Short-term physiological responses to moderate heat stress in grazing dairy cows in temperate climate.

Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit short-term physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals' vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na+, K+, and Cl- concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 °C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows' maximal daily vaginal temperature (VTMAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VTMAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na+ in milk was lower, and the concentration of K+ in milk tended to be higher in cows with increased VTMAX. For beta-hydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na+ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle.

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Effects of dexamethasone and growth hormone treatment on hepatic gluconeogenic enzymes in calves.

The hypothesis was tested that dexamethasone (DX) and bovine somatotropin (bST) alter expression or activity of gluconeogenic enzymes in neonatal calves. Holstein dairy calves (n = 24) were randomly divided in 4 groups and were treated with saline (control group), with DX at 30 microg/kg body weight per d (CDX), with 500 mg of sustained-release recombinant bST every 14 d (CbST), and with the combination of DX and bST from d 3 through 42 of life (CbSTDX). Plasma glucose and insulin concentrations were elevated throughout the study in CbSTDX, and insulin concentrations were elevated in CDX from d 7 to 28. Treatment with DX and the combination of DX and bST increased plasma glucagon concentrations from d 14 to 42, but decreased plasma cortisol concentrations on d 7 and 14 when compared with control calves. In liver, phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels were reduced in CDX and CbSTDX when compared with control calves or CbST. The activity of PEPCK on d 14 was higher in CbSTDX compared with control calves. Pyruvate carboxylase mRNA levels were decreased on d 7 in CDX and CbSTDX. Pyruvate carboxylase activities on d 14 and 28 were lower in CDX and CbSTDX than in control calves or CbST. These data indicate an age-dependent response to DX for blood metabolites, expression and activities of hepatic PEPCK and pyruvate carboxylase, and for effects of bST, suggesting that glucocorticoid status is important.

Effects of feeding vitamin A and lactoferrin on epithelium of lymphoid tissues of intestine of neonatal calves.

Circulating levels of vitamin A (retinol) and lactoferrin (Lf) are low in calves at birth. Bovine colostrum contains relatively high amounts of vitamin A and Lf, and both substances are intestinally absorbed by neonatal calves. There is evidence that these compounds interact with insulin-like growth factor binding proteins and thus influence the status and effects of insulin-like growth factor. The hypothesis was therefore tested that vitamin A and Lf influence epithelial growth, development, and absorptive capacity of the small and large intestine and modulate intestinal immune tissues (Peyer's patches; PP). Four groups of calves (n = 7 per group) were fed a milk-based formula with or without vitamin A and (or) Lf. Group F received formula (F) only; group F(A) was fed F supplemented with vitamin A; group F(L) was fed F supplemented with Lf, and group F(AL) received F plus vitamin A plus Lf. An additional group of calves (group C; n = 7) served as positive control and was fed colostrum (C) from pooled milk obtained on d 1, 2, and 3 of lactation. Amounts of nutritive components in formula and colostrum were similar. Blood samples were taken to measure vitamin A and Lf, and plasma xylose (added on d 4 to feeds) was measured postprandially for 8 h as a marker of intestinal absorptive capacity. Plasma vitamin A was low at birth and further decreased in groups F and F(L), but increased in groups F(A), F(AL), and C. Plasma Lf was low at birth and transiently increased up to 4 h after the first meal in group C. Xylose absorption was higher in group C than in other groups. Incorporation of 5-bromo-2'-deoxyuridine into DNA (as a measure of cell proliferation rate) was enhanced in intestinal crypts in groups F and F(L) at all intestinal sites. Ileum villus heights of groups F and F(L) were smaller than of groups F(A) and F(AL). Villus height to crypt depth ratios were smaller in F-fed groups (especially in groups F and F(L)) than in C-fed calves in the duodenum and jejunum. Incorporation of 5-bromo-2'-deoxyuridine into colon crypt cells of group F was greater than in groups F(L) and F(A). Sizes of follicles of PP in the ileum were greater in group F(A) than in group F. In the ileum, vitamin A and Lf tended to interact with PP size. In conclusion, feed supplementation of vitamin A and Lf influenced growth of the ileum and colon. Interactions were observed between vitamin A and Lf on epithelial cell maturation, villus growth, and size of follicles in PP of neonatal calves.

Abundance of mRNA encoding for components of the somatotropic axis and insulin receptor in different layers of the jejunum and ileum of neonatal calves.

Insulin-like growth factors-1 and -2, IGFBP-2 and -3, and receptors for IGF type-1 and type-2 (IGF-1R, IGF-2R), growth hormone (GHR), and insulin (InsR) in neonatal calves are variably expressed among gastrointestinal sites and thought to exert site-specific physiological functions. We studied by real-time reverse-transcription PCR, whether there are differences in the abundance of mRNA coding for IGF-I, IGF-2, IGFBP-2, IGFBP-3, IGF-1R, IGF-2R, GHR, and InsR in compartmentalized layers (fractions) of jejunum and ileum of 5-d-old calves fed colostrum. Samples of jejunum consisted primarily of villi and crypts; samples from ileum consisted mainly of villus tips, crypts, and lamina propria (LP; containing mainly Peyer's patches). After slaughter, segments of middle areas of jejunum and ileum were flushed with 154 mM NaCl. Pieces (5 mm x 5 mm) of jejunal (n = 9) and ileal walls (n = 5) were placed on glass slides and snap-frozen in liquid N before being cut horizontally into 10-mum-deep slices using a cryotome at -20 degrees C. Fifteen consecutive and morphologically similar slices were collected as fractions of villus, crypt, and LP layers, respectively. Fractions were characterized by use of 5'-bromo-2-deoxyuridine (BrdU) that labeled proliferating cells, and by expression of lactase mRNA. The BrdU-labeled cells were present in crypts and LP, but not in tips of villi. Lactase mRNA levels were greater in villus than crypt fractions, but lactase mRNA was absent in LP. In jejunum, mRNA levels, relative to levels of housekeeping genes (sum of levels of mRNA coding for ubiquitin, glyceraldehyde phosphate dehydrogenase, beta-actin, and ribosomal RNA), differed (P < 0.05) between fractions for InsR (crypts > villi), IGFBP-2 (crypts > villi), and IGFBP-3 (crypts > villi), and total RNA levels were greater (P < 0.05) in crypt than villus fractions. In ileum, mRNA levels, expressed relative to housekeeping genes, differed (P < 0.05) between fractions for IGF-I (LP > villi, crypts), IGF-2, and IGFBP-3 (villi > crypts, LP), GHR and InsR (crypts > LP), IGFBP-2 (crypts > villi, LP), and total RNA levels were greater (P < 0.05) in LP and crypt than in villus fractions. In conclusion, the tested fractionation technique is quite applicable for gene expression studies in the intestine of calves. Members of the somatotropic axis and of the insulin receptor are not equally expressed in different jejunal and ileal layers of neonatal calves.

Preterm as compared with full-term neonatal calves are characterized by morphological and functional immaturity of the small intestine.

Intestinal diseases in neonatal calves may be due to morphological and functional immaturity. We have studied histomorphology, crypt cell proliferation rates (based on incorporation of 5-bromo-2'-deoxyuridine into DNA), presence of apoptotic cells (based on terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling), and brush border enzyme activities in preterm calves (277 d of gestation), euthanized on d 1 (P0) or 8 (P8), and in full-term calves (290 d of gestation), euthanized on d 1 (F0) or 8 (F8). Vacuolated epithelial cells were present in ileum of P0 and F0 but not in P8 and F8. During the first 8 d, villus sizes, crypt depths, and proliferation rates of crypt cells in the small intestine of preterm calves did not significantly change. In contrast, in full-term calves during the first 8 d, villus sizes in jejunum decreased, crypt depths increased in small intestine and colon, and crypt cell proliferation increased in duodenum and jejunum. Submucosal thickness in jejunum was highest in P0, but in ileum it increased with gestational age and feeding. Gestational age x feeding interactions indicated increased activities of aminopeptidase N and reduced lactase activities only in F8 and reduced dipeptidylpeptidase IV activities only in P8. In conclusion, in preterm calves the small intestinal epithelium was immature and brush border enzyme activities differed in part from those in full-term calves.

Intestinal development in neonatal calves: effects of glucocorticoids and dependence of colostrum feeding.

The neonatal development of the gastrointestinal tract around parturition in precocious mammals is greatly affected by endocrine factors like glucocorticoids as well as by nutritional factors. We have studied the effects of glucocorticoids and colostrum supply on intestinal morphology, cell proliferation, digestive enzyme activities, and xylose absorption in neonatal calves to test the hypothesis that the intestinal development in neonatal calves is influenced by glucocorticoids, dependent on colostrum feeding. Calves designated GrFD(-) and GrFD(+) were fed a milk-based formula, whereas those designated GrCD(-) and GrCD(+) received colostrum. Dexamethasone (DEXA; 30 microg/kg/day) was injected at feeding times to calves of GrFD(+) and GrCD(+). On day 3, the D-xylose absorption was measured. The calves were euthanized on day 5 of life. Colostrum feeding increased villus sizes in jejunum and ileum, enhanced xylose absorption capacity, and increased peptidase activities in the ileum. DEXA treatment diminished sizes and cell proliferation rates of Peyer's patches in the ileum, yet increased proliferation of crypt cells in the ileum of formula-fed calves. DEXA reduced aminopeptidase N activities in the jejunum of formula-fed calves, but increased the peptidase activities mainly of colostrum-fed calves in the ileum. Thus, DEXA effects depended on intestinal segment and on different feeding, resulting in stimulation of crypt cell proliferation in the less mature ileum (of formula-fed calves) and in stimulation of peptidase activities in the more mature ileum (of colostrum-fed calves). We conclude that the effects of DEXA were related to the developmental stage of the neonatal intestine and promoted the intestinal development, depending on the developmental stage.

Dexamethasone and colostrum feeding affect hepatic gluconeogenic enzymes differently in neonatal calves.

Plasma glucose concentrations in neonates are influenced by colostrum feeding and by glucocorticoids. We have tested whether a high-glucocorticoid status after birth, as well as colostrum feeding, influences glucose metabolism in association with changes of hepatic expression and activities of gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and pyruvate carboxylase (PC; EC 6.4.1.1) in neonatal calves. Calves (n = 14 per group) were fed either colostrum or a milk-based formula with nutrient and energy contents similar to colostrum. Half the calves in each feeding group were treated with dexamethasone (DEXA; 30 microg/[kg BW x d]). Pre- and postprandial blood samples were taken on d 1, 2, 4, and 5 and liver samples were collected on d 5 of life. Dexamethasone treatment increased (P < or = 0.05) plasma concentrations of glucose, insulin, and glucagon more in colostrum-fed than in formula-fed calves but increased (P < or = 0.05) urea concentrations and decreased (P < or = 0.05) concentrations of NEFA, ACTH, and cortisol independent of colostrum vs. formula feeding. Colostrum feeding increased (P < 0.05) plasma glucose, but decreased (P < 0.05) plasma urea concentrations. Glucagon-to-insulin ratios in DEXA-treated and colostrum-fed calves were decreased (P < 0.05). Dexamethasone treatment decreased hepatic mRNA levels and activities of PC (P < 0.001 and P < 0.10) and activities of PEPCK (P < 0.001) but increased (P < 0.001) the glycogen content. Colostrum feeding increased (P < 0.05) mitochondrial PEPCK mRNA levels and PEPCK activities in calves not treated with DEXA but decreased (P < 0.1) amounts of PC mRNA. In conclusion, increased plasma glucose concentrations after DEXA treatment were not associated with a stimulation of hepatic gluconeogenic enzyme activities; however, colostrum feeding probably raised plasma glucose concentrations because of increased hepatic gluconeogenic activities.

Physiological traits in preterm calves during their first week of life.

Morbidity and mortality of preterm neonatal calves are higher than of calves born at normal term, possibly and in part due to immaturity of physiological functions. Physiological parameters were therefore studied during the first week of life in seven preterm calves, born on day 277 of gestation after dams were injected prostaglandin F2alpha and flumethason. Calves were fed colostrum of the first milking for the first 3 days and from day 4 to day 7 the same colostrum diluted with milk replacer. Body weight increased during the first week of life by 2.2 kg. Heart rate and respiratory rate were always relatively high, whereas values of rectal temperature, blood gases, haematological, metabolic and endocrine traits were in the range and behaved similarly as is the experience in full-term neonatal calves. Major exceptions were glucose and insulin, the concentrations of which barely rose postprandially, and growth hormone, the responses of which to growth hormone releasing factor analogue 1-29 were extremely variable and in part very small. In conclusion, calves born 2 week before normal term that survived the first week of life, although physiologically immature, were well able to handle ingested nutrients and to control their metabolism.

Estimation of energy balance at the individual and herd level using blood and milk traits in high-yielding dairy cows.

This study aimed to estimate individual and herd-level energy balance (EB) using blood and milk traits in 90 multiparous high-yielding Holstein cows, held on a research farm, from wk 1 to 10 postpartum (p.p.) and to investigate the precision of prediction with successively decreased data sets simulating smaller herd sizes and with pooled samples. Dry matter intake, milk yield, and BW were measured daily from parturition through wk 10 p.p. Milk composition was determined 4 times per week, and milk acetone was measured weekly. Blood samples for the determination of metabolites, hormones, electrolytes, and enzyme activities were taken weekly from wk 1 to 10 p.p. between 0730 and 0900. Body condition scores and ultrasonic measurements of backfat thickness and fat depth in the pelvic area were evaluated in wk 1, 4, and 8 p.p. Concentrations of glucose, cholesterol, urea, insulin, insulin-like growth factor-1, triiodothyronine, and thyroxine (T4) in blood plasma and of lactose and urea in milk were positively correlated with EB, whereas concentrations of nonesterified fatty acids (NEFA), creatinine, albumin, beta-hydroxybutyrate, and growth hormone and enzyme activities in blood, and concentrations of fat, protein, fat:lactose ratio, and acetone in milk were negatively correlated with EB. Leptin concentration was not correlated to EB over the first 10 wk p.p. To estimate EB linear mixed-effects, models were developed by backward selection procedures. The most informative traits for estimation of EB were the fat:lactose ratio in milk and NEFA and T4 concentrations in blood. The precision of estimation of EB in individual cows was low. Using blood in addition to milk traits did not result in higher precision of estimation of herd-level EB, and decreasing sample sizes considerably lowered the precision of EB prediction. Estimation of overall mean herd-level EB over the first 10 wk p.p. using pooled samples was precise even with small sample sizes, but does not consider the level of EB in particular weeks. In conclusion, estimation of herd-level EB at individual weeks using milk traits only has practical implication with herd sizes of > or = 100 cows if calving is highly seasonal and of or = 400 cows if calving is uniformly distributed. Using blood in addition to milk traits does not improve precision of estimation of herd-level EB, regardless of sample size.

A scanning near-field optical microscope approach to biomolecule patterning.

In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.

Feeding colostrum, its composition and feeding duration variably modify proliferation and morphology of the intestine and digestive enzyme activities of neonatal calves.

We studied the effects of amounts of colostrum consumed on intestinal morphology and proliferation and digestive enzyme activities in neonatal calves. Group GrCmax calves were fed colostrum from the first milking undiluted on d 1-3 and diluted with 25, 50, 75 and 75 parts of a milk replacer on d 4-7. Group GrC1-3 calves were fed colostrum from milkings 1-6 up to d 3 and then a milk replacer up to d 7. Group GrF1-3 calves were fed a milk-based formula (containing only traces of growth factors and hormones) up to d 3 and then a milk replacer up to d 7. Calves were killed on d 8. Differences in feeding affected villus sizes and villus height/crypt depth ratios in the duodenum (GrCmax > GrC1-3), villus areas and villus height/crypt depth ratios in the jejunum (GrC1-3 > GrF1-3) and crypt depths in the colon (GrF1-3 > GrC1-3). Furthermore, different feeding protocols affected the proliferation rates of epithelial cells in the duodenum (GrC1-3 > GrCmax; GrC1-3 > GrF1-3) and the jejunum (GrF1-3 > GrC1-3; based on Ki-67 labeling). Lipase activities in the pancreas were influenced by colostrum feeding (GrC(max) > GrC(1-3)). Colostrum intake differentially affected intestinal epithelial surface and proliferation and enzyme activities. Feeding high amounts of first colostrum seemed to enhance the survival of mature mucosal epithelial cells in selected parts of the small intestine, whereas the lack of colostrum seemed to decrease epithelial growth.

Chemically etched fiber tips for near-field optical microscopy: a process for smoother tips.

An improved method for producing fiber tips for scanning near-field optical microscopy is presented. The improvement consists of chemically etching quartz optical fibers through their acrylate jacket. This new method is compared with the previous one in which bare fibers were etched. With the new process the meniscus formed by the acid along the fiber does not move during etching, leading to a much smoother surface of the tip cone. Subsequent metallization is thus improved, resulting in better coverage of the tip with an aluminum opaque layer. Our results show that leakage can be avoided along the cone, and light transmission through the tip is spatially limited to an optical aperture of a 100-nm dimension.