Metabolic Changes Associated with Different Levels of Energy Deficits in Mediterranean Buffaloes during the Early Lactation Stage: Type and Role of the Main Lipid Fractions Involved.

Cell function and energy redistribution are influenced by lipid classes (phospholipids (PLs), free fatty acids (FFAs), triglycerides (TGs), and cholesterol esters (CEs)). The aim of this study was to investigate metabolic alterations that are related to changes in lipid classes according to different levels of energy deficits in early lactating Mediterranean buffaloes (MBs). Sixty-three MBs were enrolled at the beginning of lactation using an observational study with a cross-sectional experimental design. Serum ß-hydroxybutyrate (BHB) levels were used to group the animals into a healthy group (Group H; n = 38; BHB < 0.70 mmol/L) and hyperketonemia risk group (Group K; n = 25; BHB ≥ 0.70 mmol/L). Statistical analysis was performed using a linear model that included the effect of the group and body condition score to assess differences in fatty acid (FA) concentrations. A total of 40 plasma FAs were assessed in each lipid class. Among the FAs, eight PLs, seven FFAs, four TGs, and four CEs increased according to BHB levels, while three FFAs, three TGs, and one CE decreased. The changes among lipid class profiles suggested the influence of inflammatory response, liver metabolism, and the state of body lipid reserves. In addition, the possible similarities of buffaloes at risk of hyperketonemia with ketotic cows suggest the necessity of further investigations in these ruminants.

Changes in plasma fatty acids profile in hyperketonemic ewes during early lactation: a preliminary study.

The transition from late pregnancy to early lactation is characterized by marked changes in energy balance of dairy ruminants. The mobilization of adipose tissue led to an increase in plasma non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). The aim of this study was to analyze the total plasma fatty acids of healthy and hyperketonemic dairy ewes in early lactation through gas chromatography (GC) to evaluate metabolic alterations. An observational study was used with a cross-sectional experimental design. Forty-six Sarda dairy ewes were enrolled in the immediate post-partum (7 ± 3 days in milk) and divided into two groups according to serum BHB concentration: non-hyperketonemic group (n = 28; BHB < 0.86 mmol/L) and hyperketonemic group (n = 18; BHB ≥ 0.86 mmol/L). A two-way ANOVA included the effect of group and parity was used to evaluate differences in fatty acids (FA) concentrations. A total of 34 plasma FA was assessed using GC. 12 out of 34 FA showed a significant different between groups and 3 out of 34 were tended to significance. Only NEFA concentration and stearic acid were influenced by parity. The results may suggest possible links with lipid metabolism, inflammatory and immune responses in hyperketonemic group. In conclusion, GC represents a useful tool in the study of hyperketonemia and primiparous dairy ewes might show a greater risk to develop this condition.

Analytical and clinical performance of a fluorescence enzyme immunoassay for progesterone and determination of ovulation day in bitches.

We evaluated the performance of a third-generation fluorescence enzyme immunoassay kit (FEIA; Tosoh Bioscience) for progesterone (P4) measurement in canine serum to identify the day of luteinizing hormone (LH) peak and ovulation in bitches. We conducted P4 assays on 54 serum samples using a FEIA and a chemiluminescence immunoassay (CLIA; Siemens). For the FEIA kit, the linearity test, recovery test, inter- and intra-assay CVs, and total error observed (TEo) were calculated. Serum samples from 28 bitches were used to evaluate the association of P4 concentration with the day of LH peak and the day of ovulation based on P4 thresholds (P4 ≥ 2 ng/mL, and doubling at 4-8 ng/mL in the following 48 h), and with pregnancy length. Linearity was 75-97% and 86-94% for high (37.0 ng/mL) and medium (3.8 ng/mL) pool serum samples, respectively. Recovery was 86.4-119%. Intra-assay CVs were 2.6%, 3.3%, and 5.2% for low (0.23 ng/mL), medium (6.24 ng/mL), and high (38.3 ng/mL) pool concentrations. Inter-assay CVs were 2.22% and 2.53% for P4 concentrations of 2.70 ng/mL and 8.2 ng/mL, respectively. TEo was 8.72% and 22.7% for P4 concentrations of 2 and 8 ng/mL, respectively. The mean pregnancy length from the day of LH peak and ovulation were 64.6 ± 1.7 and 63.3 ± 1.1 d, respectively. The third-generation FEIA kit that we tested was highly reliable.

Differences of the Plasma Total Lipid Fraction from Pre-Foaling to Post-Foaling Period in Donkeys.

The period from late gestation to early lactation is characterized by changes in energy metabolism. The aim of the current study was to analyze the plasma total lipid fraction using gas chromatography (GC) analysis, in order to highlight metabolic changes from the pre-foaling to post-foaling periods. Eleven pluriparous dairy jennies (mean age of 11.88 ± 3.79 years) belonging to the Amiata donkey breed were enrolled. Blood sampling was performed at 15 days before foaling (T0), and 15 (T1), 30 (T2), 60 (T3), and 90 (T4) days after foaling, for biochemical and GC analysis. A total of 37 fatty acids were identified in plasma samples: 4 medium chain (MCFA), 24 long chain (LCFA), and 9 very-long chain (VLCFA) fatty acids. Among them, 20 fatty acids changed significantly, and two fatty acid showed a trend toward significance. Furthermore, the LCFA, saturated, unsaturated, monounsaturated, and polyunsaturated ω-3 fatty acids changed significantly during the study period. The main alterations were between T0 and the other time points and appeared to be related to lipid metabolism, cellular structure and function, and inflammatory and immune responses. Our findings reveal greater energy requirements at the end of gestation compared to early lactation in donkeys.

Milk Fatty Acids Composition Changes According to ß-Hydroxybutyrate Concentrations in Ewes during Early Lactation.

Ketosis is a metabolic disease of pregnant and lactating ewes linked to a negative energy balance which can cause different economic losses. The aims of this study were to understand the metabolic status of the early-lactating ewes and to identify biomarkers for early diagnosis of subclinical ketosis. Forty-six Sarda ewes were selected in the immediate post-partum for the collection of the biological samples. A blood sample from the jugular vein was used to determine ß-Hydroxybutyrate (BHB) concentrations. Animals were divided into two groups: BHB 0 or healthy group (n = 28) with BHB concentration < 0.86 mmol/L; and BHB 1 or subclinical ketosis (n = 18) with a BHB concentration ≥ 0.86 mmol/L. Ten mL of pool milk were collected at the morning milking for the analyses. The concentration of 34 milk fatty acids was evaluated using gas chromatography. Two biochemical parameters and 11 milk fatty acids of the total lipid fraction presented a p-value ≤ 0.05. The study revealed different relationships with tricarboxylic acid cycle, blood flows, immune and nervous systems, cell functions, inflammatory response, and oxidative stress status. Eight parameters were significant for the receiver operating characteristic (ROC) analysis with an area under the curve greater than 0.70.

Autologous Platelet-Rich Plasma Enhances the Healing of Large Cutaneous Wounds in Dogs.

Platelet-rich plasma (PRP) is known to play a crucial role in skin wound healing, in both Human and Veterinary Medicine. Remarkably, until now, no studies have reported PRP treatment in subacute full-thickness skin wounds of the dog. The aim of this study was to evaluate the effects of two consecutive applications of autologous PRP, with the second application after 15 days, in 6 dogs showing large subacute skin wounds. The percentage of contraction, re-epithelialization and healing in all treated patients indicated that no complications or side effects, associated with consecutive PRP treatments, occurred in any patient and all wounds achieved complete closure and re-epithelialization. Our results suggest a positive effect of repeated autologous topical PRP treatments in large cutaneous subacute wounds of different etiology. Therefore, this PRP treatment could represent a simple, cost-effective, and valid alternative to promote healing processes in subacute large wounds cases in dogs.

Evaluation of an automated immunoturbidimetric assay for detecting canine C-reactive protein.

C-reactive protein (CRP) is a major acute-phase protein, and it is produced by the liver in response to a pro-inflammatory stimulus. Given that human and canine CRP have a similar molecular structure, the assays used for human CRP detection have been used to measure CRP concentrations in dogs. We evaluated the use of a human CRP assay (Biotecnica CRP assay) and validated its application in dogs. We analyzed 91 canine serum samples with a fully automated analyzer. Our validation was based on the evaluation of imprecision, limits of linearity, limits of quantification, and an evaluation of interferences. The new assay was also compared with the Randox CRP assay, a validated assay for the measurement of CRP. Intra- and inter-assay repeatability were <8% and <11%, respectively. The tested assay proportionally measured canine CRP in an analytical range up to 60 mg/L; however, hemoglobin, triglycerides, and bilirubin interfered with the determination. Good agreement, with the presence of proportional systematic bias, was observed between Biotecnica and Randox assays. The Biotecnica CRP assay provides reliable measurement of CRP in canine serum, provided that samples are free of interferents.

Changes of Plasma Fatty Acids in Four Lipid Classes to Understand Energy Metabolism at Different Levels of Non-Esterified Fatty Acid (NEFA) in Dairy Cows.

The transition period is a central moment in dairy cows breeding because metabolic disorders may occur in relation to a dramatic increase in energy demand. This research aimed to identify new biomarkers for the diagnosis of hyperketonemia in bovine in early lactation phase with different value of plasmatic non-esterified fatty acid (NEFA). The profile of plasma fatty acids (FAs) divided into four lipid classes was evaluated using thin layer chromatography and gas chromatographic techniques (TLC-GC). A group of 60 multiparous Holstein-Friesian dairy cows were recruited in the present study. Blood samples were collected from the coccygeal vein and NEFA and the ß-hydroxybutyrate (BHB) were evaluated. All animals were divided in 2 groups based on NEFA, NEFA0 group had as mean value 0.24 ± 0.12 mEq/L and NEFA1 group had as mean value 0.87 ± 0.23 mEq/L. Plasma FA concentrations were analyzed separately in free fatty acids, cholesterol esters, phospholipids and triglycerides. Six FAs demonstrated a predictive value in the hyperketonemic dairy cows. In the free fatty acid class, the predictive FAs were C14:0 (AUC = 0.77), C18:1 ω 9 (AUC = 0.72), C18:1 ω 7 (AUC = 0.70) and C18:3 ω 3 (AUC = 0.68). In the phospholipids class the predictive parameters were C12:0 (AUC = 0.78) and C8:0 (AUC = 0.73). In cholesterol, esters and triglycerides lipidic classes no FA had a predictive function.

Changes of milk fatty acid composition in four lipid classes as biomarkers for the diagnosis of bovine ketosis using bioanalytical Thin Layer Chromatography and Gas Chromatographic techniques (TLC-GC).

The aim of this study was to extend the limited research available on the association between the concentration of milk fatty acids and the elevated plasmatic value of ß-hydroxybutyrate (BHB) in early lactation of dairy cows. Fifty-four Holstein Friesian dairy cows were enrolled in the study. All animals were classified on the basis of their blood BHB concentration: BHB ≥ 1.0 mmol/L (BHB-1, sick group) and BHB ≤ 0.99 mmol/L (BHB-0, healthy group). Using Thin Layer Chromatography (TLC), four lipid classes (cholesterol esters -CE-, phospholipids -PL-, free fatty acids -FFA- and triacylglycerols -TAG-) were separated, and then the fatty acid (FA) composition was determined by High Resolution Gas Chromatography coupled with Flame Ionization Detector/Mass Spectrometer (HRGC-FID/MS). The FA profiles were used to investigate the diagnostic potential value of milk fatty acids for the correct classification of cows with BHB concentration above the established threshold (BHB < 1.0 mmol/L). Boruta Test and Receiver Operating Characteristic curves (ROC) were used to identify which FA and their thresholds of concentration could be used when animals presented hyperketonemia. The research has identified fourteen FA, belonging to CE, FFA, and TAG classes, useful for an association with BHB-1. These compounds, with predictive value for the development of hyperketonemia, could be considered valuable biomarkers. Further studies on a wider sampling, based on clinical and therapeutic approach, will be necessary to confirm, by bioanalytical chromatographic approaches, if these predictive FA will change between healthy and sick animals. New approaches in relation on the administration of different diets or supplements, and administration of drugs might improve the prevention of hyperketonemia.

Identification of Plasma Fatty Acids in Four Lipid Classes to Understand Energy Metabolism at Different Levels of Ketonemia in Dairy Cows Using Thin Layer Chromatography and Gas Chromatographic Techniques (TLC-GC).

Excessive mobilization of adipose tissue in high milk producing dairy cows predisposes to metabolic diseases. The aim of this research was to identify the plasma fatty acids in four lipid classes as biomarkers for the diagnosis of hyperketonemia in bovines using thin layer chromatography and gas chromatographic techniques (TLC-GC). Sixty multiparous Holstein-Friesian dairy cows were enrolled in the study. Blood samples from the coccygeal vein were collected and ß-hydroxybutyrate (BHB) was evaluated. All animals were divided into three groups on the basis of ketonemia: BHB < 0.50 mmol/L, 0.50 < BHB < 1.0 mmol/L, and BHB > 1.0 mmol/L. Plasma fatty acid concentrations were evaluated in four lipid classes: Free Fatty Acids (FFA), Triglycerides (TG), Cholesterol Esters (CE) And Phospholipids (PL). The concentration of fatty acids was analyzed using TLC-GC. The results showed the following significance in the lipid classes: 19 fatty acids were significant (p < 0.053) in FFA, nine fatty acids were significant (p < 0.050) in TG, eight fatty acids were significant (p < 0.050) in CE and three fatty acids were significant (p < 0.049) in PL. Eleven parameters were considered as predictive fatty acids related to animals in hyperketonemia. The FFA increased simultaneously with blood BHB levels, although the identified predictive fatty acids related to the TG and CE lipid classes decreased, meanwhile the BHB values increased. In the PL lipid class, no fatty acids were predictive.

Serum haptoglobin and protein electrophoretic fraction modifications in buffaloes (Bubalus bubalis) around calving and during early lactation.

Serum protein distribution and concentration can be affected by different physiological and pathological conditions. The aim of this study was to evaluate the changes in the concentration of serum protein fractions and haptoglobin in clinically healthy dairy buffaloes during late pregnancy and early lactation. Blood and milk samples were collected from 30 buffaloes at around 7 d before expected calving (blood only) and 7, 30 and 50 d after calving. In serum samples, the total protein, haptoglobin, albumin, α1-, α2-, ß1-, ß2-, γ-globulins, and albumin/globulin ratio (A/G) values were evaluated. In milk, fat%, protein%, lactose%, somatic cell score (SCS) were assessed, along with milk yield (MY) and daily milk production (DMP). The peripartum period significantly influenced (P < 0.005) total protein, albumin, haptoglobin, α2-, ß2- and γ-globulins (P < 0.005). Milk yield, DMP and fat% changed significantly throughout the monitoring period (P < 0.005). Milk yield and DMP were positively correlated with total protein, albumin, ß2-globulins and A/G ratio, and negatively correlated with haptoglobin and α2-globulins. These results provide new knowledge about the serum protein electrophoretic pattern in Italian Mediterranean Buffaloes during the last phase of pregnancy and early stages of lactation.

Length of efficacy and effect of implant location in adult tom cats treated with a 9.4 mg deslorelin subcutaneous implant.

OBJECTIVES: The objective of this study was to assess duration of efficacy, side effects and return to fertility following use of the 9.4 mg deslorelin implant (Suprelorin 12; Virbac) in cats, and test whether efficacy and duration of action are influenced by implantation site (interscapular vs periumbilical). METHODS: Sixteen healthy adult tom cats were checked with (1) reproductive examination, (2) gonadotropin-releasing hormone stimulation test and (3) semen collection until achievement of sterility, then with (1) and (2) only at 2, 4, 6 and 12 months, and every 6 months thereafter until treatment effect disappeared. RESULTS: Serum testosterone reached basal levels by 7 days post-treatment. Semen quality improved initially then started to worsen by 1 month post-treatment and after 70 days post-treatment all cats were sterile. Early in the third month post-treatment there was a significant decrease in testicular volume and penile spikes. Testicular histology was normal upon neutering performed after resumption of fertility. No injection site lesions or treatment-related side effects were observed. There was no difference between periumbilical and interscapular placement for all criteria, but there was a trend for the decrease in testicular volume to last longer and for the regression of penile spikes to start sooner after interscapular administration. One of 16 cats did not respond to treatment. Six cats were lost at variable times during the study while fully responding to treatment. In the cats that completed the study, normal fertility was regained after 805 days, on average, but with a variable duration of effect from 750-850 days. CONCLUSIONS AND RELEVANCE: Treatment with a 9.4 mg deslorelin implant in male cats was effective for a period of 750-850 days, which is 1.5-2 times longer than the effect of the 4.7 mg deslorelin implant. Fertility (based on serum testosterone production and the presence of penile spikes) was regained at the end of the study. Placing implants in the intrascapular vs periumbilical location did not affect duration of suppression of testosterone production. The interscapular location may be characterised by a better efficacy, although further studies are needed to clarify this issue.

Metabolic and hormonal adaptation in Bubalus bubalis around calving and early lactation.

Pregnancy and lactation are physiological periods that result in an increased metabolic demand that, if not satisfied, could provoke a threat to homeostasis. In this study changes in the values of Non-Esterified Fatty Acids (NEFA) and ß-hydroxybutyrate (BHB), glucose, insulin, thyroid hormones, milk composition and yield were investigated in buffaloes during the late pregnancy and early lactation. From a total of 50 buffaloes, blood samples were collected -7±5 days before expected calving; +7±5; +30±5 and +50±5 days post-partum; milk samples were collected at the same post-partum time points. On serum samples, the values of Non-Esterified Fatty Acids (NEFA), ß-hydroxybutyrate (BHB), glucose, insulin, Triiodothyronine (T3), Thyroxine (T4) and Thyroid-stimulating hormone (TSH) were evaluated. On milk, fat %, protein %, lactose %, somatic cells score (SCS), milk yield and daily milk production (DMP) were assessed. Peripartum period significantly influenced all studied parameters (P<0.05). Milk constituents and productivity statistically changed throughout monitoring period (P<0.005). Milk yield resulted positively correlated with insulin and TSH values, negatively correlated with NEFA and BHB. Insulin was negatively correlated with lactose % and positively correlated with SCS. The obtained results showed that the peripartum period and lactation are accompanied by marked changes in some biochemical variables and in the thyroid hormones values in Italian Mediterranean Buffaloes. Moreover, the relationship found between TSH values and milk yield seems to suggest a possible role of thyroid gland on the maintenance of lactogenesis. This study underlines the importance of monitoring the hormonal status of buffalo during the transition period in order to understand when adjustments of regulatory mechanisms break through physiological limits predisposing the buffalo to metabolic problems.

Salivary pH, calcium, phosphorus and selected enzymes in healthy dogs: a pilot study.

BACKGROUND: Saliva in dogs, as in humans, is a complex fluid secreted by different salivary glands in the oral cavity to protect the oral mucosa and teeth. The use of saliva as a substitute for blood in diagnosing and prognosticating disease in humans is widely accepted. Salivary biochemistry has also been used as a marker for periodontal disease in humans. No studies have as yet investigated the relation between salivary biochemistry and periodontal disease in dogs, however; neither has the salivary composition of healthy dogs with no oral disease been assessed. The purpose of this study was to obtain an overview on pH distribution and a set of salivary biochemical analytes (calcium, phosphorus, lactate dehydrogenase, lysozyme and amylase) commonly related to oral health in humans in a subset population of healthy young dogs with no periodontal disease or previous oral disease. Data were analyzed to gather salivary reference ranges for pH and each parameter and to assess a possible correlation between salivary and serum analytes. RESULTS: Twenty-nine adult client-owned dogs were recruited for the study. Lactate dehydrogenase and lysozyme showed higher concentrations in saliva than in serum, whereas amylase showed the contrary. Salivary biochemistry values did not differ between males and females or between non-neutered and neutered individuals. No significant correlations between salivary and serum calcium, phosphorus, lactate dehydrogenase, amylase and lysozyme were identified in this study. Data allowed intervals for the salivary pH and other analytes investigated to be obtained from healthy dogs with healthy oral conditions. CONCLUSIONS: These preliminary data can contribute to enlarge our understanding of the functional role of saliva and its relation to oral health in dogs.

Surfactant-associated protein B kinetics in vivo in newborn infants by stable isotopes.

Surfactant-associated protein B (SP-B) is critical to the biophysical function of pulmonary surfactant. No information is available on SP-B synthesis and kinetics in humans. We administered a 24-h i.v. infusion of 13C-valine as metabolic precursor of SP-B to six newborn infants (weight 3.5+/-0.5 kg; age 12 d, range 1-43 d). Three of the study infants also received i.v. 2H-palmitate to label surfactant disaturated phosphatidylcholine (DSPC). SP-B and DSPC were isolated from tracheal aspirates, and their respective 13C and 2H enrichments were measured by gas chromatography-mass spectrometry. SP-B kinetics was measured successfully in all six infants. SP-B median (range) fractional synthesis rate was 30% per day (20-78% per day), secretion time was 4.5 h (1-9 h), time to peak was 24 h (12-36 h), and half-life was 21 h (8-35 h). The ascending part of the SP-B kinetic curve was similar to the DSPC curve, suggesting similar secretion pathways. SP-B half-life seemed to be shorter than DSPC half-life. These results agree with existing animal data. We conclude that the measurement of SP-B kinetics is feasible in vivo in humans by stable isotope technology.

Surfactant kinetics in preterm infants on mechanical ventilation who did and did not develop bronchopulmonary dysplasia.

OBJECTIVE: To characterize surfactant kinetics in vivo in two groups of premature infants on different levels of mechanical ventilation and at different risk of developing bronchopulmonary dysplasia. DESIGN: Controlled observational study in two independent groups of infants. SETTING: Neonatal intensive care unit. PATIENTS: Thirteen preterm infants (26 +/- 0.5 wks, birth weight 801 +/- 64 g) on high ventilatory setting and who finally all developed bronchopulmonary dysplasia (MechVentBPD), and eight (26 +/- 0.5 wks, birth weight 887 +/- 103 g) who had minimal or no lung disease and of whom none developed bronchopulmonary dysplasia (MechVentNoBPD). MEASUREMENTS AND MAIN RESULTS: Endotracheal 13C-labeled dipalmitoyl-phosphatidylcholine was administered and subsequent measurements of the 13C enrichment of surfactant-disaturated phosphatidylcholine (DSPC) from serial tracheal aspirates were made by gas chromatography-mass spectrometry. We calculated disaturated phosphatidylcholine pharmacokinetic variables in terms of half-life and apparent pool size from the enrichment decay curves over time. DSPC concentration from tracheal aspirates was expressed as milligrams/milliliter epithelial lining fluid (ELF-DSPC). Data are presented as mean +/- se. In MechVentBPD infants vs. MechVentNoBPD, ELF-DSPC was much reduced, 2.9 +/- 0.6 vs. 9.4 +/- 3.0 mg/mL ELF (p =.03), half-life was shorter, 19.4 +/- 2.8 vs. 42.5 +/- 6.3 hrs (p =.002), and apparent pool size larger, 136 +/- 21 vs. 65.8 +/- 16.0 mg/kg (p =.057). In MechVentBPD, apparent DSPC pool size positively correlated with mean airway pressure x Fio(2) and inversely correlated with ELF-DSPC. ELF-DSPC was inversely correlated with mean airway pressure x Fio(2). No significant correlations were found in the MechVentNoBPD group. CONCLUSIONS: MechVentBPD infants showed profound alteration of surfactant kinetics compared with preterm infants with minimal lung disease, and these alterations were correlated with severity of ventilatory support.