Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Cowphilosophy; the art of practical dairy practice

Mode of access: Internet.

Herdbook.

1911-16 as British Holstein-Friesian Cattle Society.

Friesch Rundvee-Stamboek.

English title: Frisian herdbook.

Feeding strategies for grain-fed cattle in a hot environment

Six Bos taurus (Hereford) steers (body weight 324 22 kg) were used in a 45-day study with a replicated 3 x 3 Latin-square design. Three treatments [ad libitum feeding (ADLIB); limit feeding, 85% of ad libitum (LIMIT); bunk management feeding where steers were only given access to feed from 1600 to 0800 hours the following day (BUNK)] were imposed over 3 periods, with 2 steers assigned to each treatment in each period. Cattle were managed in a temperature-controlled metabolism unit and were exposed to both thermoneutral (17.7degreesC-26.1degreesC) and hot (16.7degreesC-32.9degreesC) environmental conditions. By design, during the thermoneutral period, the ADLIB cattle displayed greater intake (P < 0.05) than the LIMIT group, with the BUNK group being intermediate. However, during the hot period, both the LIMIT and BUNK treatment groups increased feed intake 4-5%, whereas feed intake of the ADLIB treatment group declined nearly 2%. During both periods respiration rate (RR, breath/min) followed the same pattern that was observed for feed intake, with the greatest (P < 0.05) RR found in the ADLIB treatment group (81.09 and 109.55, thermoneutral and hot, respectively) and lowest (P < 0.05) RR in the LIMIT treatment group (74.47 and 102.76, thermoneutral and hot, respectively). Rectal temperature (RT) did not differ among treatments during the thermoneutral period or the first hot day, although during the thermoneutral period the ADLIB treatment group did tend to display a lower RT, possibly as a result of other physiological processes (pulse rate and RR) aiding to keep RT lower. During the hot period, differences in RT were found on Day 5, with the LIMIT cattle having lower (P < 0.10) RT (38.92degreesC) than the ADLIB (39.18degreesC) cattle, with BUNK cattle RT (39.14degreesC) being intermediate. However, when hourly data were examined, the ADLIB cattle had greater(P < 0.05) RT than the BUNK and LIMIT at 1800 hours and greater RT (P < 0.05) than the LIMIT group at 1400, 1500, and 1600 hours. Clearly, a change in diurnal RT pattern was obtained by using the LIMIT and BUNK feeding regimen. Both of these groups displayed a peak RT during the hot conditions, between 2100 and 2200 hours, whereas the ADLIB group displayed a peak RT between 1400 and 1500 hours, a time very close to when peak climatic stress occurs. Based on these results it is apparent that feedlot managers could alleviate the effects of adverse hot weather on cattle by utilising either a limit-feeding regimen or altering bunk management practices to prevent feed from being consumed several hours prior to the hottest portion of the day.

Monensin supplementation of lactating cows fed tropical grasses and cane molasses or grain

Effects of monensin (Mon) on performance of Holstein-Friesian cows fed tropical grasses and cane molasses (M) or cereal grain were examined in three experiments. In experiment I (incomplete 4 x 4 Latin square), three rumen-fistulated cows [188 I I days in milk (DIM)] were fed mixed diets based on rhodes grass (Chloris gayana cv. Callide) bay where M was substituted for wheat grain (W) at rates of 0 (MO), 125 (M 125) or 250 (M250) g/kg dry matter (DM). A fourth diet contained M250 plus 0.02 g Mon/kg DM (M250 + Mon). Substituting M for W tended (P < 0.10) to decrease the ratio of rumen molar proportions of acetate+butyrate (Bu):propionate (Pr) (4.3 versus 3.8 and 4.0 for M0, M125 and M250, respectively). There were no treatment effects (P> 0.10) on intake, organic matter digestibility, milk production or liveweight (LW) change. In experiment 2, 48 cows (173 &PLUSMN; 28.3 DIM) grazing kikuyu (Pennisetum clandestinum cv. common) pastures and supplemented with maize silage and a grain-based concentrate were offered either M (2.6 kg DM/(cow day)) or barley grain (B) (2.7 kg DM/(cow day)). Within each supplement type, half were fed 0 or 320 mg of Mon/(cow day). There were Mon x supplement interactions (Mon x S; P < 0.05) on the rumen molar proportion of Pr and Bu at 15:00 h, with B + Mon having the highest value for Pr (0.259 mmol/mmol) and lowest value for Bu (0.121 mmol/mmol). A Mon x S effect (P < 0.05) on milk fat content was noted with Mon causing a lower value regardless of energy source (31 and 36 g/l versus 40 and 38 g/l for B + Mon, M + Mon, B - Mon and M - Mon, respectively). As a main effect, M as opposed to B, reduced yields of milk (P < 0.05; 16.21/(cow day) versus 18.01/(cow day)) and protein (P < 0.05; 479 g/(cow day) versus 538 g/(cow day)). Monensin reduced milk fat yield (P < 0.05; 669 g/(cow day) versus 562 g/(cow day)), raised milk protein concentration (P < 0.05; 31 g/l versus 29 g/l) and caused LW gain rather than loss (P < 0.05; +0.06 kg/(cow day) versus -0.30 kg/(cow day)). No treatment effects on pasture intake were noted. In experiment 3, 48 cows (91 &PLUSMN; 16.1 DIM) grazing kikuyu pasture and supplemented with grain-based concentrate, sugar cane silage and 2.7 kg DM(cow day) of M were supplemented with either 0 or 320 mg Mon/(cow day). Monensin reduced (P < 0.05) milk fat content (33 g/l versus 30 g/l) and tended (P < 0.10) to reduce milk protein content (29 g/l versus 28 g/l). No effects of Mon on other milk production parameters, LW change or pasture intake were noted. Feeding monensin to mid-lactation Holstein-Friesian cows offered diets based on tropical grasses, and cane molasses or grain, improves rumen fermentation efficiency, thereby improving energy efficiency resulting in higher LW gain. Monensin had no effect on milk yield, but reduced milk fat concentration.

Impacts of feeding system and season on milk composition and Cheddar cheese yield in a subtropical environment

Milk obtained from cows on 2 subtropical dairy feeding systems were compared for their suitability for Cheddar cheese manufacture. Cheeses were made in a small-scale cheesemaking plant capable of making 2 blocks ( about 2 kg each) of Cheddar cheese concurrently. Its repeatability was tested over 10 separate cheesemaking days with no significant differences being found between the 2 vats in cheesemaking parameters or cheese characteristics. In the feeding trial, 16 pairs of Holstein - Friesian cows were used in 2 feeding systems (M1, rain-grown tropical grass pastures and oats; and M5, a feedlot, based on maize/barley silage and lucerne hay) over 2 seasons ( spring and autumn corresponding to early and late lactation, respectively). Total dry matter, crude protein (kg/cow. day) and metabolisable energy (MJ/cow.day) intakes were 17, 2.7, and 187 for M1 and 24, 4, 260 for M5, respectively. M5 cows produced higher milk yields and milk with higher protein and casein levels than the M1 cows, but the total solids and fat levels were similar (P > 0.05) for both M1 and M5 cows. The yield and yield efficiency of cheese produced from the 2 feeding systems were also not significantly different. The results suggest that intensive tropical pasture systems can produce milk suitable for Cheddar cheese manufacture when cows are supplemented with a high energy concentrate. Season and stage of lactation had a much greater effect than feeding system on milk and cheesemaking characteristics with autumn ( late lactation) milk having higher protein and fat contents and producing higher cheese yields.

A comparison of the excretion rate of endogenous purine derivatives in the urine of Bos indicus and Bos taurus steers

Estimates of microbial crude protein (MCP) production by ruminants, using a method based on the excretion of purine derivatives in urine, require an estimate of the excretion of endogenous purine derivatives (PD) by the animal. Current methods allocate a single value to all cattle. An experiment was carried out to compare the endogenous PD excretion in Bos taurus and high-content B. indicus ( hereafter, B. indicus) cattle. Five Holstein - Friesian ( B. taurus) and 5 Brahman (> 75% B. indicus) steers ( mean liveweight 326 +/- 3.0 kg) were used in a fasting study. Steers were fed a low-quality buffel grass (Cenchrus ciliaris; 59.4 g crude protein/kg dry matter) hay at estimated maintenance requirements for 19 days, after which hay intake was incrementally reduced for 2 days and the steers were fasted for 7 days. The excretion of PD in urine was measured daily for the last 6 days of the fasting period and the mean represented the daily endogenous PD excretion. Excretion of endogenous PD in the urine of B. indicus steers was less than half that of the B. taurus steers ( 190 mu mol/kg W-0.75. day v. 414 mu mol/kg W-0.75. day; combined s.e. 37.2 mu mol/kg W-0.75. day; P< 0.001). It was concluded that the use of a single value for endogenous PD excretion is inappropriate for use in MCP estimations and that subspecies-specific values would improve precision.

Extent and economic effect of heat loads on dairy cattle production in Australia

Objective To investigate the extent of heat load problems, caused by the combination of excessive temperature and humidity, in Holstein-Friesian cows in Australia. Also, to outline how milk production losses and consequent costs from this can be estimated and minimised. Procedures Long-term meteorological data for Australia were analysed to determine the distribution of hot conditions over space and time. Fifteen dairy production regions were identified for higher-resolution data analysis. Both the raw meteorological data and their integration into a temperature-humidity thermal index were compiled onto a computer program. This mapping software displays the distribution of climatic patterns, both Australia-wide and within the selected dairying regions. Graphical displays of the variation in historical records for 200 locations in the 15 dairying regions are also available. As a separate study, production data from research stations, on-farm trials and milk factory records were statistically analysed and correlated with the climatic indices, to estimate production losses due to hot conditions. Results Both milk yields and milk constituents declined with increases in the temperature-humidity index. The onset and rate of this decline are dependent on a number of factors, including location, level of production, adaptation, and management regime. These results have been integrated into a farm-level economic analysis for managers of dairy properties. Conclusion By considering the historical patterns of hot conditions over time and space, along with expected production losses, managers of dairy farms can now conduct an economic evaluation of investment strategies to alleviate heat loads. These strategies include the provision of sprinklers, shade structures, or combinations of these.

Clinical approach of atresia coli in a calf

Atresia coli is a congenital defect with disruption of the intestinal flow. Its etiopathogenesis is still unknown and the prognosis is reserved. This paper describes the case of a one week old Holstein Friesian calf that was referred to the HCVC, and was diagnosed with atresia coli. This confirmation was made by contrast radiography and the lesion was detected in dorsal portion of the spiral colon. A cecostomy was performed followed by a marsupialization of the intestinal cecum to the abdominal wall. After 36 hours of surgery, calf’s state worsens dramatically and for reasons of welfare, it was proceeded to sacrifice. The post-operative recovery is possible, even though the mortality rate is high. As in this case, this mortality rate increases when early diagnosis of atresia coli does not take place and there is peritonitis.

Prevalencia de brucelosis bovina en el periodo 2004-2012 y tuberculosis bovina en el periodo 2006-2012 en hatos lecheros del Cantón Mejía

Brucelosis y tuberculosis bovina son dos enfermedades que causan importantes pérdidas productivas y reproductivas en la ganadería local, nacional e internacional. En el Ecuador, desde hace varios años se viene trabajando en un programa para el control y erradicación de estas dos enfermedades. En el año 2006, por primera vez, siete predios de producción lechera consiguieron la certificación de predio libre de Brucelosis bovina, otorgada por el Servicio Ecuatoriano de Sanidad Animal (SESA) de ese entonces (Paredes 2014, comunicación personal). La Asociación Holstein Friesian del Ecuador (AHFE) inicia en el año 2004 el Programa de Control y Erradicación de Brucelosis bovina y en el año 2006 el Programa de Control y Erradicación de Tuberculosis bovina. Sin embargo, los datos de ambos programas no han sido analizados para determinar el estado de estas dos enfermedades en los periodos comprendidos entre los años 2004 a 2012 y periodo 2006 a 2012, respectivamente.

Efecto de la levadura Saccharomyces cerevisiae en condición corporal, alzada, ganancia diaria de peso, parámetros hematológicos y metabólicos con terneros de remplazo criados al pastoreo en la Hacienda Nero

En la presente investigación se evaluó el efecto de la levadura Saccharomyces cerevisiae adicionada a la dieta basal de terneros sobre la Condición Corporal (CC), Alzada, Ganancia Diaria de Peso (GDP), Parámetros Hematológicos y Metabólicos en terneros de reemplazo. Se realizó en la granja Nero de la Universidad de Cuenca con 18 terneros Holstein Friesian de 4 a 6 meses de edad, entre 100 a 200 kg/PV, criados en pastoreo, todos con las mismas condiciones de suplementación y manejo nutricional; divididos en dos grupos, un control T1 (n1=9) alimentados con dieta basal y un experimental T2 (n2=9) adicionado a la dieta 15 g/ternero/día de levadura. Se usó un diseño de bloques completamente al azar (DBA) y los resultados fueron analizados con el programa SPSS. El peso y alzada se registró semanalmente para evaluar su GDP (gramos) y talla (cm), respectivamente. La CC y los parámetros sanguíneos fueron realizados en 5 momentos. Los resultados de GDP y Alzada fueron analizados con las pruebas estadísticas de Shapiro – Wilk y Levene al 5%, al realizar el (ADEVA) no se encontró diferencias estadísticas. La variable CC presento diferencia significativa (p <0.05). Al analizar los parámetros hematológicos se encontró diferencia estadística en glucosa (p <0.05). En el análisis financiero, se apreció que el grupo control tiene un menor costo por Kg de peso producido 0,84 USD vs 0,96 USD del grupo experimental. En conclusión, se pone en manifiesto que el empleo de S. cerevisiae como aditivo nutricional de terneros de remplazo criados al pastoreo, puede constituir como una alternativa que incrementa los parámetros de salud expresados en glucosa y condición corporal

Effects of forage type and extruded linseed supplementation on methane production and milk fatty acid composition of lactating dairy cows

Replacing dietary grass silage (GS) with maize silage (MS) and dietary fat supplements may reduce milk concentration of specific saturated fatty acids (SFA) and can reduce methane production by dairy cows. The present study investigated the effect of feeding an extruded linseed supplement on milk fatty acid (FA) composition and methane production of lactating dairy cows, and whether basal forage type, in diets formulated for similar neutral detergent fiber and starch, altered the response to the extruded linseed supplement. Four mid-lactation Holstein-Friesian cows were fed diets as total mixed rations, containing either high proportions of MS or GS, both with or without extruded linseed supplement, in a 4×4 Latin square design experiment with 28-d periods. Diets contained 500 g of forage/kg of dry matter (DM) containing MS and GS in proportions (DM basis) of either 75:25 or 25:75 for high MS or high GS diets, respectively. Extruded linseed supplement (275 g/kg ether extract, DM basis) was included in treatment diets at 50 g/kg of DM. Milk yields, DM intake, milk composition, and methane production were measured at the end of each experimental period when cows were housed in respiration chambers. Whereas DM intake was higher for the MS-based diet, forage type and extruded linseed had no significant effect on milk yield, milk fat, protein, or lactose concentration, methane production, or methane per kilogram of DM intake or milk yield. Total milk fat SFA concentrations were lower with MS compared with GS-based diets (65.4 vs. 68.4 g/100 g of FA, respectively) and with extruded linseed compared with no extruded linseed (65.2 vs. 68.6 g/100 g of FA, respectively), and these effects were additive. Concentrations of total trans FA were higher with MS compared with GS-based diets (7.0 vs. 5.4 g/100 g of FA, respectively) and when extruded linseed was fed (6.8 vs. 5. 6g/100 g of FA, respectively). Total n-3 FA were higher when extruded linseed was fed compared with no extruded linseed (1.2 vs. 0.8 g/100 g of FA, respectively), whereas total n-6 polyunsaturated FA were higher when feeding MS compared with GS (2.5 vs. 2.1 g/100 g of FA, respectively). Feeding extruded linseed and MS both provided potentially beneficial decreases in SFA concentration of milk, and no significant interactions were found between extruded linseed supplementation and forage type. However, both MS and extruded linseed increased trans FA concentration in milk fat. Neither MS nor extruded linseed had significant effects on methane production or yield, but the amounts of supplemental lipid provided by extruded linseed were relatively small.

Studies on serum progesterone levels in Zebu × Holstein heifers during pre- and peripubertal periods

The peripheral serum progesterone levels of six normal Zebu × Holstein heifers (75% Holstein inheritance) during the prepubertal period (−150 days) were low (0.23 ± 0.06 ng/ml). They reached maximal values (0.73 ± 0.06 ng/ml) by −45 days (P<0.05 for progesterone values on −90th vs. −45th days) and thereafter decreased to the base level at the time of puberty. The mean (± SEM) age and body weight at puberty of these six heifers were 720 ± 20.70 days and 260.67 ± 8.82 kg, respectively. The serum progesterone levels remained low (0.38 ± 0.17 ng/ml) during early oestrus (up to 28 h) and gradually increased to 2.3 ± 0.84 ng/ml by the 15th day of the cycle.