 6 weeks of age . experiment 2 : thirty six heifer calves were randomly allotted to control ( no addition ), rfs or frc treatments ( 12 calves / treatment ). treated - calves received 8 ml rfs or rfc / day in the colostrum or morning feeding for 42 days . all 3 groups of calves were fed equal amounts of a commercially produced milk replacer ( excelerate , 30 % protein , 20 % fat , milk specialties company , dundee , ill .) three times / day ( approximately 8 hours interval between feedings , 7 . 5 kg / day ). the calves were weaned at 6 weeks of age . calves were weighed at birth and at 2 , 4 and 6 weeks of age . experiment 3 : a total of 24 heifer calves were randomly allotted to control ( no addition ) or the arf treatment ( 12 calves / treatment ). treated calves received 8 ml arf / day in the in the colostrum or morning milk feeding for 42 day . both groups of calves were fed equal amounts of whole milk ( 6 kg / day , 2 feedings / day ). the calves were weaned at 6 weeks of age . calves were weighed at birth and at 2 , 4 and 6 weeks of age . experiment 4 : a total of 24 heifer calves was randomly allotted to control ( no addition ) or the arf treatment ( 12 calves / treatment ). treated calves received 8 ml arf / day in the colostrum or morning milk feeding for the first 5 days of life . both groups of calves were fed equal amounts of milk replacer ( 7 . 5 kg / day , 3 feedings / day ). the calves were weaned at 6 weeks of age . calves were weighed at birth and at 2 , 4 and 6 weeks of age . experiment 5 : a total of 24 heifer calves was randomly allotted to control ( no addition ) or the arf treatment ( 12 calves / treatment ). treated calves received 4 ml arf immediately after birth . both groups of calves were fed equal amounts of milk replacer ( 7 . 5 kg / day , 3 feedings / day ). the calves were weaned at 6 weeks of age . calves were weighed at birth and at 2 , 4 and 6 weeks of age . the statistical analyses were performed by sas ( sas inst ., cary , n . c .). in the first analysis , the glm procedure was used to analyze the weight gain in a completely randomized design ( crd ) with analysis of covariance ( ancova ), as described by kuehl ( 2000 ) for each period of growth . initial body weight was used as the covariate . the interaction between treatments and the covariate was used to check the uniformity of the slopes among treatments using the sequential sum of squares ( littell et al ., 1991 ); the interaction and ( or ) the covariate were removed from the statistical model if not significant at p & lt ; 0 . 05 . the partial sum of squares was used in the ancova to test treatments ( littell et al ., 1991 ). the statistical model is shown below : y ij − μ + τ i + β ( x ij −{ overscore ( x )} . . . )+ e j where y is the body weight gain in each period , μ is the overall body weight gain mean , τ i is the fixed effect of treatments , μ 1 is the coefficient for the linear regression of y on x , x is the initial body weight , and eij is the independent , identical , and normally distributed random experimental error . in the second analysis , the weight gain of each growth period was analyzed as a repeated measure design ( rmd ) for all experiments ( except experiment 1 ). the mauchly sphericity test of the proc glm ( sas inst ., cary , n . c .) was used to test the variance - covariance matrix and a univariate analysis of variance was performed if p & gt ; 0 . 05 . the treatment comparison was performed by contrast analysis . the split - plot statistical model used is described below ( kuehl , 2000 ). y ijk = μ + α i + d i , k + β j +( αβ ) j + e ijk where μ is the general mean , α i is the fixed effect of treatment , d i , k is the random experimental error for calves within treatments to test treatment effect , β j is the effect of time ( period of growth ), ( αβ ) ij is the interaction between treatment and time , and e ijk is the normally distributed random experimental error . in the third statistical analysis , all experiments were analyzed together , and treatments had two levels : control and treated , which consisted of calves that received any form of rf . experiments were considered as blocks . experiments 2 , 3 , 4 and 5 were used to investigate the effect of rf on weight gain of each week period . all experiments were used in the overall weight gain ( 0 to 6 weeks ). in this overall analysis , milk intake was used as a covariate , and the analysis was similar to that described above . because a preliminary study indicated an interaction between treatment and the covariate ( milk intake ), a model with unequal slopes was used ( littell et al ., 1999 ). all analyses were performed by proc mixed ( sas inst ., cary , n . c . ), and the statistical model is shown below . where α i is the intercept of the i th treatment effect , β i is the slope of the regression of weight gain on milk intake of the i th treatment , b j is the random effect of experiments , and e ij is the experimental random error . the plot of studentized residues against the predicted values from the analysis of covariance was used to identify outliers , and the plot of the studentized residues against treatments was analyzed to test the assumption of identical variance ( kuehl , 2000 ). the normal distribution was also investigated ( not shown ). because a preliminary analysis of the number of day that calves had scours ( scour days ) was not normally distributed , a non - parametric test using the proc npar1way of sas ( sas inst ., cary , n . c .) was selected to compare the distributions of each treatment ( snedecor and cochran , 1971 ). treatment comparisons were done by the wilcoxon score and kruskal - wallis tests ( sas inst ., cary , n . c .) without the continuity correction . experiment 1 : a preliminary experiment was conducted to ascertain the effect of frf on the body weight gain and incidence of scours . control calves that did not receive frf had an initial body weight of 41 . 7 + 1 . 3 kg , and the total weight gain ( 0 to 6 weeks ) was 16 . 5 + 1 . 0 kg . treated calves ( n = 12 ) that were given the same amounts of milk and frf for 42 day had an initial body weight of 43 . 4 + 4 . 9 kg , and the total weight gain ( 0 to 6 weeks ) was 24 . 3 + 1 . 1 kg . the interaction between treatment ( frf ) and the covariate ( initial body weight ) was not significant ( p & gt ; 0 . 05 ), the slope of the observed variable on initial weight ( covariate ) was similar , outliers were not identified , and treated - calves gained more weight than the controls ( p & lt ; 0 . 05 ). none of these control calves died , but most of them ( 10 out 12 ) had scours during the first two weeks ( fig2 ). the incidence of scours declined during the second and third periods of the trial ( 2 to 4 and 4 to 6 weeks ), but the average number of day that each calf had scours was 2 . 67 . some frf - treated calves scoured , but the average number of scour day was 3 - fold less ( p & lt ; 0 . 05 ). experiment 2 : because results from experiment 1 indicated that frf had a positive impact , we fractionated the rf and determined body weights at 2 week intervals . control calves gained more weight in the second ( 2 to 4 weeks ) and third growth periods ( 4 to 6 weeks ) than the first period ( 0 to 2 weeks ), and the total gain was 23 . 7 kg ( fig1 ). all of the control calves ( 12 out of 12 ) scoured during the first two - week period , but the incidence of scours declined as the calves became older ( fig2 ). control calves had 2 . 75 scour days / calf . calves given rfs gained more weight than the untreated controls ( p & lt ; 0 . 05 ), and this overall advantage ( 0 to 6 weeks ) could be explained by an improvement in the first 2 weeks ( p & lt ; 0 . 05 ). during the second and third growth periods , rfs - treated calves did not gain more weight than the untreated calves ( p & gt ; 0 . 05 ). rfs - treated calves had fewer scours than untreated calves ( fig2 , p & lt ; 0 . 05 ). calves given rfc that had been resuspended in a similar volume of sodium chloride gained more weight in the first growth period ( 0 to 2 weeks ) ( p & lt ; 0 . 05 ), but the overall gain ( 0 to 6 weeks ) was not statistically improved relative to the control calves ( p = 0 . 06 ). the rfc - treated calves had fewer scours than untreated calves ( fig4 , p & lt ; 0 . 05 ). experiment 3 : because results from experiment 2 indicated even rfs could have a positive impact , we then examined the effect of arf . control calves had a total gain of 21 . 5 kg ( fig1 ), and the average number of scour day was 1 . 83 ( fig2 ). calves given arf gained more weight than untreated controls ( 0 to 6 weeks , p & lt ; 0 . 05 ), and this advantage can be explained by an improvement in the first time period ( p & lt ; 0 . 05 , fig1 ). calves that received arf had fewer scours than untreated controls ( fig2 , p & lt ; 0 . 05 ). experiment 4 : because results from experiment 3 indicated even arf could have a positive impact , we then decided to decrease the treatment period from 42 to 5 days . control calves had a total gain of 22 . 0 kg ( fig1 ), and the average number of scour days was 3 . 67 ( fig2 ). calves given arf for only 5 days gained more weight ( p & lt ; 0 . 05 ) in the first growth period ( 0 to 2 weeks ), but the overall gain ( 0 to 6 weeks ) was not statistically improved ( p = 0 . 14 ). calves that received arf for 5 days had fewer scours than untreated controls ( p & lt ; 0 . 05 ). experiment 5 : because results from experiment 4 indicated that 8 ml of arf for 5 days has a positive impact on calf health , we then decided to decrease the arf to 4 ml and administered only one dose , immediately after birth . control calves had a total gain of 22 . 2 ± 4 . 05 kg , and the gains for the first ( 0 to 2 weeks ) second ( 2 to 4 weeks ) and third ( 4 to 6 weeks ) periods were 2 . 84 ± 1 . 85 , 9 . 21 ± 3 . 41 and 9 . 77 ± 3 . 23 kg , respectively . control calves had an average number of scour days of 1 . 81 ± 1 . 99 days . treated calves had a total gain of 25 . 3 ± 4 . 25 kg , and the gains for the first ( 0 to 2 weeks ), second ( 2 to 4 weeks ) and third ( 4 to 6 weeks ) periods were 5 . 34 ± 2 . 66 , 9 . 02 ± 3 . 20 and 10 . 95 ± 1 . 85 kg , respectively . treated calves had an average number of scour days of 0 . 05 ± 0 . 80 days . the overall increase in gain ( 0 to 6 weeks ) and gains during the first period ( 0 to 2 weeks ) were statistically significant ( p & lt ; 0 . 10 and p & lt ; 0 . 05 , respectively ), but the gains during the second ( 2 to 4 weeks ) and third ( 4 to 6 weeks ) periods were not significantly different ( p & gt ; 0 . 05 or 0 . 10 ). the decrease in scour days was statistically significant ( p & lt ; 0 . 05 ). pooled experiments : the analysis of pooled body weight gains from experiments 2 , 3 and 4 are shown in fig3 . similar to the individual analysis of each experiment , the analysis of pooled experiments indicated the administration of rf affected the first 2 weeks of growth ( p & lt ; 0 . 05 ), but rf had no effect on gain in subsequent time periods ( p & gt ; 0 . 05 ). the experiments described above were conducted over a 4 year period , and during this time the standard management procedures at the cornell research center changed : 1 ) calves in the first and third studies were given whole milk , but experiments 2 and 4 were conducted with commercial milk replacers , 2 ) the intake of milk or milk replacer was varied from 4 . 5 to 7 . 5 kg / day , and 3 ) calves in experiments 1 and 3 were fed twice per day , but calves in experiment 2 , 4 and 5 were fed 3 times per day . however , within each experiment , control and rf - treated calves always were given the same amount and type of milk . it has long been recognized that calf growth experiments often have an inherently high degree of variation ( kertz et al ., 1984 ), therefore , we used relatively large numbers of calves ( n = 12 per treatment ). however , rf - dependent improvements in body weight gain were much greater for experiment 1 than experiments 2 through 4 ( fig2 ). the rf - dependent improvements in body weight gain for experiments 2 through 4 were 13 , 20 and 12 %, respectively ( fig2 ), but experiment 1 seemed to have a 49 % increase in growth . to examine if the control group of experiment 1 was abnormal , we compared it to another independent group of 12 calves . these other calves were fed the same amount of milk ( 4 . 5 g / day ), were raised during the same period , had identical management and did not receive rf , however , they gained more weight than the original controls ( 19 . 7 + 1 . 5 versus 16 . 5 + 1 . 0 kg , respectively , p & lt ; 0 . 05 ), even though the number of scour days was similar ( 2 . 51 versus 2 . 67 , respectively , p & gt ; 0 . 05 ). when this independent control was compared to the rf - treatment group the improvement was only 23 %, a value that more closely resembled the effects seen in the other experiments . previous workers used fecal appearance as an index of calf scours and health , and we used a similar scoring system ( larson et al ., 1977 ). kertz et al . ( 1984 ) reported that 95 % of their neonate calves had scours , the average number of scour d / calf for the control group was 4 . 5 . in our studies ( experiments 2 to 4 ), 86 % of the control calves had scours , the average scour days were 2 . 8 . we did not determine the cause of scours in our calves , but specific causes of diarrhea in calves are “ difficult to establish ” and are confounded by the fact that : 1 ) scours can be caused by many different viruses , bacteria and protozoa , 2 ) the mere presence of a potential pathogen in the feces does not always mean that it is the disease causing agent , 3 ) pathogens can operate in a synergistic and unpredictable fashion , and 4 ) scours are sometimes caused by digestive upsets rather than pathogens per se ( waltner - toews et al ., 1987 ; steiner et al ., 1997 ; davis and drackley , 1998 ). all of our calves were given colostrum immediately after birth via a stomach tube , and this practice should have ensured passive immunity . when colostrum is fed immediately after birth , igg concentrations in calf blood are proportional to the amount of colostrum fed ( morin et al ., 1997 ). some researchers have fortified colostrum by vaccinating cows with viral and bacterial antigens prior to calving ( acres et al ., 1979 ; snodgrass et al ., 1982 ; saif et al ., 1983 ), and calves given the same pathogens have sometimes had better weight gains and less diarrhea . however , in practice , fortified colostrum is often without effect ( tizard , 1996 ). based on the previous literature ( pounden and hibbs , 1949 a , b )., we had originally hypothesized that frf might act as a probiotic , and frf decreased the incidence and duration of scours ( p & lt ; 0 . 05 ) and increased body weight gain ( p & lt ; 0 . 05 ). however , subsequent experiments indicated that cells harvested by centrifugation , re - suspended in sodium chloride and frozen aerobically without a cryo - protectant could promote growth ( p & lt ; 0 . 05 ) and decrease scours ( p & lt ; 0 . 05 ). the idea that frf was a probiotic was further contradicted by the observation that rfs or even arf increased body weight gain ( p & lt ; 0 . 05 ) and decreased scours ( p & lt ; 0 . 05 ). ruminal fluid contains microbial proteins , volatile fatty acids and vitamins , but it is very unlikely that our response was nutritional ( fig4 ). the amount of bacterial protein was very small ( approximately 8 mg / d ), and even rfs that had been centrifuged to remove virtually all of the bacteria had activity ( p & lt ; 0 . 05 ). because rfc also had activity ( p & lt ; 0 . 05 ), the benefit could not be explained by volatile fatty acids from the fluid phase . when frf was harvested by centrifugation , there was a distinct layer of polysaccharide ( slime ) directly above the cell pellet , and rfs was clear . subsequent work , however , indicated that even rfs had an abundance of bps that could be precipitated by ctab ( fig4 ), an anionic detergent that has been used to precipitate bps and “ cleanup ” dna preparations ( ausubel et al ., 1997 ). because bps &# 39 ; s are potent antigens and retain activity after autoclaving ( tizard , 1996 ), it appears that bps is the active ingredient in rf . in the 1990 &# 39 ; s , nosky and worthington developed a product based on mycobacterium cell walls under the trade name immunoboost ( veterphram research inc , athens , ga . and chino corona veterinary services , chino , calif .). their work indicated that calves given intravenous , intramuscular and subcutaneous injections of immunoboost during the first 24 hours of life had fewer scours and higher adg than untreated controls . oral administration was not tested , but the authors noted that immunoboost - treated calves required 17 % less antibiotic treatment . previous attempts to improve calf health have tried to enhance passive immunity ( acres et al ., 1979 ; snodgrass et al ., 1982 ; saif et al , 1983 ), but it should be noted that calves usually die from dehydration rather than microbial infection per se ( tizard , 1996 ; davis and drackley , 1998 ). newborns are very prone to diarrhea , and this condition is triggered by agents that irritate the intestine ( guyton , 1971 ). intestinal irritation increases secretion , motility , and stool volume . as the animal becomes older and the intestine is repeatedly exposed to irritants and antigens , the intestinal tissues become desensitized , and the frequency of diarrhea declines ( ernst et al ., 1988 ). intestinal de - sensitization ( sometimes called oral tolerance ) is a localized phenomenon that is mediated by circulating immunoglobulins and the macrophages ( fahmi and chaby , 1993 , 1994 ). when macrophages are presented with antigens bound to immunoglobulins , they secrete cytokines that can directly affect mammalian cells ( kaufman et al ., 2000 ). cytokines appear to accelerate intestinal maturation and de - sensitization , and this process is dose - dependent . studies with food allergens have shown that low doses invoke limited suppression , but large doses can provoke clonal anergy and immunotolerance ( roitt , 1998 , tizard , 1996 ). because rf has a highly diverse population of bacteria and other microorganisms ( krause and russell , 1996 ), it would contain dozens , perhaps hundreds , of different bps molecules . the activity of rf does not seem to be highly diet - dependent . the frf ( experiment 1 ) was obtained from a cow fed a typical dairy cattle ration , but the cow that served as a donor for experiments 2 , 3 and 4 was fed only timothy hay . we originally gave the calves rf preparations each day until weaning ( 6 weeks ), but the improvement in body weight gain and decrease in scours was greatest during the first 2 weeks of life ( p & lt ; 0 . 05 , fig2 ). because the improvement in gain merely carried over into subsequent time periods ( fig2 and 3 ), we decided to decrease the dosage time from 42 to 5 days ( experiment 4 ). calves given arf for 5 days or even once on the first day of life also responded , and this result is consistent with the idea that rf is most beneficial to newborn calves that do not have a fully developed immune system . newborn dairy calves that are given daily doses of rf gain more weight and have fewer scours than untreated controls . because even autoclaved preparations give a positive response , rf preparations do not act as a probiotic . the observation that autoclaved rf preparations decrease scours as well as increase body weight gain has practical relevance : 1 ) rf preparations can be given orally via the milk , 2 ) rf contains naturally occurring non - pathogenic bacteria , 3 ) rf can be autoclaved to eliminate the chance of disease transmission , and 4 ) the time needed to demonstrate a response is relatively short ( as little as 5 days ). accordingly , it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention . reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims , which themselves recite those features regarded as essential to the invention . acres , s . d ., r . e . isaacson , and k . babiuk . 1979 . immunization of calves against enterotoxigenic colibacillosis by vaccinating dams with purified k99 antigen and whole cell bacterins . infect . immun . 25 : 121 – 126 . ashwell , g . 1966 . the phenol sulfuric acid reaction for carbohydrates . method in enzymol . 8 : 93 – 95 . ausubel , f ., r . brent , r . e . kinston , d . d . moore , j . g . seidman , j . a . smith and k . struhl , ed . 1997 . short protocols in molecular biology . 3rd ed ., page 2 – 13 . john wiley & amp ; 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