Ana Backovic PhD  ICGEB (Trieste), D.V.M. Tanja Roganovic (Sava KovacevicSerbia), Prof. Anka Popovic Vranjes PhD Univ. of Novi Sad (Serbia)
Ana Backovic, PhD^{1,2}, Dr.Vet.med Tanja Roganovic ^{3}, prof. Anka Popovic Vranjes, PhD^{4 }
1. Postdoctoral researcher; International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
2. Scientific Consultant and East European Coordinator; Turval Biotechnologies, Udine, Italy.
3. Doctor of Vet.Medicine and general chief menager for the cattle farm at Agricultural enterprise "Sava Kovacevic"
4. Full professor at Faculty of Agriculture, University of Novi Sad, Republic of Serbia.
Effects of supplementing Turval B0399 in the diet of the milking cows in the first production phaseearly lactation (aprox.60th day of lactation); influence upon production parameteres and milk quality.
The administration of TURVAL 0sp/50 additive in the daily feed significantly increased the daily milk production during the treatment period by 3.5% with respect to the group that did not receive dried lactic yeast, Turval B0399 (fig.1, Stat.T1, P=0.0003)
The feed intake was not changed by the supplementation with Turval B0399 (fig.2, Stat.T2, P=0.5629), but the feed was better converted to milk produced. Indeed, the feed conversion index was significantly reduced by 8.3% (fig.3, Stat.T3, P=0.399) in the experimental group.
Finally, While at the beginning of the trial the average number of somatic cells/ml of milk was similar for the two test groups (fig.4A, Stat.T4A, P=0.5339) at the end of the trial (3 days after the administration of Turval ceased) the number of somatic cells/ml was significantly reduced by ≈36% in the treated group when compared to the control group (fig.4B, Stat.T4B, P=0.0485) which remained similar to the beginning of the trial. This parameter testifies about better milk quality of the treated group upon Turval treatment and indicates better health condition of these animals.
I Experimental conditions and generalities.
Methodology and statistical aspects:
The test was carried out in a farm: Sava Kovacevic – Vrbas, Vojvodina region, Serbia.
On the beginning of the trial 59 female Holstein Friesian calves were divided in two homogeneous groups and placed in two separate spaces (picture1). 30 cows were randomly assigned to the "treated" group that received experimental treatment  8 gr. of feed supplement, TURVAL 0sp/50 per head/day for 31 days. The rest of the animals  29 were assigned to the control group, which did not receive the supplement. On the 3rd day of the trial one animal suddenly died in the control group and thus the data concerning this animal was eliminated from the final statistical analysis. Number of animals on the test site, in both groups, slightly varied on daytoday basis during the trial period due to regular activities and procedures on the farm (unavoidable translocations from one farm to another, insemination, blood sampling, sudden health issues, injures etc.). These changes were accurately documented and taken into consideration when performance and production parameters were analysed and compared statistically between the two groups.
All the calves were fed with a UNIFEED technique from a mixing wagon.
Animals were fed in such a way that the feed was never missing in front animals (ad libitum) and the feed consumed by each group was registered every day as the difference between input (the feed given to the group in the morning) and output (the remaining feed collected on the following morning before subsequent feeding).
The feed was not supplemented with any antimicrobial additive.
Breed and Study conditions:
Trial period: Start: December 14, 2013; End: January 13, 2014
Climate/temperature conditions during the trial period are shown in the SupplementaryTable1
Duration of trial: 31 days
F: unifeed feed
Breed: Holstein Friesian
Study place: Farm Sava Kovacevic (http://www.savakovacevic.rs); Vrbas, Serbia
Effective N° of animals per group, in 2 stall compartments as shown on the picture1; selected homogeneously with regard to the distance from calving:
Treated : 2530 animals
Control: 2528 animals
Dosage: Turval 0sp/50( Dried concentrated yeast Kluyveromyces marxianus fragilis B0399 – Raw material for fodderszootechnical use.)  8g/per head/day;
Picture1: The place of the study on the farm Sava Kovacevic
Feed:
Unifeed method; Composition determined directly by the veterinary, and including corn silage, hay from permanent meadows, extruded full fat soybeans, soy flour, dried sugar beet pulp, corn and barley meal, mineral and vitamin supplements“premix”, TURVAL 0sp/50(dried concentrate yeastsprayed).
Statistical analysis of the data:
The data for each experimental group (considered as an experimental unit) were collected on the daily basis and converted to averages individual data per one animal of each test group, depending on the number of subjects per each group on a certain day of the trial period. The number of subjects in both groups was retained statistically sufficient throughout the whole study. The obtained average values for two independent samples, control and treated group, representing: i)daily milk production, ii)daily feed intake and iii)daily feed conversion rate, were statistically compared as individual values. All data are presented as MEAN values, the standard deviation (SD) of the individual values and ± standard error of the mean (S.E.M.). To evaluate the significance of the difference in mean values between the two groups, without the importance of the direction of the difference, we used the unpaired, twotailed Student’s Ttest, provided by statistical GraphPad software, Prism  version 5.
In the test, P refers to significance: probability of retaining the null hypothesis of equal means.
Means differences are considered statistically significant when P < 0.05. Means and s.e.m. are shown on the histograms, bellow in the Results section, where * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001 level of significance.
Effect of Turval B0399 on milk production parameters during the trial period
The following production  performance parameters of each trial group were followed during the study: i) Daily milk production, ii) Daily feed intake and iii) Daily Conversion Rate. Each of these parameters is expressed as an average value per a single animal of each group and their change upon the influence of Turval B0399 was followed over timeTable1.
The statistical significance of this change was verified by statistical comparison of the parameters' changes between treated and nontreated (control) group over the whole 31 day trial period and is further explained in detail.
i) Daily milk production for each group is the average amount of milk produced per one animal of the group on a certain day. It is calculated as the ration between the total amount of milk produced by a group (obtained by summarizing all exact amounts of milk produced by each cow of the group represented in Supplementary Table2A and B) on a certain day and the exact number of animals in that group on that day. To test the influence of Kluyveromyces administration on the milk production of the treated group we compared MEANs of this parameter for the whole treatment period between treated and control group of animals. The statistical test shows very significant difference between control and treated groups, evaluated by a low P value (p <<0.05), thus confirming a positive effect Turval B0399 administration on the milk production  Figure1, Statisticstable1.
Figure1  Statisticstable1  
Analyzed: Table1 PRODUCTION PARAMETERS 
 
Column: Treated group  Daily milk production  treated group  
vs  vs  
Column: Control group  Daily milk production  control group  

 
Unpaired t test 
 
P value  0.0003  
P value summary  ***  
Are means signif. different? (P < 0.05)  Yes  
One or twotailed P value?  Twotailed  
t, df  t=3.834 df=60  

 
How big is the difference? 
 
Mean ± SEM of column A  40.04 ± 0.2803 N=31  
Mean ± SEM of column B  38.70 ± 0.2066 N=31  
Difference between means  1.335 ± 0.3482  
95% confidence interval  0.6384 to 2.031  
R square  0.1967  

 
F test to compare variances 
 
F,DFn, Dfd  1.842, 30, 30  
P value  0.0998  
P value summary  ns  
Are variances significantly different?  No 
In order to better describe/visualize the change of the average milk production under the influence of Kluyveromyces, we graphically presented how this parameter changed over the trial period in both test groups  Graph1. In correspondence, the increasing difference between the average daily milk productions between the treated group and the control group increased along the treatment period, which can be represented by an approximate linear line on the Graph2 whose angle shows the tendency of this difference / the increase in time.
Graph1: Average daily milk production change along the trail period for the two test groups
Graph2: Increasing difference between the average daily milk production of the treated group and the control group along the trail period.
ii) Daily feed intake for each group is the average mass of the feed consumed by one animal of the group on a certain day. It is expressed as the ration between the mass of food eaten by that group on a certain day and the exact number of animals in that group on that day. Just as for the daily milk production evaluation, to evaluate weather yeast had an influence on feed intake during the trial period we compared the MENAS of individual daily measurement between treated and control group of animals during the whole trial period (31 day)  Figure2, statisticstable2.
Figure2  Statisticstable2  
Analyzed: Table1 PRODUCTION PARAMETERS 
 
Column: Treated group  Daily feed intake  treated group  
vs  vs  
Column: Control group  Daily feed intake  control group  

 
Unpaired t test 
 
P value  0.5629  
P value summary  ns  
Are means signif. different? (P < 0.05)  No  
One or twotailed P value?  Twotailed  
t, df  t=0.5818 df=60  

 
How big is the difference? 
 
Mean ± SEM of column A  49.15 ± 1.560 N=31  
Mean ± SEM of column B  50.34 ± 1.324 N=31  
Difference between means  1.191 ± 2.047  
95% confidence interval  5.284 to 2.902  
R square  0.005610  

 
F test to compare variances 
 
F,DFn, Dfd  1.388, 30, 30  
P value  0.3744  
P value summary  ns  
Are variances significantly different?  No 
Daily Feed Conversion Rate (FCR) (or average, daily FCR) is a daily production parameter obtained when the average daily feed intake for a group is divided by the average amount of milk (daily milk production) produced by that group of that day. In order to evaluate the influence of TURVAL B0399 yeast on FCR we compared MEAN values for average daily FCRs between the treated and untreated (control) group over the whole trial period. As shown in the Figure3 and statisticstable3, treated group of animals showed significantly better average FCR over the trial period. Besides, the farm staff reported (even though this parameter was not part of the protocol and thus was not regularly measured) that the treated animals ate more frequently than the control ones. In other words the intermeal interval was reduced in the treated group.
Figure3  Statisticstable3  
Analyzed: Table1 PRODUCTION PARAMETERS 
 
Column: Treated group  Daily FCR  treated group  
vs  vs  
Column: Control group  Daily FCR  control group  

 
P value  0.0399  
P value summary  *  
Are means signif. different? (P < 0.05)  Yes  
One or twotailed P value?  Twotailed  
t, df  t=2.101 df=60  

 
How big is the difference? 
 
Mean ± SEM of column A  1.229 ± 0.03999 N=31  
Mean ± SEM of column B  1.340 ± 0.03495 N=31  
Difference between means  0.1116 ± 0.05311  
95% confidence interval  0.2178 to 0.005357  
R square  0.06852  

 
F test to compare variances 
 
F,DFn, Dfd  1.309, 30, 30  
P value  0.4653  
P value summary  ns  
Are variances significantly different?  No  
P value  0.0399 
PART B: Effect of Turval B0399 during on the milk quality expressed as number of somatic cells/ml
As shown in the Figure4A, statisticstable4A the number of somatic cells before the study was similar (P=0.7209) for the control and treated group and it was little less than 400.000/ml. This measurement was done 4 days before the "trial start" by milk sampling from 20 randomly selected animals from each groupTable2. On the contrary, by the end of the trial the number of somatic cells in the treated group significantly decreased when referred to the control group Figure4B, statisticstable4B. Similarly, milk samples were taken 3 days after the "trial end" by milk sampling from 20 randomly selected animals from each groupTable2.
Figure4: Average number of somatic cells for treated and control group; Aat the beginning of the study; Bat the end of the study.
A  B 
Statisticstable4A  Statisticstable4B  
Column A  tretaed start  Column A  treated  end 
vs  vs  vs  vs 
Column B  control start  Column B  control  end 




Unpaired t test 
 Unpaired t test 

P value  0.5339  P value  0.0494 
P value summary  ns  P value summary  * 
Are means signif. different? (P < 0.05)  No  Are means signif. different? (P < 0.05)  Yes 
One or twotailed P value?  Twotailed  One or twotailed P value?  Twotailed 
t, df  t=0.6278 df=38  t, df  t=2.030 df=38 




How big is the difference? 
 How big is the difference? 

Mean ± SEM of column A  331450 ± 60760 N=20  Mean ± SEM of column A  220600 ± 30864 N=20 
Mean ± SEM of column B  386450 ± 63119 N=20  Mean ± SEM of column B  347350 ± 54284 N=20 
Difference between means  55000 ± 87611  Difference between means  126750 ± 62445 
95% confidence interval  232430 to 122430  95% confidence interval  253213 to 287.2 
R square  0.01026  R square  0.09782 




F test to compare variances 
 F test to compare variances 

F,DFn, Dfd  1.079, 19, 19  F,DFn, Dfd  3.093, 19, 19 
P value  0.8698  P value  0.0178 
P value summary  ns  P value summary  * 
Are variances significantly different?  No  Are variances significantly different?  Yes 
The purpose of this test was to test whether the administration of the lactic yeast for cattle can improve the quantity and quality of produced milk on the farm Sava Kovacevic. This study confirmed significantly improved feedtomilk conversion, important increase in the milk production as well as significant improvement in milk quality indicated by a significant drop in the number of somatic cells/ml of milk. The latter testifies to an improvement in the general health of the animals. Together these date indicate significant economical benefit both in terms of increased milk production for the same food quantity and in terms of the medical expenses reduction in case of mastitis, implying the profitability of including TURVAL 0^{sp/50 }in the regular cattle meal.

Supplementary
Supplementary Table1
Supplementary Table2 A
Supplementary Table2 B