Single-step production of autologous bovine platelet concentrate for clinical applications in cattle.

Platelet concentrate (PC) is an alternative therapy to treat mastitis in dairy cattle and is an alternative treatment for reproduction problems such as endometritis. Unfortunately, double-centrifugation processing methods described are time-consuming, require specialized laboratory equipment, and are usually done in a heterologous way, which risks herd health. To overcome this limitation, we evaluated single-step bovine PC processing methods readily applicable to a farm setting using an autologous conditioned plasma (ACP) production system. We investigated the hematologic findings, cytokines, and growth factors of the obtained PC samples. Autologous conditioned plasma was prepared using whole blood (WB) from 4 cows (group 1) using single-step centrifugation and 16 different processing methods. The 2 protocols that yielded the highest ratio of platelet to white blood cell (WBC) concentration were ACP-1 [720 × g (2,200 rpm), 5 min] and ACP-2 [929 × g (2,500 rpm), 3 min]. They were subsequently reproduced and compared using WB from 8 cows (group 2). Hematologic findings were quantified, IL-1ß (cytokine) and growth factors [platelet-derived growth factor (PDGF), transforming growth factor (TGF)-ß, bovine fibroblast growth factor (b-FGF)] were measured, and enrichment factors were compared between samples and processing methods. Hematological characteristics and platelet enrichment varied markedly among tested protocols and all were statistically different from WB. Protocol ACP-2 resulted in significantly greater platelet enrichment (mean 169% of WB) than ACP-1 (125% of WB). We found no significant difference between the 2 ACP preparation protocols with regard to leukocyte reduction (7.53-9.75% WBC compared with WB) or growth factor enrichment (124-125% PDGF, 95-100% TGF-ß, 102-104% b-FGF, and 56-74% IL-1ß compared with WB). In conclusion, both ACP protocols yielded a platelet concentration shown to promote healing for clinical applications in cattle, and the ACP-2 protocol resulted in a greater degree of platelet enrichment. Therefore, this protocol could be used for ACP production for clinical applications in cattle.

In vitro biomechanical properties of sole tissues: Comparison between healthy and ulcerated bovine claws.

Sole ulcers are reportedly one of the most prevalent diseases associated with lameness in dairy cattle, significantly affecting animal welfare and farm profitability. The degree to which sole soft tissues, healthy or ulcerated, are able to maintain their structure and function when subjected to compressive forces remains unknown. Therefore, the aims of the present study were to assess sole tissue biomechanics in healthy and ulcerated claws and to describe correlated histology. Cylindrical samples were harvested from zones 4 and 6, as described by the international foot map, from hind lateral healthy (n = 12) and ulcerated bovine claws (n = 8; animals n = 12). Tissue biomechanics and morphology were evaluated via compressive tests and hematoxylin-eosin-phloxine-saffron staining, respectively. A 2-sample t-test was used to compare zones' mechanical properties between healthy and ulcerated tissues, and the Cochran-Mantel-Haenszel test was used to measure the effect of claw zone on histology. The fibril modulus (Ef) and permeability (k) respectively increased and decreased in ulcerated claws (Ef = 0.201 ± 0.104 MPa; k = 0.128 ± 0.069 mm2/MPa·s) compared with healthy claws (Ef = 0.105 ± 0.050 MPa; k = 0.452 ± 0.365 mm2/MPa·s) only for zone 6. Histology scores equal to or greater than 3 were associated with macroscopic presence of ulceration. A higher proportion of adipose tissue (30% or more) was associated with zone 6 compared with zone 4, but no difference was seen between healthy and ulcerated claws. Ulcerated claws had a higher prevalence of exostoses compared with healthy ones (33% vs. 8%). Sole soft tissues showed, as hypothesized, a viscoelastic behavior using unconfined compression testing, which, however, may not reflect in vivo loading conditions. Clinical and histological signs of sole ulceration were not associated with decreased strength of the supportive apparatus of the distal phalanx in zone 4 in this study.

Non-invasive acquisition of fetal ECG from the maternal xyphoid process: a feasibility study in pregnant sheep and a call for open data sets.

OBJECTIVE: The utility of fetal heart rate (FHR) monitoring can only be achieved with an acquisition sampling rate that preserves the underlying physiological information on the millisecond time scale (1000 Hz rather than 4 Hz). For such acquisition, fetal ECG (fECG) is required, rather than the ultrasound to derive FHR. We tested one recently developed algorithm, SAVER, and two widely applied algorithms to extract fECG from a single-channel maternal ECG signal recorded over the xyphoid process rather than the routine abdominal signal. APPROACH: At 126dG, ECG was attached to near-term ewe and fetal shoulders, manubrium and xyphoid processes (n = 12). fECG served as the ground-truth to which the fetal ECG signal extracted from the simultaneously-acquired maternal ECG was compared. All fetuses were in good health during surgery (pH 7.29 ± 0.03, pO2 33.2 ± 8.4, pCO2 56.0 ± 7.8, O2Sat 78.3 ± 7.6, lactate 2.8 ± 0.6, BE -0.3 ± 2.4). MAIN RESULT: In all animals, single lead fECG extraction algorithm could not extract fECG from the maternal ECG signal over the xyphoid process with the F1 less than 50%. SIGNIFICANCE: The applied fECG extraction algorithms might be unsuitable for the maternal ECG signal over the xyphoid process, or the latter does not contain strong enough fECG signal, although the lead is near the mother's abdomen. Fetal sheep model is widely used to mimic various fetal conditions, yet ECG recordings in a public data set form are not available to test the predictive ability of fECG and FHR. We are making this data set openly available to other researchers to foster non-invasive fECG acquisition in this animal model.

α7 nicotinic acetylcholine receptor signaling modulates the inflammatory phenotype of fetal brain microglia: first evidence of interference by iron homeostasis.

Neuroinflammation in utero may result in life-long neurological disabilities. Microglia play a pivotal role, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of microglia. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal microglia will augment their neuroprotective transcriptome profile, while the antagonistic stimulation of α7nAChR will achieve the opposite. Using an in vivo - in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep microglia cultures re-exposed to LPS in presence of a selective α7nAChR agonist or antagonist. Our RNAseq and protein level findings show that a pro-inflammatory microglial phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, antagonistic α7nAChR stimulation potentiates the pro-inflammatory microglial phenotype. Surprisingly, under conditions of LPS double-hit an interference of a postulated α7nAChR - ferroportin signaling pathway may impede this mechanism. These results suggest a therapeutic potential of α7nAChR agonists in early re-programming of microglia in neonates exposed to in utero inflammation via an endogenous cerebral cholinergic anti-inflammatory pathway. Future studies will assess the role of interactions between inflammation-triggered microglial iron sequestering and α7nAChR signaling in neurodevelopment.

Cerebrospinal Fluid Analysis in Recumbent Adult Dairy Cows With or Without Spinal Cord Lesions.

BACKGROUND: Diagnosis of central nervous system (CNS) lesions in recumbent dairy cattle (RDC) is challenging because neurologic examination is limited and medical imaging often is challenging or unrewarding. Cerebrospinal fluid (CSF) analysis is useful in the diagnosis of CNS disorders in cattle. However, its utility in identifying spinal cord lesions in RDC remains to be evaluated. HYPOTHESIS/OBJECTIVES: We hypothesized that CSF analysis would discriminate between RDC with and without spinal cord lesions. ANIMALS: Twenty-one RDC with spinal cord lesions (RDC+) and 19 without (RDC-) were evaluated. METHODS: Spinal cord lesions were confirmed at necropsy. Signalment, clinical findings, and CSF results were compared retrospectively. Total nucleated cell count and differential, protein concentration, and red blood cell count in RDC+ and RDC- were compared. RESULTS: Neoplasia, trauma, and infectious processes were the most frequent spinal cord lesions identified. Cerebrospinal fluid protein concentrations and TNCC were significantly higher in RDC+ compared to RDC- (P = .0092 and P = .0103, respectively). Additionally, CSF protein concentrations and TNCC in RDC- were lower than previously published reference ranges. Using an interpretation rule based on CSF protein concentration and TNCC, it was possible to accurately identify 13 RDC with spinal cord lesions and 6 RDC without lesions. It was not possible to determine spinal cord status in the remaining 18 RDC. CONCLUSIONS AND CLINICAL IMPORTANCE: Cerebrospinal fluid analysis is valuable in the evaluation of spinal cord status in RDC. The prognosis associated with these findings remains to be determined.

Cholangiohepatitis in Dairy Cattle: 13 Cases.

BACKGROUND: The signalment, clinicopathologic, bacteriological, histopathological, ultrasonographic characteristics, and the treatment and outcomes of adult cattle with cholangiohepatitis are poorly defined. ANIMALS: Thirteen Holstein cows with cholangiohepatitis. METHODS: Retrospective study of medical records of cattle admitted to the CHUV and the AVC between 1992 and 2012 and 2000 and 2012, respectively, for cattle older than 3 months of age with a histopathological diagnosis of cholangiohepatitis. Cholangiohepatitis was defined as the presence of portal inflammation surrounding or infiltrating bile ducts, with or without epithelial damage, and extending into the adjacent lobules. RESULTS: At the time of diagnosis of cholangiohepatitis, cows had decreased appetite (n = 7) or were anorectic (n = 6), had fever (n = 5), and had tachycardia (n = 8). Icterus was detected in 5 cows. Yellow discoloration was identified on the skin of the udder (n = 3), conjunctiva (n = 2), and vulva (n = 1). There was leukocytosis (n = 6), neutrophilia (n = 9), and hyperfibrinogenemia (n = 8). Alteration in the serum biochemistry profile included hyperglobulinemia (n = 8), hypoalbuminemia (n = 10), increased activity of GGT (n = 12), AST (n = 8), and ALP (n = 10), and hyperbilirubinemia (n = 10). Histopathological diagnosis included mild, subacute, nonsuppurative cholangiohepatitis (n = 4), mild suppurative cholangiohepatitis (n = 4), mild mixed (neutrophilic and lymphocytic) cholangiohepatitis (n = 3), and moderate, chronic, nonsuppurative cholangiohepatitis (n = 1). Six cows were discharged from the hospital, and 7 were euthanized. CONCLUSIONS AND CLINICAL IMPORTANCE: Cholangiohepatitis is a rare condition in adult cattle. Antemortem diagnosis can be challenging because clinical signs are unspecific.

RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain.

BACKGROUND: The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. NEW METHOD: Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. RESULTS: We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1ß) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. COMPARISON WITH EXISTING METHODS: This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. CONCLUSIONS: The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.

Glucocorticoid receptor density and binding affinity in healthy horses and horses with systemic inflammatory response syndrome.

BACKGROUND: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in horses with systemic inflammatory response syndrome (SIRS). Peripheral resistance to glucocorticoids has not been investigated in horses. OBJECTIVE: To determine if glucocorticoid receptor (GR) function in horses can be measured using flow cytometry, and to use this information to evaluate HPA axis dynamics. ANIMALS: Eleven healthy adult horses in parts 1 and 2. Ten horses with SIRS and 10 age and sex matched controls in part 3. METHODS: Flow cytometry was used to evaluate GR density and binding affinity (BA) in 3 healthy horses in part 1. In part 2, exogenous ACTH was administered to eight healthy horses. Their cortisol response and GR properties were measured. In part 3, CBC, serum biochemistry, cortisol and ACTH, and GR properties were compared between controls without SIRS (n = 10) and horses with SIRS (n = 10), and between survivors and nonsurvivors (n = 4 and n = 6 respectively). RESULTS: Flow cytometry can be used to measure GR properties in equine PBMCs. No correlation was observed between plasma cortisol concentration and GR density or BA in healthy horses (r = -0.145, P = .428 and r = 0.046, P = .802 respectively). Nonsurvivors with SIRS had significantly decreased GR BA (P = .008). Horses with triglyceride concentration > 28.5 mg/dL had increased odds of nonsurvival (OR=117; 95% CI, 1.94-7,060). GR BA <35.79% was associated with nonsurvival (OR = 30.33; 95% CI, 0.96-960.5). CONCLUSIONS AND CLINICAL IMPORTANCE: Tissue resistance to glucocorticoids contributes to HPA axis dysfunction in adult horses with SIRS. These horses might benefit from treatment with exogenous glucocorticoids.

Neonatal morbidity and mortality of 31 calves derived from somatic cloning.

BACKGROUND: The neonatal period is associated with high morbidity and mortality in cloned calves. OBJECTIVE: To describe morbidity and mortality in cloned calves from birth to 2 years of age. ANIMALS: Thirty-one somatic cell-derived Holstein calves delivered at a veterinary teaching hospital. METHODS: Medical files were retrospectively analyzed. RESULTS: Four calves were stillborn. Five calves born alive had physical congenital defects. Twenty-three calves had an enlarged umbilical cord. Laboratory abnormalities included acidemia, respiratory acidosis, hyperlactatemia, anemia, stress leukogram, decreased total protein, albumin and globulins, and increased creatinine. Twenty-five calves survived the 1st hour of life. Among them, 11 stood without assistance within 6 hours of birth, 10 calves took longer than 6 hours to stand, and 4 never stood. Twenty-two calves suffered from anorexia. Twelve calves had complications arising from umbilical cord infections. Three calves developed idiopathic hyperthermia (>40°C). Eight calves suffered from gastrointestinal problems, including ruminal distension, abomasal ulcers, neonatal enteritis, intussusception, and abomasal displacement. Mortality between birth and 3 weeks of age was 32% (10/31). Causes of death and reasons for euthanasia included stillbirths, respiratory failure, and limb deformities. Mortality between 3 weeks and 2 years of age was 19% (4/21), with deaths in this group attributed to generalized peritonitis and complications arising from umbilical infections. Overall, mortality rate within 2 years of age was 14/31 (45%). CONCLUSION AND CLINICAL IMPORTANCE: Respiratory problems, limb deformities, and umbilical infections were the most common causes of morbidity and mortality in these cloned calves.

Morphological response of songbirds to 100 years of landscape change in North America.

Major landscape changes caused by humans may create strong selection pressures and induce rapid evolution in natural populations. In the last 100 years, eastern North America has experienced extensive clear-cutting in boreal areas, while afforestation has occurred in most temperate areas. Based on museum specimens, I show that wings of several boreal forest songbirds and temperate songbirds of non-forest habitats have become more pointed over the last 100 years. In contrast, wings of most temperate forest and early-successional boreal forests species have become less pointed over the same period. In contrast to wing shape, the bill length of most species did not change significantly through time. These results are consistent with the "habitat isolation hypothesis", i.e., songbirds evolved in response to recent changes in the amount of available habitat and associated implications for mobility. Rapid morphological evolution may mitigate, without necessarily preventing, negative consequences of habitat loss caused by humans through direct exploitation or climate change.

Effects of supplementary folic acid and vitamin B(12) on hepatic metabolism of dairy cows according to methionine supply.

The present experiment was undertaken to study the interactions between dietary supplements of rumen-protected methionine (RPM) and intramuscular injections of folic acid and vitamin B(12), given from 3 wk before calving to 16 wk of lactation, on hepatic metabolism of lactating dairy cows. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply Met as 1.83% of metabolizable protein, whereas the 3 other cows were fed the same diet supplemented with 18g of RPM calculated to provide Met as 2.23% of metabolizable protein. Within each level of Met, the cows received no vitamin supplement or weekly intramuscular injections of 160mg of folic acid alone or combined with 10mg of vitamin B(12). Liver biopsies were taken at 2, 4, 8, and 16 wk of lactation. Liver concentrations of folates and vitamin B(12) were increased by their respective supplements but this response to vitamin supplements was altered by methionine supply. Concentrations of total lipids and triglycerides increased in livers of cows fed RPM, whereas concentrations of cholesterol ester, cholesterol, diglycerides, phosphatidylethanolamine, and phosphatidylcholine were not affected. Folic acid, alone or combined with vitamin B(12), tended to increase the ratio of phosphatidylcholine to phosphatidylethanolamine. Gene expression of 5,10-methylene-tetrahydrofolate reductase, microsomal transfer protein, and phosphatidylethanolamine methyltransferase were higher in liver of cows fed RPM supplements. The relative mRNA abundance of 5,10-methylene-tetrahydrofolate reductase and methylmalonyl-CoA mutase were increased by the combined injections of folic acid and vitamin B(12), whereas those of methionine synthase and methionine synthase reductase were not affected by treatments. These results suggest that increasing supply of methyl groups, as preformed labile methyl groups or through methylneogenesis, affected the methylation cycle but had a limited effect on dairy cow performance. The observed effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows probably result from an improvement of energy metabolism during early lactation.

Net flux of nutrients across splanchnic tissues of lactating dairy cows as influenced by dietary supplements of biotin and vitamin B12.

Biotin and vitamin B(12) are coenzymes in reactions that are essential to propionate metabolism in dairy cows. The objective of the present studies was to determine whether an increased dietary supply of these vitamins would change the net flux of nutrients through the rumen, the portal-drained viscera (PDV), the total splanchnic tissues (TSP), and the liver. Four lactating cows equipped with ultrasonic flow probes around the right ruminal artery and the portal vein and catheters in the right ruminal vein, the portal vein, one hepatic vein, and one mesenteric artery were fed 12 times per day a mixed ration at 95% of ad libitum dry matter intake. Daily supplements of 500 mg of vitamin B(12)+20mg of biotin or no vitamin supplement (study 1) or 500 mg of vitamin B(12) alone or with 20mg of biotin (study 2) were fed according to a crossover design with two 4-wk periods in each study. On the last day of each period, blood flow was recorded and blood samples were collected every 30 min for 4h. In study 1, biotin and vitamin B(12) given together increased milk production and milk protein yields compared with the control diet. The supplement increased appearance of the 2 vitamins across the PDV and TSP. It also reduced the net portal appearance of ammonia and total volatile fatty acids across the PDV. In study 2, compared with the 2 vitamins together, vitamin B(12) alone increased glucose flux across PDV and TSP as well as its arterial concentration and PDV flux of ammonia. With the diet used in the present experiment, the major effects of the vitamin supplements seem to be mediated through changes in ruminal fermentation and gastrointestinal tract metabolism rather than by effects on hepatic metabolism.

Net flux of nutrients across the rumen wall of lactating dairy cows as influenced by dietary supplements of folic acid.

The objective of the present study was to determine whether a dietary supplementation of folic acid, at levels used in our previous studies, would affect ruminal fermentation and the net flux of nutrients across the rumen wall of lactating dairy cows. Approximately 4 wk after calving, 5 lactating multiparous cows were surgically equipped with a ruminal cannula, an ultrasonic flow probe around the right ruminal artery, and indwelling catheters in the right ruminal vein and the ileocolic artery. Cows were fed a total mixed ration served in 7 equal meals per d (i.e., every 3.4 h). The experimental design was an unbalanced crossover arrangement with 3 periods of 4 wk each. The vitamin supplement, incorporated in equal amounts into each meal, was supplied at 0, 3, or 6 mg of folic acid per kg of BW per d. During the last week of each experimental period, blood samples were taken simultaneously from the 2 catheters every 30 min and rumen fluid was collected every 60 min during 2 consecutive meal intervals. Dietary supplementation with folic acid had no effect on milk production (27.2 +/- 1.3 kg/d) or DMI (19.9 +/- 0.7 kg/d), but milk concentrations and yields of total solids, fat, and protein increased linearly with increasing doses of folic acid ingested. Concentrations of folates in rumen fluid and arterial plasma, averaged over time, increased linearly with the dose of folic acid ingested but the net flux of folates across the rumen wall was not different from zero. Concentrations of butyrate in ruminal fluid decreased quadratically with the daily supply in folic acid. Dietary supplements of folic acid had no effect on pH and osmolality of ruminal fluid, nor on ruminal concentrations of lactate, ammonia, acetate, or propionate, total VFA, or microbial counts. The uptake of urea-N by the rumen wall tended to increase quadratically with the dose ingested but net fluxes of other nutrients were not affected by treatments. These results suggest that the effects of folic acid supplements on lactational performance cannot be explained by effects on rumen metabolism.

Behavioural and cardiorespiratory effects of a constant rate infusion of medetomidine and morphine for sedation during standing laparoscopy in horses.

REASONS FOR PERFORMING STUDY: Standing surgical procedures are performed commonly in horses under sedation. This approach minimises the morbidity/mortality risks associated with general anaesthesia. The use of a medetomidine and morphine combination has not been investigated in horses despite the usefulness of each drug individually. OBJECTIVE: To determine the efficacy of a medetomidine and morphine combination to produce standing sedation with minimal cardiorespiratory changes and adequate analgesia for exploratory laparascopy in mature horses. HYPOTHESIS: The combination of medetomidine and morphine will induce reliable sedation with minimal cardiorespiratory changes. METHODS: Medetomidine (5 microg/kg bwt i.v.) followed in 10 min by morphine (50 microg/kg bwt i.v.) and 10 min later by a constant rate infusion (CRI) of medetomidine and morphine (5 and 30 microg/kg bwt/h, respectively) was administered in 7 horses undergoing standing exploratory laparoscopy. Quality of sedation and cardiorespiratory function were assessed. RESULTS: Sedation was satisfactory after the medetomidine and morphine bolus. The CRI of both drugs enhanced sedation and ataxia. Mean visual analogue scores on a scale of 10 varied between 7.8 and 8.8 and were similar between anaesthesiologists and surgeons. Heart rate, respiratory rate and packed cell volume (PCV) decreased significantly after medetomidine and, at some periods, during the CRI. Blood pressure only increased significantly at 5 min and arterial O2 decreased significantly at 10 min post medetomidine. PCV remained significantly lower and total protein decreased post morphine and CRI administration. Arterial CO2 increased towards the end of the CRI. Cardiac output did not change significantly over time. CONCLUSION AND CLINICAL RELEVANCE: The combination of medetomidine and morphine results in reliable sedation and stable cardiorespiratory function in horses undergoing exploratory laparascopy.

Influence of methionine supply on the response of lactational performance of dairy cows to supplementary folic acid and vitamin B12.

The present experiment was undertaken to determine if the effects of supplementary folic acid on lactational performance were caused by improved methylneogenesis and if the supply in vitamin B(12) could affect this metabolic pathway. In this eventuality, supplementary Met, a major source of preformed methyl groups, should reduce the requirements for these vitamins. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet estimated to supply Met as 1.83% metabolizable protein and 3 cows were fed the same diet supplemented with 18 g of rumen-protected methionine (RPM) to supply Met as 2.23% of metabolizable protein. Within each level of Met, cows received no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid alone or combined with 10 mg of vitamin B(12) from 3 wk before to 16 wk after calving. There was no treatment effect on dry matter intake during pre- and postcalving periods: 13.4 +/- 0.4 and 21.8 +/- 0.4 kg/d, respectively. Milk production was not affected by RPM supplementation. Folic acid and vitamin B(12) given together tended to increase milk production during the 16 wk of lactation. This effect was more pronounced during the first 4 wk of lactation: 37.5, 37.7, and 40.3 +/- 0.9 kg/d for cows receiving no vitamin supplement, folic acid alone, or folic acid combined with vitamin B(12), respectively. Milk fat yield was not affected by treatments. Lactose, crude protein, and total solid yields were greater, in early lactation, in cows injected with folic acid and vitamin B(12) together but this effect diminished as lactation progressed. Intramuscular injections of folic acid alone or combined with vitamin B(12) tended to decrease plasma concentrations of homocysteine from 5.51 microM with no vitamin supplement to 4.54 and 4.77 +/- 0.37 microM, respectively. Results of the present experiment suggest that the effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows were not due to an improvement in methyl groups supply, because RPM supplement, a source of preformed methyl groups, did not alter the cow responsiveness to vitamin supplements.

Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.

The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B(12), given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B(12.) To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-(13)C]glucose, [(13)C]NaHCO(3) and 3[1-(13)C,(2)H(3)] methionine. Milk and plasma concentrations of folic acid and vitamin B(12) increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 +/- 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism.

Effects of dietary supplements of folic acid and vitamin B12 on metabolism of dairy cows in early lactation.

The present experiment was undertaken to determine the effects of dietary supplements of folic acid and vitamin B12 given from 3 wk before to 8 wk after calving on lactational performance and metabolism of 24 multiparous Holstein cows assigned to 6 blocks of 4 cows each according to their previous milk production. Supplementary folic acid at 0 or 2.6 g/d and vitamin B12 at 0 or 0.5 g/d were used in a 2 x 2 factorial arrangement. Supplementary folic acid increased milk production from 38.0 +/- 0.9 to 41.4 +/- 1.0 kg/d and milk crude protein yield from 1.17 +/- 0.02 to 1.25 +/- 0.03 kg/d. It also increased plasma Gly, Ser, Thr, and total sulfur AA, decreased Asp, and tended to increase plasma Met. Supplementary B12 decreased milk urea N, plasma Ile, and Leu and tended to decrease Val but increased homocysteine, Cys, and total sulfur AA. Liver concentration of phospholipids was higher in cows fed supplementary B12. Plasma and liver concentrations of folates and B12 were increased by their respective supplements, but the increase in plasma folates and plasma and liver B12 was smaller for cows fed the 2 vitamins together. In cows fed folic acid supplements, supplementary B12 increased plasma glucose and alanine, tended to decrease plasma biotin, and decreased Km of the methylmalonyl-coenzyme A mutase in hepatic tissues following addition of deoxyadenosylcobalamin, whereas it had no effect when cows were not fed folic acid supplements. There was no treatment effect on plasma nonesterified fatty acids as well as specific activity and gene expression of Met synthase and methylmalonyl-coenzyme A mutase in the liver. Ingestion of folic acid supplements by cows fed no supplementary B12 increased total lipid and triacylglycerols in liver, whereas these supplements had no effect in cows supplemented with B12. The increases in milk and milk protein yields due to folic acid supplements did not seem to be dependent on the vitamin B12 supply. However, when vitamin B12 was given in combination with folic acid, utilization of the 2 vitamins seems to be increased, probably more so in extrahepatic tissues. Metabolic efficiency seems also to be improved as suggested by similar lactational performance and dry matter intake for cows fed supplementary folic acid but increased plasma glucose and decreased hepatic lipids in cows fed folic acid and vitamin B12 together.

Net flux of folates and vitamin B12 through the gastrointestinal tract and the liver of lactating dairy cows.

In study 1, four cows had a ruminal canula, a catheter in the right ruminal vein and an ultrasonic flow probe around the right ruminal artery; a catheter was placed in the auricular artery on experimental days. Blood samples were taken every 10 min from -20 to 60 min after ruminal infusion of 5.79 mmol pteroylmonoglutamic acid and cyanocobalamin. There was a net release of these vitamins across the rumen wall following the infusion (P=0.06). In studies 2 and 3, four cows had catheters in the portal, one hepatic and two mesenteric veins and one mesenteric artery. Plasma flow was determined using p-aminohippurate. In study 2, blood samples were taken before and every 30 min for 6 h after feeding 0 or 4 mg of pteroylmonoglutamic acid. Flow of folates through the portal-drained viscera (PDV) and the total splanchnic tissues (TSP) tended to increase with the ingestion of pteroylmonoglutamic acid (P=0.19). In study 3, blood samples were collected every 30 min for the first 3 h to calculate plasma flow and basal net fluxes of folates and vitamin B12. The cows were fed 2.6 g pteroylmonoglutamic acid and 500 mg cyanocobalamin; blood samples were taken every 2 h for 24 h. Vitamin supplements increased the net release of folates and vitamin B12 from PDV (P=0.04) and TSP (P=0.13). The present results demonstrate that, in dairy cows, at doses reported to improve animal performance, passage of pteroylmonoglutamic acid to the portal blood appears during the 6 h following its ingestion, whereas for cyanocobalamin, it is a slow process, not yet completed 24 h after its ingestion.

[The development of ethics. Identifying what training in medical ethics is needed by family physicians]. / La formation en éthique. Identification des besoins par des médecins enseignants en médecine familiale.

OBJECTIVE: To identify what training in medical ethics physician teachers need. DESIGN: Qualitative research study using a modified nominal group technique. SETTING: Family practice units affiliated with the Department of Family Medicine at Laval University in Quebec. PARTICIPANTS: Fifty-three physician teachers in six family practice units. METHOD: During seven meetings, the teachers shared information on clinical situations that had posed ethical problems. Data were analyzed using Strauss and Corbin's method. MAIN OUTCOME FINDINGS: The 277 clinical situations were classified under nine themes: ethics; confidentiality; consent, refusal of treatment, and the right to information; level of care and abstention from and cessation of treatment; relationships with pharmaceutical companies and the ethics of research; ethics of teaching; allocation of resources; influence of third parties; and euthanasia and assisted suicide. Learning objectives were developed. CONCLUSION: This research forms the basis of the ethics curriculum in the family medicine residency program at Laval University. It also offers a strategy for integrating ethics into daily teaching activities because the learning objectives derive directly from the concerns of the teaching faculty.