Rumen Research Articles

Evaluation of rumen ingesta based-diet as alternative feed stuff for growing rabbits.

Trente lapins croises (lapin blanc de la Nouvelle Zelande x race locale) ont ete utilises dans une experimentation pour evaluer l\'effet des rations a base d\'ingesta du rumen sur la performance des lapins et sur les proprietes sensorielles de leur viande. Trois rations alimentaires: ingesta du rumen (RI) seches au soleil ; aliment pour volaille sous forme de pâtee (GM) et un melange de RI et de GM ( ratio 2:3 de RI /GM) ont ete servies aux lapins. La consommation alimentaire, le gain de poids et la qualite de la carcasse ont fait l\'objet d\'evaluation. Il y avait une difference significative quant a la consommation alimentaire entre les lapins nourris de RI & GM et ceux nourris de GM (p 0,05) entre les morceaux de viande de lapin et la ration alimentaire pour toutes les proprietes sensorielles evaluees par un groupe de huit membres utilisant une echelle Hedonique en dix points. On a isole Citrobacter diversus des ingesta du rumen, des echantillons d\'intestins et de bile de lapins dans tous les groupes. La viande de lapin du groupe nourri de RI & GM etait moins chere et acceptable pour la consommation humaine. Dans le contexte de recyclage de nutriment surtout en milieu urbain, les dechets d\'abattoir peuvent etre reintegres sans risque dans la chaine alimentaire sous forme de valeur ajoutee. Thirty cross-bred rabbits (New Zealand white x local breed) were used in an experiment to evaluate the effect of rumen ingesta based diets on the performance of rabbits and on sensory properties of its meat. Three diets: sun-dried rumen ingesta (RI), poultry growers mash feed (GM) and mixture of RI and GM (RIGM ratio of 2:3) were fed to the animals. Feed intake, weight gain and carcass quality were assessed. There was significant difference in feed consumption between rabbits fed RIGM and those fed GM, (p 0.05) difference between cuts of rabbit meat and feed regime for all sensory properties evaluated by an eight member trained panel using a ten-point Hedonic scale. Citrobacter diversus was isolated from rumen ingesta, intestinal and bile samples of Rabbits in all the groups. Rabbit meat from group fed RIGM was cheaper and equally acceptable for human consumption. In a nutrient recycling context especially in urban locations, abattoir waste can be safely re-integrated into the food chain in a value added form Keywords : Rabbit, Rumen ingesta, Weight gain, Carcass quality, Sensory Properties. . Bulletin of Animal Health and Production in Africa Vol. 56 (2) 2008: pp. 126-135

Robert E. Hungate's The Rumen and Its Microbes after 25 years

Robert E. Hungate's The Rumen and Its Microbes after 25 years

Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.

The present study was performed to show the molecular identity of functionally characterized Mg transport pathways in rumen epithelial cells (REC) and to investigate the effects of extracellular [Mg] changes on their expression and activity. By using RT- PCR, Western blot, flow cytometry and immunocytochemistry, TRPM7, MagT1 and a Na+/Mg2+ exchanger were found in REC. Compared with control conditions ([Mg]e = 1.2 mM), a decreased or increased MagT1 (-30%; 20%) and Na+/Mg2+ exchanger (-25%; 40%) protein abundance was observed after a 24-h incubation of REC in low (0.12 mM)- and high (5 mM)-Mg medium, respectively. To determine the Mg transport capacity, [Mg2+]i changes were measured by use of mag-fura 2. The basal [Mg2]i (0.43 +/- 0.03 mM) was not influenced by the [Mg] of the pre-incubation medium. However, compared to control cells, REC incubated in low- or high-Mg medium showed significantly reduced (59%) and elevated (57%) Mg extrusion rates, respectively. In addition, they were characterized by an increased influx capacity (30-40%). In low-Mg cells the latter results mainly from a strong TRPM7 related transport component whereas in high-Mg cells the imipramine-sensitive, the Na+/Mg2+ exchanger-mediated transport component causes this effect. In conclusion, TRPM7, MagT1 and a Na+/Mg2+ exchanger are shown to be the main Mg transport proteins in REC and their expression and functional activity is influenced by the cellular Mg status. The latter responses permit adaptation of epithelial Mg absorption and enable REC to maintain a physiological [Mg2+]i which is a prerequisite for various cell functions.

Induction of forestomach lesions in rats by oral administrations of naturally occurring antioxidants for 4 weeks.

The effects of naturally occurring antioxidants on rat forestomach epithelium were compared with those of synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), of which the former is a known forestomach carcinogen. Groups of five F344 male rats were given diet containing BHA, BHT, gallic acid, syringic acid, sesamol, caffeic acid, chlorogenic acid, ferulic acid, eugenol or esculin for 4 weeks at a level of 0.7% for BHT or 2% for other compounds. Histological examination of the forestomach showed that BHA induced hyperplasia mainly in the prefundic region near the esophageal orifice, caffeic acid induced pronounced hyperplasia throughout the forestomach epithelium, and sesamol induced large ulcers and hyperplasia in the central region. Thus, these naturally occurring antioxidants showed different toxicities and abilities to induce hyperplasia in the rat forestomach.

In Commemoriation of Acad. Rumen Tsanev

In Commemoriation of Acad. Rumen Tsanev

Intoxicação espontânea e experimental por Eupatorium tremulum (Asteraceae) em bovinos

Descreve-se a intoxicação natural e experimental por Eupatorium tremulum em bovinos. Um surto de intoxicação espontânea por esta planta foi diagnosticado no município de Lages, Santa Catarina. Em um lote de 19 bovinos, três morreram após a transferência para uma invernada aonde havia grande quantidade de E. tremulum. Os animais foram encontrados mortos, e dois foram necropsiados e coletado amostras de vísceras para exame histológico. Experimentalmente, folhas verdes de E. tremulum foram administradas a cinco bovinos, em doses únicas de 23 a 32g/kg de peso animal. Destes, três adoeceram e dois morreram. Os principais sinais clínicos observados foram anorexia, apatia, atonia ruminal, micção freqüente e em pequenos jatos, fezes pastosas e ventre flácido. As lesões macroscópicas restringiram-se aos pré-estômagos e foram idênticas, tanto para intoxicação natural como para a experimental. O rúmen e retículo externamente mostravam tonalidade levemente avermelhada, a camada córnea da mucosa estava frouxamente aderida e a mucosa tinha coloração vermelha acentuada. Ao exame histológico observou-se no rúmen e retículo, tanto na intoxicação espontânea, como experimental, necrose do epitélio da mucosa com formações de pequenas vesículas e em alguns segmentos, desprendimento da camada epitelial e leve infiltrado de neutrófilos. A intoxicação por E. tremulum tem curso clínico, lesões macro e microscópicas muito semelhantes àquelas produzidas pela intoxicação por Baccharidastrum triplinervium, Baccharis coridifolia (mio-mio) e Baccharis megapotamica var. weirii (mio-mio do banhado). O diagnóstico diferencial entre essas quatro intoxicações deve ser feito pela presença da planta e pelos dados epidemiológicos. O diagnóstico dos casos espontâneos foram confirmados pelos dados epidemiológicos e reprodução experimental das lesões macro e microscópicas.

Expression of PCNA, AKP and ACP during development of mouse fore stomach carcinoma

To find out the relationship of the expressions of proliferating cell nuclear antigen (proliferating cell nuclear antigen, PCNA), alkaline phosphotase (alkaline phosphotase, AKP) and acid phosphotase (acid phosphotase, ACP) with the development of mouse fore stomach cancerization. The animal models, including the various stages during the development of NIH mouse fore stomach carcinoma, were made by N-Nitrososarcosineethylester (N-Nitrososarcosineethylester, NSEE). The mice were sacrificed on the 14th, 28th, 42nd, 56th, 70th and 84th days respectively after mice were irrigated with NSEE. The fore stomach was taken out and dissected. The methods of histopathology, immunohistochemistry and isoenzyme electrophoresis were adopted to study the dynamic changes of cell shape and expression of PCNA, AKP and ACP. On the 42nd and 56th days after NSEE treatment, the expression of PCNA increased gradually along with the cancerization. Comparing with the control, there were significant differences (P<0.05). On the 70th and 84th days, the expression of PCNA increased further (compared with the control P<0.01). The activity of AKP increased gradually along with the cancerization. On the 14th, 28th, 42nd and 56th days, there were significant differences (P<0.05); on the 70th and 84th days, the activity of AKP increased further (P<0.01). The activity of ACP also increased on the 14th, 28th, 42nd and 56th days, and there were significant differences on the 70th days (P<0.05) and on the 84th days (P<0.01) compared with the control. During the carcinogenesis of NIH mouse fore stomach, the expressions of PCNA, AKP and ACP increased gradually and were consisted with the changes of cell shapes.

Early Descriptions of Parkinson Disease in Ancient China

Ahistorical search of traditional Chinese medical sources revealed several ancient descriptions suggestive of Parkinson disease dating back to 425 BC. Zhang Zihe (1156-1228) provided the first description of a typical case of Parkinson disease in the book Ru Men Shi Qin. Traditional Chinese medicine also recommended an antitremor pill that to this day appears to provide reasonable symptomatic treatment for tremor and rigidity. Based on this evidence, we conclude that Parkinson disease (PD) was first described more than 2400 years ago in China.

The effect of dent versus flint maize genotype on site and the extent of starch and protein digestion, ruminal fermentation and microbial protein synthesis in the rumen of sheep

La digestibilite des proteines, de l'amidon et des constituants parietaux dans l'ensemble du tractus digestif, le rendement de synthese des proteines microbiennes dans le rumen et les proportions molaires des acides gras volatils (AGV) dans le jus de rumen et dans les feces ont ete determinees sur des ovins qui ont recu de l'ensilage de mais de type dente (DTS) ou corne (FTS). Les degradabilites dans le rumen ont ete mesurees par la methode in sacco sur trois ovins alors que le rendement de synthese des proteines microbiennes a ete determine a partir de l'excretion urinaire des derives de purines en utilisant quatre ovins. L'importance de la degradation de l'amidon et des proteines a ete significativement (P < 0,001) plus faible pour FTS que pour DTS (respectivement, 718 vs. 913 et 704 vs. 767 g.kg -1 ). Les differences entre les digestibilites de l'amidon et des proteines dans l'ensemble du tractus digestif ont ete faibles (respectivement, 986 vs. 997 g.kg -1 , P < 0,01 et 939 vs. 931 g.kg -1 , P < 0,05 pour FTS et DTS). La concentration en AGV totaux dans le jus de rumen n'a pas ete affectee par le type d'ensilage (94,5 et 94,7 mmol.L -1 ), pourtant, FTS a induit des proportions molaires plus elevees de propionate (22,6 vs. 20,7 %, P < 0,05 ) et DTS des proportions de butyrate superieures (13,5 vs. 10,8 %, P<0,05). La synthese des proteines microbiennes pour FTS a ete significativement plus elevee que pour DTS (109,7 vs. 96,0 g.kg -1 de MS ingeree, P < 0,05). Quand elles sont exprimees par rapport a l'ingestion de la matiere organique fermentescible (MOF), les differences entre les hybrides sont plus importantes (215,6 vs. 158,6 g de proteines microbiennes par kg MOF, P < 0,01 ). L'efficacite plus faible de la synthese de proteines microbiennes pour DTS est probablement due a la valeur plus faible du pH ruminai (6,21 vs. 6,33, P < 0,05). Les FTS ont presente les conditions les plus favorables pour la degradation de la fraction fibreuse dans le rumen. Les feces des ovins qui ont recu FTS etaient moins riches en MS (304 vs. 371 g.kg -1 , P < 0,05) et tendaient a etre plus riche en AGV (383 et 235 mmol.kg -1 DM, P < 0,1); ce qui laisse supposer que, pour FTS, une partie de l'amidon, non degrade dans le rumen, n'a pas ete digere dans l'intestin grele et a ete fermente dans le gros intestin. En conclusion, FTS semble fournir approximativement trois fois plus d'amidon digere dans l'intestin et 20 % de proteines metabolisables en plus que DTS.

Nutrient digestibility, Haematological indices and carcass measurements of rabbits fed graded levels of goat rumen content

No abstract Keywords: nutrient digestibility; carcass data; blood parameters; rabbits; goat rumen content Global Journal of Agricultural Sciences Vol. 4(1) 2005: 1-6

Effects of feeding different levels of rumen contents and palm oil vitamins on egg production and quality

EFFETS DE DIVERS NIVEAUX DE VITAMINE DANS LE CONTENU DU RUMEN ET DANS L'HUILE DE PALME SUR LA PRODUCTION ET LA QUALITE DES OEUFS Resume Deux essais ont ete conduits en vue de determiner les effets de divers niveaux de vitamine dans le contenu du rumen et dans l'huile de palme sur la production et la qualite des oeufs. Dans le premier essai qui a porte sur le contenu du rumen, 160 poules pondeuses de souche Hisex blanche âgees de neuf mois ont ete reparties au hasard en quatre groupes et nourries avec des aliments experimentaux contenant quatre niveaux de contenu du rumen : 0 ; 5 ; 10 et 15%. Le dispositif experimental est un plan completement randomise avec 40 poules pondeuses par traitement. Les poules ont ete elevees sur un plancher en bambou sureleve de 57 cm par rapport au sol. Dans le second essai, 160 poules pondeuses de souche Hisex blanche, âgees de 10 mois reparties au hasard en quatre lots ont ete utilisees. Les pondeuses ont ete elevees sur un plancher en bambou suspendu a 57cm au-dessus du sol. Ils ont recu quatre aliments experimentaux contenant de l'huile de palme a des taux de 0 ; 0,5 ; 1 ; 1,5 et 2% respectivement. Le dispositif experimental etait un plan completement randomise avec 40 pondeuses par traitement. L'eau et l'aliment etaient servis a volonte. Les resultats de ces essais ont montre que le contenu du rumen n'a pas d'effet majeur sur la consommation alimentaire, la production journaliere d'oeufs et la masse d'oeufs exportee par poule, mais augmente de facon significative (p L'huile de palme a peu d'effets sur la consommation alimentaire, la production d'oeufs, la masse d'oeufs exportee et la fertilite, a l'exception de la couleur du jaune d'oeuf. La couleur du jaune d'oeuf a 2% (4,1) et a 1,5% (3,8) d'huile de palme dans l'aliment experimental etait significativement mieux coloree qu'aux taux de 0,5% (1,8) et 1% (2,6) d'huile de palme. Summary Two experiments were carried out in order to determine the effects of rumen contents and palm oil vitamins on egg production and quality. The first experiment on rumen contents used 160 nine-month old Hisex white layers randomly placed into four groups and fed test diets containing four levels, 0, 5, 10 and 15%of rumen content. The experimental design was completely randomized with 40 hens per treatment. The birds were managed on a suspended bamboo floor, 57 cm high. In the second experiment, 160 Hisex white ten-month old birds randomly grouped in four lots were used.The birds were managed in a suspended bamboo floor, 57 cm from the ground and fed four test diets containing palm oil at 0;0.5;1.5 and 2% levels respectively. The experimental design was a completely randomized design with 40 birds per treatment. Water and feed were given ad libitum. The results showed that rumen contents non-significantly increased feed consumption, hen day production and egg mass but significantly(p Palm oil had little effect on feed intake, egg production, egg mass, fertility except egg yolk colour. Egg yolk colour at 2.0%(4.1) and 1.5%(3.8) levels of palm oil in the diet was significantly more colourful than at the 0.5%(1.8) and 1.0% (2.6) levels of palm Bull Anim. Hlth. Prod. Afr. (2004) 53, 69-75

Intake, ingesta retention, particle size distribution and digestibility in the hippopotamidae

Although several aspects of the digestive physiology of the hippopotamidae—non-ruminating foregut fermenters—have been described, ingesta kinetics and passage characteristics of these species are not well understood. The most outstanding feature of the hippo digestive physiology reported so far is the very long mean ingesta retention times (MRTs) measured by Foose [Foose, T., 1982. Trophic strategies of ruminant versus nonruminant ungulates. PhD dissertation, University of Chicago, Chicago.]. Since those data had been investigated with animals without water access, we intended to measure MRT in hippos which were allowed to enter water pools during the night. MRT parameters as well as dry matter (DM) digestibility were determined in four common ( Hippopotamus amphibius) and four pygmy hippos ( Hexaprotodon liberiensis) on two different diets each using cobalt ethylendiamintetraacetate (Co-EDTA) as a fluid, chromium (Cr)-mordanted fibre (<2 mm) as a particle and acid detergent lignin (ADL) as an internal digestibility marker. Four of the animals additionally received cerium (Ce)-mordanted fibres (2–10 mm) as particle markers. Total MRTs for fluids and particles ranged between 20–35 and 48–106 h in the common and between 13–39 and 32–107 h in the pygmy hippos. The difference between fluid and particle retention was greater than usually reported in ruminants. Excretion patterns of the markers differed from those usually observed in ruminants but resembled those reported for macropods (kangaroos), indicating a plug-flow reactor-like physiology in the hippo forestomach (FRST). This finding complements other described similarities between the macropod and the hippo forestomach. The measurements of larger particle retention profiles suggest that in the hippo, larger particles might be excreted either faster or at the same rate as smaller particles, indicating a general difference between ruminants and hippos with respect to differential particle retention. The digestive physiology of hippos is characterised by a generally low food intake, long ingesta retention times and dry matter digestibilities lower than reported in ruminants. Moderate digestibilities in spite of long retention times might be the result of the generally high average ingesta particle size in hippos. The comparatively easy management of pygmy hippos, together with the significant correlations between food intake, MRT and digestibility in the pygmy hippos of this study, recommends this species for further studies on the interplay of these parameters in herbivore digestive physiology.

Effects of Feed Quality on Rumen Microbial Protein Synthesis in Sheep

Abstract dalam bentuk hard copy

Farmer's Respone to Rumen Fill Transfer Technique

Abstract dalam bentuk hard copy

The Effect of Different Rice Straw and Concentrate Content of The Ration on Hemicellulase Activity in The Rumen Fluid of Carabao, Cattle, Goat and Sheep

Abstrak dalam bentuk hard copy

Rumen fermentation in hereford steers grazing ryegrass and supplemented with whole or ground maize

The experiment investigated rumen fermentation in Hereford steers grazing high quality pastures, supplemented daily with whole or ground maize grain (1% of body weight), or grazing without supplementation, during autumn. Six rumen cannulated steers were allotted at random, to 1 of the 3 treatments. Grain was offered each morning. Dry matter degradability was measured using the nylon bag technique. Samples of rumen fluid were collected for analysis of pH, volatile fatty acids (VFA) and ammonia. Supplementation with maize did not affect (P>0.05) degradation rate of forage in the rumen, rumen pH, or rumen VFA concentration. It was concluded that neither grain supplement adversely affected the rumen environment of cattle grazing lush pastures during autumn.

Skatole and indole formation in comparison to rumen ammonia and volatile fatty acids in sheep fed fresh forages

Skatole and indole concentrations in the rumen were compared with the rumen concentrations of ammonia and volatile fatty acids (VFA) in sheep fed fresh white clover (WC; Trifolium repens), perennial ryegrass (PRG; Lolium perenne) or the condensed tannin-containing forage, Lotus corniculatus (LC). Six rumen fistulated Romney wethers were fed the cut forages over two 2-hour meal periods in the morning and afternoon, with rumen contents sampled at intervals after the start of the morning meal. Maximum concentrations of skatole, indole and ammonia were highest when feeding WC (P<0.05). Ammonia accumulates in the rumen when there is rapid protein degradation with unsynchronised energy supply. Condensed tannins in LC slowed protein degradation and consequently maximal ammonia, indole and skatole concentration were lower when feeding this forage (P<0.05). This indicates that skatole and indole formation will be greater when there is high protein degradability. Ammonia, indole and skatole concentrations in sheep fed PRG were intermediate, and not significantly different from either WC or LC. Maximum concentration of straight-chain volatile fatty acids (VFA) were not significantly different for the forages, but branchedchain VFA concentrations tended to be higher for WC (P=0.103) compared with LC. Straight-chain VFAs are a by-product of microbial carbohydrate fermentation in the rumen, but branched-chain VFAs are formed from deamination of amino acids. This suggests that skatole and indole production are not related to carbohydrate digestion in the rumen. In order for meat and milk producers in pastoral systems to ameliorate undesirable flavours and increase demand in certain markets, they will need to consider using alternative forages to reduce protein solubility and degradability.

Effect of white clover (Trifolium repens), perennial ryegrass (Lolium perenne) and Lotus corniculatus on in vitro skatole and indole formation

Consumers accustomed to eating meat and dairy products from animals raised on grain often describe products from New Zealand pasture-raised animals as having an undesirable flavour. This could adversely affect the acceptability and returns for meat and dairy products in certain oversea markets. This flavour, often described as pastoral, grassy or faecal, has been attributed in part to the high concentration of indole and skatole present in the meat of animals raised on fresh perennial ryegrass/white clover pasture. Skatole and indole are formed in the rumen from the microbial degradation of tryptophan. A pasture diet is high in protein that is both soluble and rapidly degraded, resulting in an abundance of free peptides and amino acids in the rumen. Condensed tannins (CT) in Lotus corniculatus have been shown to reduce the solubility and degradability of plant protein in the rumen. This study investigated the in vitro rumen fermentation characteristics of white clover (Trifolium repens; WC), perennial ryegrass (Lolium perenne; PRG) and Lotus corniculatus (LC) to determine their effects on skatole and indole formation. All incubations were carried out in the presence and absence of polyethylene glycol (PEG), which inactivates CT. In the absence of PEG, maximal skatole and ammonia concentrations were greater when incubating WC compared with PRG and LC (P<0.05). A similar trend was evident for indole concentrations, but statistical significance was not reached. Adding PEG to the incubations with LC increased the concentration of indole, skatole and ammonia (P<0.05), but did not affect those incubations with WC or PRG. Differences in peak concentrations by the in vitro method were comparable with those found in an in vivo study using the same forages. Thus, the in vitro method can be used to screen a range of forages to determine their effect on skatole and indole formation in ruminants, and to identify candidate forages for managing the flavour of products from forage-fed ruminants.

The potential of sulla in pasture-based systems

A feed evaluation trial compared lamb growth, rumen function and protein synthesis from either pasture (perennial ryegrass (Lolium perenne)/white clover (Trifolium repens)-dominant), lucerne (Medicago sativa), sulla (Hedysarum coronarium, which contains condensed tannins (CT)) or a lucerne:sulla mixture. The 8 week trial involved 32 weaned ram lambs allocated to either pasture (80% ryegrass and 20% white clover), lucerne, sulla, or a 50:50 lucerne:sulla mixture (DM basis; 8 per diet). Measurements included feed intake, liveweight (LW) gain, rumen ammonia (NH3) and volatile fatty acid (VFA) concentrations. A sub-group of 16 lambs (4 from each treatment) were given a continuous infusion of 35S-cysteine and 35S-sulphate to measure the effect of diet on cysteine irreversible loss and absolute whole body protein synthesis (WBPS). Lambs fed pasture had the slowest (P<0.05) daily gains (116 g/day) and absolute WBPS (93 g/day) compared with lambs fed sulla (308 g LW and 152 g WBPS/day) or lucerne:sulla (281 g LW and 180 g WBPS/day). Lambs fed sulla had lower rumen NH3 concentrations (13.6 mmol/L), acetate:propionate ratios (2.6) and minor VFA concentrations (2.1%) suggesting less rumen proteolysis than lambs fed the other diets. Lamb performance was improved by feeding sulla or lucerne compared to pasture, and the lucerne:sulla mixture resulted in better performance than the average of the 2 species fed alone.

Using the Cornell Net Carbohydrate and Protein System to predict ruminal pH in dairy cows grazing high quality pasture

Predicting milk production is complex due to interactions between the requirement for nutrients by the cow, the characteristics of different feeds and the contributions from body reserves towards the animal's requirements. Grazing introduces more complexity because of the variation in the characteristics of the forage, variation in nutrient intake between cows, diurnal patterns of eating, and further energy demands due to the effort of grazing. Ruminal pH is a critical determinant of metabolisable energy supply because of effects on the growth of different groups of microorganisms in the rumen. Our objective was to use the Cornell Net Carbohydrate and Protein System model to determine whether it was possible to predict ruminal pH for a range of pasture-based diets, based on inputs of nutrient intake, milk composition, liveweight and body condition. There was no (P>0.05) relationship between observed and predicted ruminal pH. The average daily ruminal pH for observed and predicted pH were similar and the mean bias was small and not different to zero. However, when the residuals were regressed against the predicted pH, a positive (P<0.05) systematic bias was demonstrated with pH over-predicted at low pH (5.7 to 6.0) and under-predicted at higher pH (6.1 to 6.2).