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  • Growth inhibition of selected microorganisms by an association of dairy starter cultures and probiotics | Cenci-Goga | Italian Journal of Animal Science
    probiotics Beniamino T Cenci Goga Musafiri Karama Paola Sechi Maria F Iulietto Sara Novelli Roberto Selvaggini Sara Mattei DOI http dx doi org 10 4081 ijas 2015 3745 Beniamino T Cenci Goga Dipartimento di Medicina Veterinaria Università di Perugia Italy beniamino cencigoga unipg it Musafiri Karama Department of Paraclinical Sciences University of Pretoria Onderstepoort South Africa Paola Sechi Dipartimento di Medicina Veterinaria Università di Perugia Italy Maria F Iulietto Dipartimento di Medicina Veterinaria Università di Perugia Italy Sara Novelli Dipartimento di Medicina Veterinaria Università di Perugia Italy Roberto Selvaggini Dipartimento di Scienze Agrarie Alimentari e Ambientali Università di Perugia Italy Sara Mattei Dipartimento di Medicina Veterinaria Università di Perugia Italy Abstract Several growth curves for selected pathogens and hygiene indicators alone and vs selected dairy starter cultures LAB and commercial probiotics have been performed All strains for LAB and commercial probiotics were inoculated as pure cultures into skim milk to get an initial cocci bacilli enterocci ratio of 2 1 1 and a concentration of approximately 10 7 cfu mL 1 until challenge vs selected pathogens and hygiene indicators Selected pathogens came from the collection of the Laboratorio di Ispezione degli Alimenti di O A or were reference strains Escherichia coli CSH26 K12 Staphylococcus aureus 27R Salmonella Derby 27 Pseudomonas fluorescens ATCC 13525 Listeria innocua ATCC 33090 Each strain was inoculated into skim milk to get an initial concentration of approximately 10 6 cfu mL 1 Growth curves in skim milk for the following challenges were studied i sterility control ii association LAB iii association of LAB vs each selected pathogen or hygiene indicator iv selected pathogen or hygiene indicator alone The challenges were carried out in BHI broth and in skim milk at 37 C The highest reduction was observed in milk but in general the association of LAB

    Original URL path: http://ijas.pagepress.org/index.php/ijas/article/view/ijas.2015.3745 (2016-04-25)
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  • Growth inhibition of selected microorganisms by an association of dairy starter cultures and probiotics | Cenci-Goga | Italian Journal of Animal Science
    C on air for 24 h at 24 h was approximately 1x10 9 cfu mL 1 For the test each strain was inoculated into skim milk to get an initial concentration of approx 10 6 cfu mL Characterization of starter cultures with acidification and challenge growth curves Acidification All strains were inoculated as pure cultures into skim milk BD Difco to get an initial concentration of about 10 7 cfu mL 1 All strains were incubated at 37 C The association was inoculated into skim milk BD Difco to get an initial cocci bacilli enterocci ratio of 2 1 1 and a concentration of approx 10 7 cfu mL 1 The association was incubated at 37 C and pH was measured with a Foodtrode electrode Hamilton Company Reno NV USA hooked to an Eutech pH 2700 Eutech Instrument Europe B V Nijkerk Netherlands which recorded pH values continuously with CyberComm 6000 Eutech Instrument every minute The following fourth degree polynomial was used as an empirical model to fit the experimental data eq 1 where y is pH x is time a c d e are the generic regression coefficients of the polynomial under study as determined by the statistical package Stat Graphics ver 6 1 Manugistics Inc Rockville MD USA The first derivative of the equation gives the instantaneous acidification rate and its maximum value V m which corresponds to the inflection point of the acidification curve whereas the second derivative gives the acceleration and its roots give the x values t m at the inflection points Challenge test Several growth curves for selected pathogens and hygiene indicators alone and vs the selected dairy starter cultures and commercial probiotics have been performed All strains for the selected dairy starter cultures and commercial probiotics were inoculated as pure cultures into skim milk BD Difco and incubated at 37 C to get an initial concentration of about 10 9 cfu mL 1 after 24 h of incubation The association was then inoculated into skim milk BD Difco to get an initial cocci bacilli enterocci ratio of 2 1 1 and a concentration of approx 10 7 cfu mL 1 until challenge vs selected pathogens and hygienic indicators All strains for the selected pathogens came from the collection of the Laboratorio di Ispezione degli Alimenti di Origine Animale Escherichia coli strain CSH26 K12 Staphylococcus aureus strain 27R Salmonella Derby 27 Pseudomonas fluorescens ATCC 13525 Listeria innocua ATCC 33090 Strains were inoculated as pure cultures into BHI BD Difco and incubated at 37 C 30 C for Pseudomonas fluorescens to get an initial concentration of about 10 9 cfu mL 1 after 24 h of incubation Each strain was then inoculated into skim milk BD Difco to get an initial concentration of approximately 10 6 cfu mL 1 Growth curves for the following challenges in triplicates were studied i sterility control ii association of starter cultures LAB iii association of LAB vs each selected pathogen or hygiene indicator iv selected pathogen or hygiene indicator alone The challenges were carried out in BHI broth BD Difco and in skim milk BD Difco at 37 C Bacterial counts were recorded at time 0 12 h 24 h 30 h 48 h 72 h 120 h 168 h 240 h Microbiological analysis were conducted using the methods described in paragraph 2 4 Microbiological analysis For the analysis about 25 mL of sample were transferred aseptically to 225 mL of sterile buffered peptone water Oxoid CM1049 and homogenised in a stomacher PBI International for 1 min at low speed and 1 min at high speed at room temperature Serial decimal dilutions in buffered peptone water were prepared and triplicate 1 mL or 0 1 mL samples of appropriate dilutions were poured or spread on total count and selective agar plates Total lactic microbiota was determined on de Man Rogosa Sharpe MRS Agar Oxoid acidified to pH 5 5 incubated at 30 C for 72 h under anaerobic conditions Gas generating kit Oxoid BR0038 Enterococcus faecium on Slanetz and Bartley SB Agar Oxoid CM0377 incubated at 37 C for 48 h Pseudomonas fluorescens on Pseudomonas agar base Oxoid CM0559 added with Pseudomonas CFC Supplement Oxoid SR0103 incubated at 25 C for 48 h S aureus were determined on Rabbit Plasma Fibrinogen RFP Agar Oxoid CM0961 with RFP Supplement Oxoid SR0122 incubated at 37 C for 48 h Escherichia coli CSH26 K12 on Sorbitol MacConkey agar McS Agar Oxoid CM0813 overlaid with 5 ml of the same medium and incubated at 30 C for 24 h The ISO 6579 method was used ISO 2007 for Salmonella Derby 27 Briefly after a pre enrichment step buffered peptone water Oxoid 18 h at 37 C and the inoculum onto two selective enrichments Rappaport Vassiliadis Soya peptone broth RVS Oxoid CM0866 24 h at 42 and Muller Kaufmann Tetrathionate Novobiocin Broth MKTTn Oxoid CM1048 24 h at 37 C a loopful of broth was spread onto Xylose Lysine Desoxycolate Agar XLD Oxoid CM0469 and Salmonella Chromogenic Agar Oxoid CM1007 with Salmonella selective supplement Oxoid SR0194 The ISO 11290 method ISO 1996 was used for Listeria innocua ATCC 33090 Briefly after a primary Fraser Oxoid CM 0895 with half Fraser selective supplement Oxoid SR0166 24 h at 30 C and a secondary enrichment medium Fraser with Fraser supplement Oxoid SR0156 48 h at 35 C a loopful of broth was spread onto Oxford agar Listeria selective agar Oxoid CM0856 with Listeria selective supplement SR0140 and PALCAM agar PALCAM agar base Oxoid CM0877 with PALCAM selective supplement Oxoid SR0150 Sensitivity for spread plate was 10 2 cfu g 1 and for pour plate was 10 cfu g 1 and the 95 confidence limit as given by the classic formula 2s 2 x Adams and Moss 2000 ranged between 37 and 12 i e plates with a number of cfu ranging from 30 to 300 Therefore no plates with less than 30 cfu were used for data analysis and when this applied to the lowest dilution the results were recorded as 300 for pour plate and 3000 for spread plate Samples without typical colonies were recorded as negative and samples with at least one typical colony in the lowest dilution were recorded as positive regardless the number of colonies Cenci Goga et al 2005 Analysis of the results For each sampling the arithmetic means of the three subsamples of each of the three sausages was calculated and then all the values converted to log for microbiological analyses were analysed using GraphPad InStat version 3 0b for Mac OS X and graphs were obtained with GraphPad Prism version 6 0d for Mac OS X Results and discussion Characterization of starter cultures with acidification and challenge growth curves Acidifying activity of selected starter cultures Three species of lactic acid bacteria previously isolated from among the most representative ones from sheep milk curd and cheese in cheese making trials carried out previously Cenci Goga et al 1995 along with a commercial probiotic were chosen to be used as a starter These strains had been previously identified by API 50 CHL and some of them had already been used as a starter in the manufacturing of salami Cenci Goga et al 2008 2012 The acidifying activity of the different species had been preliminarily tested Cenci Goga et al 1995 Clementi et al 1998 as pure cultures and as different associations in sterilized cow milk to assess their suitability to be used as a starter The parameters describing the acidification kinetics are reported in Table 1 together with the regression coefficients of the fourth degree polynomial equation 1 which was used as an empirical model This adequately fitted the experimental data since the r 2 values varied from 0 997 to 0 9998 and the actual values were almost exactly superimposed on the empirical model curves Figure 1 The values of the maximum instantaneous acidification rate V m were of the same order for all strains except for Lc lactis ssp lactis strain 16 which had the highest V m 3 14 10 3 ΔpH min and the lowest t m 350 min values These values were not very different from those 5 5 l0 3 ΔpH min and 330 min observed for Lc lactis SL03 in a study evaluating the acidifying activity of several lactic acid bacteria with a similar procedure Picque et al 1992 The final pH values of milk inoculated with the different strains as pure cultures Figure 1 were lower than those previously obtained in cow milk Cenci Goga et al 1995 Clementi et al 1998 except for Lb casei spp casei which reached the same value in both types of milk After 900 min the two strains of Lc lactis ssp lactis reached pH values equal to or lower than those obtained in cow and sheep milk inoculated with Lc lactis ssp lactis strains isolated from cheeses Ledda et al 1994 Enterococcus faecium gave a one fold pH decrease within about 500 min like some commercial thermophilic lactobacilli Chamba and Prost 1989 The association of the four strains performed well with a final pH of 3 74 and showed the fastest pH drop within the first 24 h along with a steady descent subsequently The association mimicked the behaviour of the two Lc lactis ssp lactis strains in the first 16 h and then when the lactococci acidification activity decreased steady pH values after 24 h mirrored the Lb casei spp casei performance for a continuous pH drop up to 70 hrs This synergism is of the utmost importance in the production of fermented food such as cheeses and dry salami because the LAB activity is desirable throughout the whole fermentation process Cenci Goga et al 2012 Cizeikiene et al 2013 Challenge growth curve The results are shown in Figure 2 which displays the different growth curves of pathogens or hygiene indicators vs the association of selected dairy starter cultures and commercial probiotics Figure 2 a shows the evolution of E coli CSH26 K12 panel 2b shows the evolution of Salmonella Derby strain 27 panel 2c shows Staphylococcus aureus strain 27R panel 2d shows Listeria innocua ATCC 33090 panel 2e shows Pseudomonas fluorescens ATCC 12983 and panel 2f shows the evolution of LAB and E faecium For the challenge E coli vs LAB level of E coli in milk dropped after 48 h below 10 4 cfu mL 1 and was no longer detectable at 72 h while in BHI the growth curve of E coli was parallel to control indicating an effect of pH rather than a direct activity of LAB and or bacteriocins A similar behaviour was shown by Salmonella Derby with an even faster drop 10 4 cfu mL 1 after 30 h S aureus proved to be more resilient and a substantial reduction in total viable cells was observed between 72 and 120 h 10 3 cfu mL 1 after 120 h with a complete elimination after 168 hrs L innocua was no longer detectable in milk after 120 h with a steady drop after 24 hrs 10 4 cfu mL 1 after 72 h LAB had an effect on L innocua also in BHI with levels below 10 4 cfu mL 1 just after 30 h The direct effect of certain LAB strains on Listeria spp rather than the indirect effect of pH drop has been described Winkelströter and De Martinis 2015 P fluorescens had a similar drop both in milk and in BHI and was no longer detectable after only 24 h proving that the LAB formulation tested in this study is able to limit the growth of this typical spoilage microorganism even in absence of pH drop Folawe Okorhi 2014 Figure 2 f shows the correct evolution of the LAB formulation and of the probiotic strain both in milk and in BHI The probiotic strain of E faecium showed a certain susceptibility to pH drop in fact after 48 h of incubation in milk as pure culture a rapid descent was recorded and counts reached values below 10 6 cfu mL 1 after 72 h and below 10 4 cfu mL 1 after 120 h when pH had reached levels below 5 5 A similar behaviour has been described in cheese Reale et al 2010 Conclusions There are certain criteria that lactic acid bacteria starter cultures should meet when being included in food products such as to compete effectively with indigenous lactic acid bacteria to produce adequate quantities of lactic acid to grow in the presence of up to 6 NaCl to tolerate NaNO 2 to grow between 10 C and 40 C with an optimum between 30 C and 37 C to be homo fermentative and catalase positive to be non proteolytic and to produce only very small quantities of H 2 O 2 to reduce nitrate to enhance flavour of the finished sausage not to produce biogenic amines not to produce slime to be antagonistic to pathogenic and other undesirable micro organisms and tolerant of or synergistic with other starter components Various species have been and are used as inoculants in sausage fermentation Campbell Platt 1987 The fermentation of carbohydrates leads to several desirable results production of organic acids which lead to the reduced pH value production of favourable organoleptic compounds coagulation of meat proteins thereby decreasing the water holding capacity and thus facilitating the drying process which affects the texture and firmness of the end product and red colour formation due to the reaction of nitrogen monoxide with the heme group in myoglobin at pH 5 4 to 5 5 The association of selected starter cultures and probiotics tested in this research was able to limit the growth of pathogens and hygiene indicators in an invitro setting According to the strong inhibitory activity our results show that the proposed formulation of LAB and probiotics could be applied as food preservative and as starter cultures The LAB application is wide ranging and most promising if one considers that the indicator microorganisms used in this experiment were suppressed These paper along with another study on the use of this formulation in salami production Cenci Goga et al 2015 highlights the possibility that food safety and food quality can dramatically be improved by using novel formulations of LAB with antimicrobial activities as starter for food fermentation It must be considered that in the production of traditional southern European style sausages the fermentation profile must phase in order to ensure the growth of the added starter culture at the expense of the background flora Additionally the acidification profile must not reach values below pH 4 8 5 0 at any time This will ensure that autochthonous microbiota maintain its activity over a longer period of time foremost reductase and flavour forming activities Lebert et al 2007 Marco et al 2008 Barbut 2010 Reale et al 2010 The starter cultures should be specifically selected for traditional fermentation profiles applying temperatures not higher than 24ºC Holley et al 1988 Scannell et al 2001 Cenci Goga et al 2012 On the other hand in the production of north European and US style sausages the fermentation profile have a very short lag phase in order to rapidly show a fast drop in pH to below 5 3 within 30 hours as a minimum This ensures an efficient inhibition of background flora and the production time is typically less than 2 weeks at the cost of a general lower consumer acceptability because the faster the acidification the less enzymes enhancing colour and aroma formation are produced Marco et al 2008 Barbut 2010 The proposed formulation is an additional tool in the production of low acid fermented dry salami that are nowadays the preferred consumer s choice given that the low speed of fermentation does not negatively affect the aroma in the product Moreover the incorporation of E faecium is a further asset because researchers postulate that enterococci may contribute to the development of the typical sensory properties of certain fermented meat products such as salami Holley et al 1988 Enterococci are in fact capable of modulating the aroma by means of the conversion of amino acids and free fatty acids González Fernández et al 2006 Leroy et al 2006 Corbiere Morot Bizot et al 2007 García Fontán et al 2007 References 1 M R Adams M O Moss 2000 Food microbiology Royal Society of Chemistry Publ Cambridge UK 2 S Barbut 2010 Color development during natural fermentation and chemical acidification of salami type products J Muscle Food 21 499 508 3 G Campbell Platt 1987 Fermented foods of the world Butterworth London UK 4 B T Cenci Goga F Clementi E Di Antonio 1995 Behaviour of lactic and non lactic microflora during production and ripening of farm manufactured Pecorino cheese Ann Microbiol 45 219 236 5 B T Cenci Goga R Ortenzi E Bartocci A Codega de Oliveira F Clementi A Vizzani 2005 Effect of the implementation of HACCP on the microbiological quality of meals at a university restaurant Foodborne Pathog Dis 2 138 145 6 B T Cenci Goga D Ranucci D Miraglia A Cioffi 2008 Use of starter cultures of dairy origin in the production of Salame nostrano an Italian dry cured sausage Meat Sci 78 381 390 7 B T Cenci Goga P V Rossitto P Sechi S Parmegiani V Cambiotti J S Cullor 2012 Effect of selected dairy starter cultures on microbiological chemical and sensory characteristics of swine and venison Dama dama nitrite free dry cured sausages Meat Sci 90 599 606 8 B T Cenci Goga M Karama P Sechi M F Iulietto S Mattei S Novelli 2015 Effect of a novel starter culture and specific ripening conditions on the characteristics of Italian dry salami Meat Sci In press 9 J F Chamba F Prost 1989 Mesure de l activité acidifiante des bactéries lactiques thermophiles utilizées pour la fabrication des fromages à pâte cuite Lait 69 417 431 10 D Cizeikiene G Juodeikiene A Paskevicius E Bartkiene 2013 Antimicrobial activity

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  • Meat quality of lambs fed different saltbush hay (Atriplex nummularia) levels | Moreno | Italian Journal of Animal Science
    Almeida Oscar Boaventura Neto DOI http dx doi org 10 4081 ijas 2015 3302 Greicy M B Moreno Colegiado de Zootecnia Universidade Federal de Alagoas Arapiraca Brazil greicymitzimoreno yahoo com br Hirasilva Borba Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Jaboticabal Brazil Gherman G L Araújo Empresa Brasileira de Pesquisa Agropecuária Petrolina Brazil Carlos Sañudo Departamento de Producción Animal y Ciencia de Alimentos Universidad de Zaragoza Spain Américo G Silva Sobrinho Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Jaboticabal Brazil Marcos E Buzanskas Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Jaboticabal Brazil Dorgival M Lima Júnior Colegiado de Zootecnia Universidade Federal de Alagoas Arapiraca Brazil Vitor V S de Almeida Colegiado de Zootecnia Universidade Federal de Alagoas Arapiraca Brazil Oscar Boaventura Neto Colegiado de Medicina Veterinária Universidade Federal de Alagoas Viçosa Brazil Abstract Climate changes have increased soil and water salinity compromising animal production especially in dry areas where scientists have become more interested in halophyte plants like saltbush The effects of saltbush hay levels 30 40 50 and 60 were evaluated based on physical chemical nutritional and sensory parameters of Santa Inês lamb meat Thirty two 8 month old castrated Santa Inês lambs with initial weights of 22 1 97 kg were used they were slaughtered after 60 days in the feedlot The pH colour moisture protein and cholesterol contents did not differ among treatments Panelists observed a greater intensity of lamb smell and flavour P 0 0035 in the meat of animals that received more concentrate in the diet An increase in the inclusion of saltbush increased ash percentage P 0 0232 total saturated P 0 0035 and polyunsaturated P 0 0287 fatty acids and reduced the lipids P 0 0055 and the n 6 n 3 ratio P 0 0058 of

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  • Meat quality of lambs fed different saltbush hay (Atriplex nummularia) levels | Moreno | Italian Journal of Animal Science
    of WHC in the Longissimus lumborum in all the studied levels of hay These results are in contrast to those reported by some authors who affirmed that with an increase in the level of concentrate in the diets the WHC of the meat also increases Puolanne and Halonen 2010 Although no significant difference was observed the WHC was 11 higher in the meat of animals fed diet with a higher amount of saltbush hay and this could be due to the activity of betain The ash content increased and the intramuscular fat content decreased in the meat as the level of saltbush hay increased in the diet Table 4 However there was no effect on moisture 74 50 protein 21 15 and cholesterol in the meat 36 76 mg 100 g Probabily the slight increase of ash is a consequence of less fat The average content of protein 21 15 indicates that the sheep meat fed saltbush hay and concentrate is nutritionally desirable and it linearly reduced the amount of intramuscular fat and increased the ash when this forage was included in the diet These results could be further studied and used as marketing tools in the systems of sheep meat production using saltbush as forage showing an improvement in the nutritional quality of this meat due to a reduction in fat content Some initiatives have already been implemented in some regions in Australia aiming at these objectives Hocquette et al 2010 The different levels of forage and concentrate affected all fat storage depots including subcutaneous Table 2 visceral Moreno 2011 and intramuscular Table 4 A higher proportion of the concentrate in the diet increased the percentage of the intramuscular fat which was also observed by other authors Luciano et al 2013 Majdoub Mathlouthi et al 2013 However other results regarding the effect of different forage concentrate ratios on the chemical composition of the sheep meat are contradictory Zeola et al 2004 Lanza et al 2011 Camilo et al 2012 Joy et al 2012 Sensory analyses High levels of saltbush hay reduced the intensity of the typical flavour of the sheep meat and meat was considered softer in the treatments with a higher amount of forage Table 5 In Australia sheep farmers along with the meat industry and the retailers state that the sheep meat from lambs fed saltbush has a higher sensory quality Anon 1996 Those empirical statements have not been proven yet by the few studies carried out on the sensory analyses of the meat from sheep receiving saltbush in the diet Pearce et al 2010 affirm that the improvement in the sensory aspects of this meat may be related to the antioxidant action of the vitamin E present in the saltbush which would inhibit the development of rancidity odour and taste especially in the aged meat which is very common in Australia The greatest ratings for flavour intensity of sheep meat were observed in this work with a lower proportion of saltbush in the diet however other studies have demonstrated that the opposite also occurs the meat may present a greater flavour intensity in animals fed more forage in the diet or in the pasture Resconi et al 2009 The greater flavour intensity may result from a greater amount of fat in the meat of animals fed more concentrate in the diet Table 4 Lipids are the precursors of most volatile compounds that by the action of the heat contribute to the flavour pallatablity and characteristic meat taste Resconi et al 2012 The main contributors in the formation of the flavour and odour in meat are the branched chain fatty acids isobutyric isovaleric 2 methyl butyric 4 methyl octanoic and 4 methyl nonanoic These fatty acids originate from the rumen propionate as derivatives of hepatic gluconeogenesis High levels of propionate in the rumen lead to the production of branched chain fatty acids and the production of propionate is higher when animals are fed high proportions of concentrate Resconi et al 2009 Luciano et al 2013 Majdoub Mathlouthi et al 2013 which may have occurred in this experiment The low energy content of saltbush may have also resulted in a lower formation of propionate and consequently a lower content of branched chain fatty acids that would be involved in the formation of odour and flavour in the assessed meat It is known that consumers in Southeast Brazil especially in São Paulo State prefer sheep meat with a softer flavour from young lambs from 4 to 6 months of age with a low fat content as opposed to most consumers in northeastern and southern Brazil who prefer animals slaughtered later The sensory panel of this study was made up only by panellists from the Southeast region of Brazil which could explain the results obtained in the study and this was also observed in other studies Osório et al 2009 Nevertheless the differences between the ratings for taste were not sufficient to reduce the whole acceptance of the meat in the different treatments The preferences of the panelists are related to their culinary and cultural habitats and by their personal characteristics moreover the sensory characteristics are a very important tool to discern the acceptability of the offered meat to a potential consumer market Regarding the juiciness consumers stated that a more juicy meat is a better meat and this quality is mostly related to the content of intramuscular fat water and protein muscle Therefore a lamb may present a less juicy meat because it has not completely reached intramuscular fat deposition Osório et al 2009 however the amount of water in muscle of young animals can also promote greater sense of meat juiciness Naturally there is a large amount of water in muscle tissue since fat and water are inversely proportional In this study even though the amount of intramuscular fat have reduced with the inclusion of saltbush hay there was no difference to the meat juiciness which may be associated with a higher relative amount of muscle and water rather than the relative amount of fat Pearce et al 2011 affirm that even though the characteristics of the myowater in general must be expected to contribute to juiciness there is only limited knowledge of how these characteristics and the temperature induced changes in these characteristics during cooking affect the sensory properties of meat Animals with more subcutaneous Table 2 and intramuscular fat Table 4 in this work also presented meat with a lower shear force Table 3 although the panelists had not found those differences in the tenderness Table 5 Hopkins and Nicholson 1999 studied the quality of lamb meat grazing saltbush and supplemented with hay or oat or fed only hay control These researchers found that there was a difference only in the flavour intensity while the meat of animals fed saltbush regardless the type of supplement presented more flavour intensity in relation to the animals fed only hay However Pearce et al 2008 did not obtain feeding effects with the use of saltbush in any of the parameters assessed in the sensory analyses of the lamb meat with average ratings of 4 8 6 0 6 2 and 6 3 for intensities of odour tenderness juiciness and whole acceptation respectively by using a 1 10 scale Fatty acid composition The fatty acids found at a higher concentration in this study were the following saturated palmitic acid C16 0 and stearic acid C18 0 and monounsaturated oleic acid C18 1n 9 Table 6 Myristic C14 0 and palmitic C16 0 acids are considered as hypercholesterolemic however the stearic acid C18 0 which represents from 10 to 20 of the fat produced by the ruminants do not show this property FAO 2010 The inclusion of saltbush hay in the diet has contributed to the reduction of myristic acid and it did not increase palmitic acid which is an interesting result with regards to its benefits for human health Conjugated linoleic acid CLA isomer C18 2 c 9 t 11 was reduced as the level of saltbush hay increased in the diet contradicting some authors who reported an increment in CLA when animals were fed higher proportions of forage or pastures Nuernberg et al 2008 Jacques et al 2011 Ekiz et al 2013 Paim et al 2014 Diets with greater forage contents would favor higher concentrations of CLA especially in animals with a completely developed rumen once the main precursor of CLA vaccenic acid Campo 2009 is formed in this environment This was not observed in this study An explanation it the reason for CLA decreasing with increasing of saltbush hay are low PUFA intake and lower intramuscular fat Table 4 The amount of CLA deposited in the muscles depends mostly of the rumen outflow of C18 1 trans 11 formed by incomplete biohydrogenation of dietary C18 PUFA and of the endogenous conversion of C18 1 trans 11 into C18 2 cis 9 trans 11 CLA in the tissues Wood et al 2008 There was an increase of linoleic acid C18 2 n 6 with the inclusion of saltbush hay in the diet The increase in linoleic with saltbush hay is directly related with lower intramuscular fat and thus higher relative proportion of phospholipids Eicosapentaenoic EPA C20 5 n 3 and docosahexaenoic DHA C22 6 n 3 acids are PUFA derivatives of linolenic acid moreover they are more propitious to oxidation than oleic or linoleic acids Elmore et al 2000 Some studies reported a greater proportion of linolenic acid and its derivatives in the fat of grazing sheep than those fed concentrate diets consistent with the results of this work Woods and Fearon 2009 Oliveira et al 2013 Although the most studies usually find higher biohydrogenation in forage fed animals than in concentrate fed animals another authors reports lower biohydrogenation in the rumen when the diet is composed of a higher level of forage Antongiovanni et al 2003 Abidi et al 2009 Buccioni et al 2009 The total amount of saturated SFA and unsaturated fatty acids UFA i e MUFA and PUFA and their main ratios are described in Table 7 The increase in the level of saltbush hay in the diet increased the total SFA and PUFA corroborating the results found by Gallo et al 2007 and Nuernberg et al 2008 who observed an increase in the concentration of these fatty acids in the meat of sheep fed a greater amount of forage There was an decrease of the n 6 n 3 ratio with the inclusion of saltbush hay in the diet In a FAO 2010 revision about fats and fatty acids in human nutrition there is no a rational specific recommendation for n 6 n 3 ratio if intakes of n6 and n3 fatty acids lie within the recommendation established Therefore the usefulness of the ratio in human nutrition is very questionable and controversial Griffin 2008 Harris 2006 Salter 2013 The greater incorporation of n3 PUFA in animals fed more forage in the diet is also associated with a greater level of α tocopherol vitamin E present in the forage which accumulates in the adipose tissue once it is a liposoluble vitamin Delgado Pertíñez et al 2013 Hou et al 2013 This fact allows for less oxidation of n 3 PUFAs which are susceptible than n 6 favoring the preservation of the final product Campo 2009 Considering that saltbush is rich in vitamin E 139 0 mg of α tocopherol kg DM Pearce et al 2005 more studies are warranted to address this potential to improve the final quality of the meat and its preservation Conclusions Saltbush provides a good quality sheep meat with a low fat content and a high ash content The saltbush hay did not cause negative effects in the sensory quality of the meat and its percentage in the diet altered the perception of lamb s flavour intensity by the panelists The inclusion of saltbush reduced the n 6 n 3 ratio and increased the total PUFA of meat which are important for human health Diets composed by saltbush hay may be used in Santa Inês finishing lambs as long as they are associated with other diets especially energy diets aiming to potentiate the voluntary intake and to promote a greater weight gain of the animals Acknowledgments The authors gratefully acknowledge funding from the Brazilian Development Bank BNDES and the São Paulo Research Foundation FAPESP References 1 S Abidi H Ben Salem V Vasta A Priolo 2009 Supplementation with barley or spineless cactus Opuntia ficus indica f inermis cladodes on digestion growth and intramuscular fatty acid composition in sheep and goats receiving oaten hay Small Ruminant Res 87 9 16 2 A N Al Owaimer S M Zahran B A Al Bassam 2008 Effect of feeding some types of Atriplex ssp in complete diet on growth performance and digestibility of growing lambs Available from http repository ksu edu sa jspui handle 123456789 7977 3 Anon 1996 Rediscovering an old treat Saltbush mutton finds gourmet market Aust Farm J 1996 32 33 4 M Antongiovanni A Buccioni F Petacchi P Secchiari M Mele A Serra 2003 Upgrading the lipid fraction of foods of animal origin by dietary means rumen activity and presence of trans fatty acids and CLA in milk and meat Ital J Anim Sci 2 3 28 5 AOAC 1995 Official methods of analysis 16th ed Association of Official Analytical Chemists International Washington DC USA 6 H Ben Salem H C Norman A Nefzaoui D E Mayberry K L Pearce D K Revell 2010 Potential use of oldman saltbush Atriplex nummularia Lindl in sheep and goat feeding Small Ruminant Res 91 13 28 7 E G Bligh W J Dyer 1959 A rapid method of total lipid extraction and purification Can J Biochem Phys 37 911 917 8 R Boccard L Buchter E Casteels E Cosentino E Dransfield D E Hood R L Joseph D B McDougall C Touraille 1981 Procedures for measuring meat quality characteristics in beef production experiments Report of a working group in the Commission of the European Communities CEC Beef Production research programme Livest Prod Sci 8 385 397 9 N Bragagnolo D B Rodriguez Amaya 1992 Teores de colesterol em carne de frango Rev Farm Bioquim Univ São Paulo 28 122 131 10 A Buccioni M Antongiovanni S Minieri S Rapaccini V Pratesi M Mele 2009 Effect of three species of herbage Medicago sativa Lolium multiflorum Avena sativa on in vitro ruminal production of conjugated linoleic and vaccenic acids Ital J Anim Sci 8 231 244 11 D A Camilo E S Pereira P G Pimentel R L Oliveira M J D Cândido M R G F Costa R M S Aquino 2012 Intake and feeding behavior of Morada Nova lambs fed different energy levels Ital J Anim Sci 11 13 19 12 M M Campo 2009 Calidad de la grasa ovina In C Sañudo R Cepero eds Ovinotecnia producción y economia en la especies ovina University of Zaragoza Publ Spain pp 337 344 13 A C Cavalcanti M R Ribeiro J C Araújo Filho F B R Silva 1994 Avaliação do potencial de terras para irrigação no Nordeste Available from http www uep cnps embrapa br zoneamentos irrigacao php 14 Commission International de l Eclairage 1976 Colorimetry official recommendations of the International Commission on Illumination CIE ed Paris France 15 S A S Craig 2014 Betaine in human nutrition Am J Clin Nutr 80 539 549 16 M Delgado Pertíñez R Gutiérrez Peña Y Mena V M Fernández Cabanás D Laberye 2013 Milk production fatty acid composition and vitamin E content of Payoya goats according to grazing level in summeron Mediterranean shrublands Small Ruminant Res 114 167 175 17 S D Dutcosky 1996 Análise sensorial de alimentos Editora Champagnat Curitiba Brazil 18 B Ekiz G Demirel A Yilmaz M Ozcan H Yalcintan O Kocak A Altinel 2013 Slaughter characteristics carcass quality and fatty acidcomposition of lambs under four different production systems Small Ruminant Res 114 26 34 19 H M El Shaer 2010 Halophytes and salt tolerant plants as potential forage for ruminants in the Near East region Small Ruminant Res 91 3 12 20 J S Elmore D S Mottram M Enser J D Wood 2000 The effects of diet and breed on volatile compounds of cooked meat Meat Sci 55 149 159 21 J B S Ferraz P E Felício 2010 Production systems An example from Brazil Meat Sci 84 238 243 22 FAO 2010 Fats and fatty acids in human nutrition Report of an expert consultation Food and Agriculture Organization of The United Nations Publ Rome Italy 23 I Font M Furnols R San Julián L Guerrero C Sañudo M M Campo J L Olleta M A Oliver V Cañeque I Álvarez M T Díaz W Branscheid M Wicke G R Nute F Montossi 2006 Acceptability of lamb meat from different producing systems and ageing time to German Spanish and British consumers Meat Sci 72 545 554 24 C R Francote H C Norman I H Williams D G Masters 2009 Cattle performed as well as sheep when grazing a river saltbush Atriplex amnicola based pasture Anim Prod Sci 49 998 1006 25 S B Gallo E R Siqueira G T Rosa 2007 Efeito da nutrição da ovelha e do cordeiro sobre o perfil de ácidos graxos do músculo Triceps brachii de cordeiros Rev Bras Zootecn 36 2069 2073 26 B A Griffin 2008 How relevant is the ratio of dietary n 6 to n 3 polyunsaturated fatty acids to cardiovascular disease risk Evidence from the OPTILIP study Curr Opin Lipidol 19 57 62 27 A Guerrero M V Valero M M Campo C Sañudo 2013 Some factors that affect ruminant meat quality from the farm to the fork Review Available from http periodicos uem br ojs index php ActaSciAnimSci article view 21756 28 R Hamm 1960 Biochemistry of meat hydratation Adv Food Res 10 335 443 29 W S Harris 2006 The omega 6 omega 3 ratio and cardiovascular disease risk uses

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  • Effects of degradable protein and non-fibre carbohydrates on microbial growth and fermentation in the rumen simulating fermenter (Rusitec) | Zhao | Italian Journal of Animal Science
    Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China Chuan B Fu Jiangxi Province Key Laboratory of Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China Chan J Liu Jiangxi Province Key Laboratory of Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China Lan J Xu Jiangxi Province Key Laboratory of Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China Ke Pan Jiangxi Province Key Laboratory of Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China Ming R Qu Jiangxi Province Key Laboratory of Animal Nutrition Engineering Research Center of Feed Development Jiangxi Agricultural University Nanchang China qumingren 126 com Abstract A rumen simulation technique Rusitec apparatus with eight 800 ml fermentation vessels was used to investigate the effects of rumen degradable protein RDP level and non fibre carbohydrate NFC type on ruminal fermentation microbial growth and populations of ruminal cellulolytic bacteria Treatments consisted of two NFC types starch and pectin supplemented with 0 g d low RDP or 1 56 g d high RDP sodium caseinate Apparent disappearance of dry matter and organic matter was greater for pectin than for starch treatment P 0 01 with low or high RDP A NFC RDP interaction was observed for neutral detergent fibre disappearance P 0 01 which was lower for pectin than for starch only under low RDP conditions Compared with starch pectin treatment increased the copy numbers of Ruminococcus albus P 0 01 and Ruminococcus flavefaciens P 0 09 the molar proportion of acetate P 0 01 the acetate propionate ratio P 0 01 and methane production P 0 01 but reduced the propionate proportion P 0 01 Increasing dietary RDP increased the production of total VFA P 0 01

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  • Effects of degradable protein and non-fibre carbohydrates on microbial growth and fermentation in the rumen simulating fermenter (Rusitec) | Zhao | Italian Journal of Animal Science
    and their metabolites xanthine and hypoxanthine Approximately 400 mL of effluent was collected for isolation of liquid associated microbial pellets LAM according to Ranilla and Carro 2003 The contents of the nylon bags removed on day 13 and 14 were collected and mixed to determine the solid associated microbial pellets SAM Ranilla and Carro 2003 Approximately 20 of solids content was frozen and lyophilized for determination of DM NAN and purines concentration The bacterial samples from LAM and SAM were lyophilized ground using a mortar and pestle and analyzed for N and total purines concentration On day 15 4 mL of fermenter fluid as liquid fraction and one feed bag containing undigested feed as solid fraction from each vessel were collected and were frozen at 80 C for deoxyribonucleic acid DNA extraction Analytical procedures Samples were analyzed for DM by drying at 135 C in an airflow type oven for 2 h AOAC 1990 method 930 15 for OM by ashing at 550 C for at least 4 h and for N using the Kjeldahl procedure AOAC 1990 method 984 13 The NDF and ADF content in all samples were analyzed according to Van Soest et al 1991 Heat stable α amylase Sigma A3306 Sigma Aldrich Shanghai China and sodium sulfite were used for NDF determination Ammonia N in samples was analysed according to Weatherburn 1967 To determine total and individual VFA acidified samples were centrifuged at 11 000 g for 10 min and the supernatant fraction was filtered through a 0 45 µm filter The VFA concentrations in the filtered samples were determined by HPLC model L 2000 Hitachi High Technologies Corporation Tokyo Japan with a reversed phase Agilent TC C18 column 4 6 mm 250 mm 5 µm Agilent Technologies Santa Clara CA according to Akalin et al 2002 Crotonic acid was used as an internal standard The concentration of CH 4 was analysed by GC model 663 30 Hitachi High Technologies Corporation equipped with a flame ionization detector Total purines in the NAN fraction of the digesta and bacterial pellets were quantified by HPLC Reynal and Broderick 2009 The volume of total gas produced was measured by the displacement of water as previously described Soliva and Hess 2007 For microbial determination total genomic DNA was extracted and purified from fermenter liquid 220 µL and digested feed 220 µg samples using the QIAamp DNA Stool Mini Kit Qiagen China Co Ltd Shanghai China The bacterial species determined were Fibrobacter succinogenes Ruminococcus albus and Ruminococcus flavefaciens as representatives of fibrolytic cellulolytic and hemicellulolytic species The 16s ribosomal deoxyribonucleic acid rDNA copy numbers of three fibrolytic bacterial species were determined according to Zhao et al 2014 Calculations and statistical analyses The proportion of digesta NAN liquid or solid of microbial origin were estimated in each fermenter by dividing the ratio total purines N of the NAN portion of digesta by the ratio total purines N in the corresponding microbial pellets LAB or SAB Daily microbial N production mg d LAM or SAM in each fermenter was calculated by multiplying the proportion of NAN of microbial origin by the amount of NAN in the corresponding digesta liquid or solid Total daily microbial production was calculated as the sum of the flows of LAM and SAM Data were analysed by the PROC MIXED procedure SAS Inst Inc Cary NC USA according to a randomized complete block design blocking by incubation Hindrichsen and Kreuzer 2009 The model included treatments NFC and RDP and the interaction between NFC and RDP as fixed effects and incubation period block as a random effect Significance was declared at P 0 05 and trends were discussed at P 0 10 When a significant effect of treatment was detected differences among means were tested using Tukey s multiple comparison tests Results and discussion Data on pH and apparent disappearance of diet are presented in Table 2 The pH before feeding was not affected by dietary treatments and averaged 6 65 across treatments A NFC RDP interaction was observed for mean pH over 12 h after the feeding P 0 01 with greater values found for pectin treatment under high RDP conditions Apparent disappearance of DM and OM was greater for fermenters fed pectin diets than those fed starch diets P 0 01 Supplementation with casein increased apparent DM OM and N disappearance P 0 01 There were NFC RDP interactions for apparent disappearance of NDF P 0 01 which was lower for pectin than for starch treatment under low RDP conditions Data on cellulolytic bacterial populations in liquid and solid fractions are shown in Table 3 The 16s rDNA copy numbers of F succinogenes both in liquid and solid fractions were not affected by dietary treatments Increased dieatry RDP increased 16s rDNA copy numbers of R albus in solid fraction P 0 01 and those of R flavefaciens both in liquid and solid fractions P 0 01 and P 0 01 respectively Compared with starch treatments 16s rDNA copy numbers of R albus and R flavefaciens both in liquid and solid fractions were greater or tended to be greater P 0 09 for pectin treatments A previous study reported that although pectin substances are readily degraded in the rumen they do not mimic the pH lowering effect of starch because they generally are not fermented to lactate Hall et al 1998 Ben Ghedalia et al 1989 observed that the sheep fed pectin rich diets maintained a higher rumen pH than the ones fed starch rich diets In the present study higher pH found for pectin treatment under high RDP conditions is in agreement with above results The apparent disappearance of DM and OM was greater for pectin treatment than for starch treatment which may relate to the differences in digestibility between pectin and starch Pectin is a rapidly degraded complex carbohydrate Van Soest et al 1991 and is almost completely degraded in the rumen Hall et al 1998 However the in situ digestibility of corn starch is only about 60 Sauvant et al 2004 Few data are available on the effect of purified pectin on ruminal NDF digestibility Rosendo et al 2003 compared the effects of starch inulin and pectins on forage NDF digestion kinetics using in vitro digestion technique and observed no difference among treatments Ben Ghedalia et al 1989 studied the effects of pectin rich vs starch rich diets on quantitative aspects of digestion in sheep and found that the cell walls in the pectin rich diet were 16 more digestible than in the starch rich ration In the present study the difference in the apparent disappearance of NDF between starch and pectin treatments was affected by dietary RDP The apparent disappearance of NDF was lower for pectin than for starch treatment when dietary RDP was low and increasing dietary RDP increased NDF disappearance for pectin and reduced that for starch treatment The results however while not unexpected remains unexplained because they were not supported by the observation of fibrolytic bacterial numbers which were only data related to the NDF disappearance in the present study The 16s rDNA copy numbers of R albus and R flavefaciens were increased by increased dietary RDP and were greater for pectin than for starch treatment Greater numbers of R albus and R flavefaciens for pectin treatment may be due to their degradation and utilization of pectin Gradel and Dehority 1972 Current results suggest that the variation in numbers of three predominant fibrolytic bacteria may be not better indicator for fibre digestibility because other ruminal microorganism such as protozoa and fungi populations Windham and Akin 1984 Coleman 1985 may also degrade and utilize feed fibre fraction The discrepancy between the population of fibrolytic bacteria and fibre digestibility was also found in other studies Barrios Urdaneta et al 2003 Data on the daily production of methane and VFA are presented in Table 4 No NFC RDP interaction was observed for those data expect for total VFA production P 0 09 which tended to be greater for pectin treatment under high RDP conditions Compared with starch treatment pectin treatment increased the molar proportion of acetate P 0 01 the acetate propionate ratio P 0 01 and methane production P 0 01 but reduced the propionate proportion P 0 01 The molar proportion of butyrate was unaffected by dietary factors Increased dietary RDP increased production of total VFA P 0 01 and methane P 0 01 Total VFA and methane production were greater for pectin and high RDP treatments which is in agreement with the observed increases of feed disappearance Compared with starch treatment pectin treatment produced greater molar proportion of acetate and lower propionate molar proportion which is consistent with results obtained in previous studies Marounek et al 1985 Strobel and Russell 1986 The methane production was greater for pectin treatment may also relate to the hydrolysis of methyl esters from pectin to produce methanol which can be exclusively converted into methane and increase the rate of methanogenesis Pol and Demeyer 1988 Data on microbial N ammonia N and NAN fractions of the digesta are presented in Table 5 As expected increased dietary RDP led to increases P 0 05 in the ammonia N production P 0 01 and the flow of total NAN P 0 01 and microbial N P 0 01 Significant NFC RDP interaction P 0 01 was observed for ammonia N production which did not differ under low RDP conditions but was lower for pectin treatment than for starch treatment under high RDP conditions Carbohydrate source did not affect daily production of NAN and efficiency of microbial synthesis EMS expressed as mg microbial N g OM fermented There were NFC RDP interaction tendencies for daily N flow of total microorganisms P 0 07 and SAM P 0 10 and the values were greater and tended to be greater for pectin than for starch treatment under high RDP conditions Daily N flow of LAM was greater P 0 02 for pectin than for starch treatment The daily production of ammonia N in the fermenters depends on the extent of CP degradation and N uptake by ruminal bacteria A large increase was observed in the production of ammonia N with supplemental RDP which is in agreement with previously reported observations Heldt et al 1999 This is mainly attributable to the direct provision of ruminally available N from supplemental casein The greater production of ammonia N in the starch treatment under high RDP conditions may have related to lower observed microbial N synthesis compared with pectin treatment Ben Ghedalia et al 1989 also found that ammonia N concentration of pectin rich diets was lower than that of starch rich diets The optimal concentrations of ruminal ammonia N for microbial growth are controversial but 5 mg dL of ammonia N maximized microbial protein synthesis in vitro in previous research Satter and Slyter 1974 The concentrations of ammonia N for starch and pectin treatments were 1 8 and 1 8 respectively under low RDP conditions and were 22 4 and 16 3 mg dL respectively under high RDP conditions which suggest that microbial growth may be limited by low supplemental RDP Greater 16s rDNA copies of R albus and R flavefaciens were observed under high RDP conditions partly supported this speculation Supplementation of RDP significantly increased microbial N flow including LAM and SAM in the present study which is in agreement with the continuous culture results obtained by Griswold et al 2003 Compared with starch pectin produced similar microbial N to several previous studies Strobel and Russell 1986 Chester Jones et al 1991 However tendencies of NFC RDP interaction were observed for microbial N flow in the present study Microbial N production did not differ under low RDP conditions but was greater for pectin treatment than for starch treatment when more supplemental RDP was provided This result may relate to the synchrony between ruminal protein and carbohydrate digestion Microbial protein synthesis depends largely on the available amount and fermentation rate of carbohydrates and N in the rumen NRC 2001 The substrate fermentation rate was roughly proportional to the rate of microbial growth with more rapidly fermented substrates yielding more microbial mass Nocek and Russell 1988 Pectin has a higher rumen degradation rate and extent compared with starch In addition diets containing high levels of pectin may have higher efficiency of available N use or create more favourable conditions for microbial utilisation of other carbohydrates cellulose in the rumen than that observed with starch diets Ariza et al 2001 However current results contradict Hall and Herejk 2001 in which maximal microbial N yield was greatest for starch compared to pectin in an in vitro fermentation with mixed ruminal microorganisms These disparities may have resulted from the fact that ammonia N could be removed from the fermenters by the effluent in the present research but not in the system used by Hall and Herejk 2001 Therefore the synchrony between ruminal protein and carbohydrate digestion may not be exhibited in the latter s research in which the net microbial synthesis between starch and pectin was compared under quiescent conditions In addition compared with starch greater apparent disappearance of OM for pectin treatment in current study also resulted in greater available carbohydrates energy for microbial synthesis The efficiency of microbial synthesis expressed as g microbial N kg OM fermented was not affected by the type of carbohydrate which is consistent with some studies on sugar beet pulp substitution for starch Chester Jones et al 1991 However Ariza et al 2001 reported that EMS was greater for citrus pulp treatment than for hominy feed treatment Supplementation of RDP tended to increase EMS in the present study which is in agreement with results obtained by Griswold et al 2003 Conclusions Compared with starch pectin treatment has greater apparent disappearance of DM and OM and produced more acetate and less propionate When rumen degradable N was deficient disappearance of fibre was lower for pectin than for starch treatment and no differences in microbial N production was observed between the two treatments When rumen degradable N was sufficient apparent disappearance of fibre did not differ but microbial N production was greater for pectin than for starch treatment These results indicate that RDP level NFC type and their interaction affected ruminal fermentation and microbial growth and under sufficient ruminal degradable N and pectin had greater advantage in microbial N synthesis than starch in vitro References 1 A Akalin S Gönç Y Akba 2002 Variation in organic acids content during ripening of pickled white cheese J Dairy Sci 85 1670 1676 2 AOAC 1990 Official methods of analysis 15th ed AOAC Arlington VA USA 3 P Ariza A Bach M Stern M Hall 2001 Effects of carbohydrates from citrus pulp and hominy feed on microbial fermentation in continuous culture J Anim Sci 79 2713 2718 4 A Barrios Urdaneta M Fondevila C Castrillo 2003 Effect of supplementation with different proportions of barley grain or citrus pulp on the digestive utilization of ammonia treated straw by sheep Anim Sci 76 309 317 5 D Ben Ghedalia E Yosef J Miron Y Est 1989 The effects of starch and pectin rich diets on quantitative aspects of digestion in sheep Anim Feed Sci Technol 24 289 298 6 D G Chamberlain S Robertson J J Choung 1993 Sugars versus starch as supplements to grass silage effects on ruminal fermentation and the supply of microbial protein to the small intestine estimated from the urinary excretion of purine derivatives in sheep J Sci Food Agric 63 189 194 7 H Chester Jones M Stern H Metwally J Linn D Ziegler 1991 Effects of dietary protein energy interrelationships on Holstein steer performance and ruminal bacterial fermentation in continuous culture J Anim Sci 69 4956 4966 8 G Coleman 1985 The cellulase content of 15 species of entodiniomorphid protozoa mixed bacteria and plant debris isolated from the ovine rumen J Agric Sci 104 349 360 9 C M Gradel B Dehority 1972 Fermentation of isolated pectin and pectin from intact forages by pure cultures of rumen bacteria Appl Microbiol 23 332 340 10 K Griswold G Apgar J Bouton J Firkins 2003 Effects of urea infusion and ruminal degradable protein concentration on microbial growth digestibility and fermentation in continuous culture J Anim Sci 81 329 336 11 M Hall 2003 Challenges with nonfiber carbohydrate methods J Anim Sci 81 3226 3232 12 M Hall C Herejk 2001 Differences in yields of microbial crude protein from in vitro fermentation of carbohydrates J Dairy Sci 84 2486 2493 13 M Hall C Larson C Wilcox 2010 Carbohydrate source and protein degradability alter lactation ruminal and blood measures J Dairy Sci 93 311 322 14 M Hall A Pell L Chase 1998 Characteristics of neutral detergent soluble fiber fermentation by mixed ruminal microbes Anim Feed Sci Technol 70 23 39 15 J Heldt R Cochran G Stokka C Farmer C Mathis E Titgemeyer T Nagaraja 1999 Effects of different supplemental sugars and starch fed in combination with degradable intake protein on low quality forage use by beef steers J Anim Sci 77 2793 2802 16 I Hindrichsen M Kreuzer 2009 High methanogenic potential of sucrose compared with starch at high ruminal pH J Anim Physiol Anim Nutr Berl 93 61 65 17 P Huhtanen 1988 The effects of barley unmolassed sugar beet pulp and molasses supplements on organic matter nitrogen and fibre digestion in the rumen of cattle given a silage diet Anim Feed Sci Technol 20 259 278 18 H Kajikawa H Jin F Terada T Suga 2003 Operation and characteristics of newly improved and marketable artificial rumen Rusitec Mem Nat Inst Livest Grassl Sci 2 1 30 19 M Marounek S Bartos P Brezina 1985 Factors influencing the production of volatile fatty acids from hemicellulose pectin and starch by mixed culture of rumen microorganisms Z Tierphysiol Tierer 53 50 58 20 E McDougall 1948 Studies on ruminant saliva 1 The composition and output of sheep s saliva Biochem J 43 99 109 21

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  • Impact of gestation and lactation stage on the dairy cow response following removal to unfamiliar housing and milking system | Broucek | Italian Journal of Animal Science
    National Agricultural and Food Centre Research Institute of Animal Production Nitra Luzianky Slovakia broucek vuzv sk Stefan Mihina National Agricultural and Food Centre Research Institute of Animal Production Nitra Luzianky Department of Structures Slovak University of Agriculture in Nitra Slovakia Michal Uhrincat National Agricultural and Food Centre Research Institute of Animal Production Nitra Luzianky Slovakia Jana Lendelova Department of Structures Slovak University of Agriculture in Nitra Slovakia Anton Hanus National Agricultural and Food Centre Research Institute of Animal Production Nitra Luzianky Slovakia Abstract The objective of this study was to test the hypotheses that milk yield and behaviour at milking after relocation are impacted by gestation and lactation stages Forty one Holstein cows were relocated from the tie stall barn with pipeline milking system into the barn with free stall housing and herringbone milking parlour Milk yield and order at the milking were recorded electronically A significant decrease in daily milk yield was detected immediately after relocation the first day compared with the last day before treatment 23 76 7 21 kg vs 30 97 7 26 kg P 0 001 However the performance drop did not last long after fourteen days milk production exceeded the mean original level 32 16 8 87 kg No significant differences were found in the comparison of cows according to gestation stages Cows in the second and first stages of lactation showed the highest declines in milk yield on the first day 36 77 6 34 kg and 33 76 7 44 kg vs 28 14 7 00 kg and 25 50 8 20 kg P 0 05 Cows in the late lactation stage came into the parlour later than the other cows equally during morning and evening milkings P 0 05 Relationships between the lactation stage and milking orders were positive and significant

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  • Impact of gestation and lactation stage on the dairy cow response following removal to unfamiliar housing and milking system | Broucek | Italian Journal of Animal Science
    was the same from the first week prior to removal through the fourth week following the move Following the relocation all cows had daily feed prepared in troughs The feeding time was set at 9 00 h in both housing systems The cows had free 24 h access to feeding except for milking time In the barn with tie stall housing cows were milked by the pipeline milking system twice a day at 05 30 and 16 30 h The last two individual milk yields were recorded during the evening and morning milkings 16 30 h 5 30 h The first milking after the removal was at the evening milking 16 30 h and the second one on the next morning 5 30 h Cows were milked in a double five herringbone parlour at the same time as before removal The cows entered the parlour individually from the holding area with dimensions of 12 6 m x 6 0 m Upon exit from the parlour the cows walked through an alley that was 7 2 m x 0 85 m and passed over a scale The cows had access to their free stall pens immediately Individual milk yield per day was calculated as the sum of evening and morning yields The individual milk yields and order from 1 to 10 were recorded electronically during the 24 milking sessions at each milking For each cow entrance orders into milking parlour where she was milked and milk yield were recorded These data were derived from the computerized identification of cows during an experimental period During this period the composition of group remained constant The data were analysed using a General Linear Model ANOVA by the statistical package STATISTIX Version 9 0 Factors of day stage of gestation 1 non gravid n 24 2 gravid from the 21 st day to five months n 10 3 from five to nine months n 7 and stage of lactation 1 from the 1 st to 100 th day n 13 2 from 101 st to 200 th day n 9 3 after 201 st day n 19 were evaluated The dependent variables included all measures of milk yield and order at milking from 1 to 10 The normality of data distribution was evaluated by the Wilk Shapiro Rankin Plot procedure Significant differences between groups were tested by Comparisons of Mean Ranks Values are expressed as means SD The correlations between entrance order at milking and observed factors gestation and lactation stages side and between entrance order at milking and milk yield in observed days were calculated using the Spearman rank correlation coefficient Results and discussion A significant decrease in daily milk yield was found immediately after relocation the first day compared with the last day before relocation 23 76 7 21 kg vs 30 97 7 26 kg P 0 001 Immediately after the relocation crowding occurs as cows explore new environments and are unable to navigate the pen This leads to an increase in stocking density It may increase feed competition and subordinate cows may not be able to access feed Another immediate effect of the removal is a reduction in the resting time which can leads to physiological changes associated with stress A study by von Keyserlingk et al 2008 shows that regrouping can disrupt behaviour and production following this interference Each regrouping exposes the cow to new individuals or new combinations of individuals Dairy cows must establish their position in the hierarchy of the new group We can assume that removal is likely a stressful event We note that all observed cows were kept in the same tie stall barn without daily exercise before relocation Also the significant milk yield reduction on the first day could be probably alleviated if cows were put into a loose housing pen or outside yard for some hours daily to acclimate them to a new social structure before relocation Sowerby and Polan 1978 highlight the importance of adaptation milk yield changed less in herds with previous experience of shifting The findings we are presenting on the significant reduction of milk production after removal are in concordance with Soch et al 1997 On the other hand other scientists have identified a lower decline Varner et al 1983 found that the milk yield of relocated cows was decreased at first milking but subsequent milk yields were similar to pre relocation yields The effects of herd relocation were negligible and these authors indicate that dairy herds can be moved without an adverse effect on production traits Brakel and Leis 1976 who measured the effect of an inter group transfer recorded a decrease in milk yield of 0 51 kg or 3 the day after cows were relocated with no evidence of an effect on milk yield after Day 1 At the present work performance drop did not last long and production increased as early as Day 3 and Day 4 but then this trend was reversed Figure 1 Also milk production exceeded the mean original level 32 16 8 87 kg after 14 days This indicates possible adaptation and improved welfare The coping of cattle to different environmental processes certainly changes neuronal and endocrine functions Huzzey et al 2012 Kovács et al 2013 The rapid return to the original milk yield was probably supported mostly by the strong adaptability of cows There are several explanations for this important result of the quick return to the original milk yield values We can attribute it to better conditions in the new barn and better well being in free stall housing The freedom of movement in tie stalls is restricted in a way that causes unnecessary suffering Cows in free stall housing have a greater freedom for movement and exercise and have more opportunity to improve production In the present study cows were not put into groups to adjust them to a new social structure before relocation The reduction in milk production commencing on the 15 th day is in line with prolonged lactation Milk production is affected more significantly by a variety of factors but the main factor is nutrition However we would not expect this result given that the total mixed ration was balanced and uniform throughout the whole trial The negative effects are not based on forage quality The methodical approach was accurate Apart from nutrition the length of daylight can also come into play since cows exposed to a long photoperiod increased milk yield Dahl et al 2000 Many studies have shown that having different photoperiods for the different stages of a cow life cycle improves milk production This is associated with an elevation of an endocrine mechanism for the galactopoietic effects However our experiment conducted during the months of November to December lasted only 25 days and could not reflect this Also we have not done any photoperiodical manipulations we did not change the length or intensity of light in the barn No significant differences were found in the comparison of cows according to gestation stages The greatest decline in the amount of milk the cow on the first day was recorded in dairy cows of 2 nd and 1 st gestation stages 25 50 6 28 kg and 24 01 8 05 kg vs 33 92 8 51 kg and 31 67 6 53 kg Daily milk yields differed only in the first gestation stage days 1 13 14 P 0 05 Open cows showed a slightly less decreased milk yield immediately after relocation compared to gravid cows 7 66 kg vs 8 42 kg Gravid cows are much more likely to be sensitive However the impact of gestation on milk yield depends on the lactation stage according to Olori et al 1997 The effect is higher in mid lactation than in late lactation Significant effects of gestation on milk yield is usually observed from the fifth month of gestation onwards Olori et al 1997 Roche 2003 Brotherstone et al 2004 We must not forget the effects of the photoperiod during the third trimesters of pregnancy on hormone prolactin although that would most likely affect future milk production Gestation also has an impact on a cow s production as it causes mammary gland regression and competition for nutrients from the developing foetus resulting in a reduction in yield The effect of gestation varies with gestation stage Olori et al 1997 Sabbioni et al 2012 the influence is small at the beginning of gestation and becomes greater at later stages of gestation Cows that conceive in the first three months in milk have lactation curves similar in shape and height to the lactation curves of non pregnant cows Brotherstone et al 2004 This may have caused a slight non significant difference between milk yield of the first and second gestation groups The slope of the milk yield curve decreases in gravid cows much more rapidly only after the 200th day of lactation Cows becoming gravid in mid lactation have a similar shape of the lactation curve as the open cows but the yield of these cows is consistently higher than the yield of the open cows This means that the shape of the lactation curve depends on the stage of lactation when pregnancy occurs The highest declines in milk yield on the first day was observed in cows in the second and first stages of lactation 36 77 6 34 kg and 33 76 7 44 kg vs 28 14 7 00 kg and 25 50 8 20 kg P 0 05 There were no significant differences in the first and second lactation stages during the experiment differences were calculated in the third lactation stage only days 14 9 10 P 0 05 14 1 20 P 0 05 That confirmed Varner et al 1983 findings that cows in late lactation tended to have the greatest decreases in milk yield after relocation The results of our study further agree with the reported decreased milk production found by Soch et al 1997 However some other authors showed an inexpressive influence of relocation Brakel and Leis 1976 Sowerby and Polan 1978 It should be noted though that our treatment was much more uncomfortable for the cows they had to cope not only with a change in housing but also in milking When we calculated the percentage of drop in milk yield after removal individually we found that some cows in the lactation stage groups one cow in each group 7 7 11 1 and 5 3 increased milk yield after removal while the others tended to maintain balanced milk production However Weiss et al 2004 found a decreased milk yield of 69 relative to the previous parlour yield during the first milking in an automated milking system The changes in milk yield could be due to multiple reasons Since the ability of an animal to change its behaviour to cope with the environment is modified by previous experience adaptation to shifting is possible Although physical contact was not quantified decreases in physical abuse of new group mates might account for later increases in milk production Whenever cows are regrouped the group s social structure becomes disrupted and a temporary increase in aggression level is typically observed until the new dominance order is established Social order is closely related to the order of milking However our experimental cows were not able to not create a social hierarchy in the previous housing and the group s social structure was a novelty for them That is why it was important to examine their behaviour when they were entering the parlour Although entry order may be affected by social dominance Gadbury 1975 Albright and Arave 1997 it may be also determined by stress from entering the milking parlour Negative past experiences may pose a problem as demonstrated by some cows that entered the milking parlour reluctantly However little is known about the stability of the milking parlour entry order in dairy cows and how it differs depending on the stages of gestation or lactation Differences found in the order at milking according to gestation stage groups were negligible No significant differences in the milking order of all cows were recorded between morning and evening milkings The highest order at milking was showed in cows after the 201th day of their lactation The highest ranking occurred in cows in the third stage of lactation including in the morning and evening milkings 6 40 2 94 6 56 2 84 Differences among lactation stage groups were significant P 0 05 Table 1 This could be related to the age and body weight although not to previous experience Hafez and Bouissou 1975 reported that entrance to the milking parlour should be considered as a voluntary movement different from forced movements as in manipulations Clark et al 1977 and Grasso et al 2007 concluded that management practices that disturb the entrance order should be removed to reduce stress In the present work cows in their first and second lactation stages entered the parlour earlier than cows in their third stage equally during morning and evening milkings Cows in their late lactation stage entered the parlour last Rathore 1982 in dairy cattle and Margetinova et al 2003 in goats proved that those animals entering first the milking parlour had higher milk yields than those entering last Other studies also showed that primiparous cows kept in the group of multiparous cows enter last into the milking parlour Micinski et al 2010 However the entrance order for milking was mostly influenced by the stage of lactation and at the least by the daily milk production according to Varlyakov et al 2011 Highly productive cows are never the majority among those that are first or last to enter for milking Relationships between the milking order during morning and evening and age or body weight were negative and often close P 0 05 Lactation stage correlated positively and significantly with the order in the morning evening and total milkings 0 3730 0 3946 0 4822 Table 2 The adaptation to the parlour was unexpectedly fast especially since this was a change from the pipeline milking system in tie stall housing to milking in the milking parlour One of the reasons highlighted by Grasso et al 2007 could be a good relationship between the milkers and the cows According to Grandin 1998 cattle with excitable temperaments must be introduced gradually to new experiences It is important that their first experiences with something new are good It is known that dairy cows with a sense of fear of humans can reduce milk production and change their behaviour at milking Albright and Arave 1997 Rushen et al 1999 Munksgaard et al 2001 and a reduction in milk yield has been reported for cows milked by different milkers Moreover fear of humans can be aggravated by the unusual noise of the milking parlour A variety of past environmental factors and previous experiences have a significant effect on emotional reactivity Boissy et al 1998 If animals are capable of social learning their level of fear could be reduced if they were handled and trained by gentle operators However experimental milkings were always done by new unfamiliar barn staff In the present study cows were not trained therefore relocation could provoke a fear of novelty and people There were noticeable effects on cows associated with removing to the new facility The reactions towards the changeover to a housing system varied widely within cows However changes after the observation of the milking session indicate that cows are able to adapt quickly to environmental change An additional study is needed in group management to determine the most efficient number of cows in groups and the optimum production for each Factors such as herd size feeding and milking system need to be considered in defining the optimum system It is necessary to improve the practical procedures that may disturb cows entering the milking parlour Conclusions The results of this study suggest that relocation from the tie stall barn with pipeline milking system into the barn with free stall housing and milking parlour caused cows to decline milk production These changes do not have a long lasting impact on the following daily milk production After fourteen days the milk production exceeded the mean original level We found that the milk yield and behaviour at milking after relocation were influenced by the stage of lactation of cows but the gestation stage had a little long term effect on the variables measured More research is needed especially in examining the methods of reducing stress associated with this type of relocation The specific type of cow relocation from the tie stall to free stall housing caused cows to decline milk production However these changes have not a long lasting impact on the following daily milk production After fourteen days the milk production exceeded the original mean level We found that the milk yield and behaviour at milking after relocation were influenced by the stage of lactation of cows but the gestation stage had a little long term effect on the variables measured Acknowledgments This study has been supported by the Slovak Research and Development Agency through projects VEGA 1 0609 12 Analysis of harmful gases production in rural area and proposal of scientifically substantiated technological measures for its minimisation TEPAG ITMS 26220220014 and APVV 14 0806 References 1 J L Albright C W Arave 1997 Rest and rumination In J L Albright C W Arave eds The behaviour of cattle 1st ed CAB International Wallingford UK pp 37 39 2 A Boissy C Terlouw P Le Neindre 1998 Presence of cues from stressed conspecifics increases reactivity to aversive events in cattle evidence for the existence of alarm substances in urine Physiol Behav 63 489 495 3 M F Bouissou A Boissy P Le Neindre I Veissier 2001 The social behaviour of cattle In L J Keeling H W Gonyou eds Social behaviour in farm animals CAB International Wallingford UK pp 113 145 4 W J

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