Chemical and nutritional characteristics of Cannabis sativa L. co-products

Cannabis sativa L. is an annual herbaceous plant. It was used for centuries to obtain different products. In the last century, hemp cultivation was forbidden due to the psychoactive effects of tetrahydrocannabinol acid (THCA). In the last years, new strains, characterized by high cannabidiolic acid (CBDA) and low THCA level, were developed renewing the interest in hemp cultivation to obtain food or to extract essential oils from flowers. All these processes produce many residues with different chemical–physical characteristics. In order to evaluate their potential use also in animal nutrition, some hemp co-products were evaluated. Two different co-products of seed processes (flour and oil) and two co-products obtained trimming the flowers, differing in granulometry were used. The samples were analysed for chemical composition and evaluated in vitro using the gas production technique with buffaloes' ruminal inoculum. All hemp co-products showed interesting nutritional characteristics, such as crude protein content always higher than 20% on a dry matter basis, and high neutral detergent fibre concentration partially lignified. The in vitro gas production parameters at 120 h of incubation showed quite low fermentability testified by the low organic matter degradability and cumulative gas volume (OMD from 28.09 to 45.64% and OMCV from 110 to 164 ml/g, respectively). Also, the methane produced after 24 h of incubation was particularly low (from 1.78 to 11.73 ml/g dOM). These results could be due to the high lipid and ash amounts or to the CBDA content that probably affected the CH₄ formation processes. According to preliminary results obtained by this study, it is possible to hypothesize that these co-products could be useful to mitigate the methane production into the rumen. Further studies are necessary in order to evaluate the correct inclusion into the diet for ruminants.     

Detecting and quantifying marijuana metabolites in serum and urine of 19 dogs affected by marijuana toxicity

Veterinarians diagnose marijuana toxicity based on clinical signs and history, or in conjunction with an over-the-counter (OTC) human urine drug screen. With the legalization of recreational marijuana use becoming more prevalent in the United States, a more accurate test to aid in the diagnosis of canine marijuana toxicity is needed. We collected urine and serum samples from 19 dogs with confirmed or suspected marijuana toxicosis from multiple veterinary hospitals and analyzed them with a novel UPLC-MS/MS method. Calibrations from 0.1 to 100 ng/mL and QC materials were prepared. Samples were extracted, purified, and eluted with solid-phase extraction. Urine samples were tested with an OTC human urine drug screen. The limit of detection (LOD) and lower limit of quantification (LLOQ) ranges for marijuana metabolites in serum were 0.05–0.25 ng/mL and 0.1–0.5 ng/mL, respectively. In urine, the LOD and LLOQ ranges for the metabolites were 0.05–0.1 ng/mL and 0.1–0.5 ng/mL, respectively. In serum, median and range of metabolite concentrations (ng/mL) detected included: THC, 65.0 (0.14–160); 11-OH-Δ⁹-THC, 4.78 (1.15–17.8); 11-nor-9-carboxy-Δ⁹-THC, 2.18 (0.71–7.79); CBD, 0.28 (0.11–82.5); and THC-glucuronide, 2.05 (0.72–18.3). In the 19 urine samples, metabolite: creatinine (ng: mg) values detected included: THC, 0.22 (0.05–0.74); 11-OH-Δ⁹-THC, 0; 11-nor-9-carboxy-Δ⁹-THC, 1.32 (0.16–11.2); CBD, 0.19 (0.12–0.26); THC-COOH-glucuronide, 0.08 (0.04–0.11); and THC-glucuronide, 0.98 (0.25–10.7). Twenty of 21 urine samples tested negative for THC on the urine drug screen. All 19 serum samples contained quantifiable concentrations of THC using our novel UPLC-MS/MS method. Utilizing a UPLC-MS/MS method can be a useful aid in the diagnosis of marijuana toxicosis in dogs, whereas using an OTC human urine drug test is not a useful test for confirming marijuana exposure in dogs because of the low concentration of THC-COOH in urine.     

Canine prostate specific esterase (CPSE) as an useful biomarker in preventive screening programme of canine prostate: CPSE threshold value assessment and its correlation with ultrasonographic prostatic abnormalities in asymptomatic dogs

Due to the increased attention that pet‐owners devote to their animals and to the improved veterinary care, investigations regarding methods to early detect prostatic disorders that might affect canine life quality have been performed. Canine prostate specific esterase (CPSE) concentration was reported to be higher in dogs suffering from prostatic diseases. This study aimed to estimate the CPSE threshold as a biomarker to early identify prostatic diseases in asymptomatic dogs. The ultrasonographic examination of the prostate was performed in 19 dogs (6–40 kg; 1–5 years) with no symptoms of prostatic diseases. Dogs were grouped according to the presence (Group A) or absence (Group B) of prostatic disorders at the ultrasound (altered appearance, the presence of cysts or irregular borders). For each dog, a venous blood sample was collected to measure serum CPSE and the ratio between calculated and normal expected prostatic volume was assessed for each dog. The CPSE data were statistically analysed (t test, p < .05), and the CPSE threshold in blood serum between groups was calculated by ROC. In 11 dogs, ultrasonography showed signs of prostatic abnormalities (Group A, 2–5 years), while no signs were detected in eight dogs (Group B, 1–3 years). The calculated/estimated volume ratio resulted greater than 1.5 in Group A dogs. The CPSE was statistically different between groups (p < .0001): higher in Group A (mean = 184.9, SD = 126 ng/ml) than in Group B (38.9 ± 22.1 ng/ml). The cut‐off CPSE threshold was 52.3 ng/ml (ROC, AUC = 0.974, SE 95.6%, SP 89.2%). This study suggests that CPSE serum concentration higher than 50 ng/ml in asymptomatic dogs is associated with ultrasonographic alterations and increased the prostatic size (volume by 1.5 times greater than the normal size). As the onset of prostatic disorders often remains asymptomatic, the rapid assessment of CPSE could be suitable for selecting preventively those animals that would require further accurate evaluation.     

Serum Levels of Cardiac Markers NT‐proANP and NT‐proBNP in Brachycephalic bitches at Different Gestational Stages

The aim of this study was to determine serum levels of natriuretic peptide precursors (NT‐proANP and NT‐proBNP) during pregnancy in brachycephalic bitches. Fifteen healthy multiparous bitches were selected for this prospective study. Serum levels of NT‐proANP and NT‐proBNP were measured during anoestrous and at 14, 35, 42, 49 and 56 days (2nd, 5th, 6th, 7th and 8th weeks) of pregnancy. Fourteen animals had normal gestations, and one bitch developed single foetus syndrome. The natriuretic peptide levels of this animal were not included in this study; however, it is important to report that its NT‐proANP levels were four times greater than those of normal patients. There was no significant difference (p = 0.072) in NT‐proBNP levels between anoestrous (0.20 ± 0.10 ng/ml) and the different pregnancy weeks (0.27 ± 0.12 ng/ml). There was a positive correlation (p < 0.0001) between NT‐proANP and gestational age, and the levels of this marker increased significantly (p < 0.0001) during the 6th (0.26 ± 0.06 ng/ml), 7th (0.28 ± 0.04 ng/ml) and 8th weeks (0.29 ± 0.05 ng/ml) when compared to anoestrous (0.18 ± 0.02 ng/ml). NT‐proANP serum levels are correlated with gestational development and may be indicative of cardiovascular adaptation in canine brachycephalic pregnancy.     

Faecal calcium excretion does not decrease during long‐term feeding of a low‐calcium diet in adult dogs

According to a previous meta‐analysis, adult dogs do not notably increase calcium absorption from the gastrointestinal tract when calcium intake is decreased. This results in a negative calcium balance even with a moderate calcium reduction. In this study we wanted to verify (i) whether a negative calcium balance occurs at a calcium intake equivalent to NRC (2006) (Nutrient requirements of dogs and cats, 2006, The National Academies Press, Washington, DC) minimal requirements, and if so (ii) whether the negative calcium balance will persist for up to 6 months on a low‐calcium diet. After a pre‐feeding period of at least 18 weeks with calcium intake slightly exceeding maintenance requirements (200 mg/kg body weight^0.75), 12 dogs (6 Beagles, 6 Foxhound crossbreds) were fed a low‐calcium diet for 28 weeks. One dog was removed from the trial for reasons unrelated to the study at week 23. Calcium intake amounted to 60 mg/kg body weight^0.75 corresponding to the minimal requirement for maintenance in dogs (NRC, 2006 (Nutrient requirements of dogs and cats, 2006, The National Academies Press, Washington, DC)). Digestion trials were carried out at week 7, 14, 21 and 28 of the low calcium feeding period. At these time points, and at week 18 of the pre‐trial, blood samples were taken and analysed for calcium, ionised calcium, phosphorus, parathyroid hormone, vitamin D, serum crosslaps and bone alkaline phosphatase. Apparent calcium digestibility was negative throughout the study, suggesting a negative calcium balance. There was no systematic decrease in faecal calcium excretion. Serum calcium, ionised calcium and phosphorus remained within the reference range. Serum crosslaps increased continuously from baseline to week 28 of trial, with averages increasing from 0.102 ng/ml to 0.279 ng/ml, suggesting osteoclastic activity, indicative of calcium mobilisation from the skeleton. The study supports the theory of a lack of adaptation of intestinal calcium absorption from diets with relatively low calcium content in dogs. This agrees with clinical findings in dogs eating low‐calcium diet.     

Plasma and platelet serotonin concentrations in healthy dogs and dogs with myxomatous mitral valve disease

ObjectivesSerotonin has been implicated in canine myxomatous mitral valve disease (MMVD); however, the sources of serotonin have not been fully elucidated. This study compared the concentration of serotonin in plasma and platelets of normal healthy small breed dogs with predisposition to MMVD and dogs with naturally occurring MMVD.Animals43 small-breed client-owned dogs with an approximate weight of <10 kg and age of 6 years or above were divided into 2 groups: a healthy control group (n = 20) and a group with echocardiographic evidence of MMVD (n = 23).Methods5 ml samples of blood were collected. Plasma and platelets were separated by centrifugation and assayed for serotonin measured by enzyme linked immunosorbent assay (ELISA).ResultsMedian plasma serotonin concentration was not significantly different (p = 0.3630) between normal healthy dogs (3.7 ng/ml) and dogs with MMVD (4.3 ng/ml). Males had higher plasma serotonin concentration than females (4.7 and 2.9 ng/ml respectively, p = 0.0043). Platelet serotonin concentration was not different between healthy dogs and dogs with MMVD (128.6 ng/10⁹ platelets and 176.6 ng/10⁹ platelets respectively, p = 0.4575). Age, echocardiographic indices and platelet count showed no correlation with plasma or platelet serotonin concentration.ConclusionsCirculating plasma serotonin is unlikely a major source of serotonin signaling in canine MMVD. Platelets could be a source of serotonin in canine MMVD through platelet adhesion to the mitral valve; however, the amount of serotonin stored in platelets of healthy dogs and dogs with MMVD is not different.     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Lack of glucuronidation products of trans‐resveratrol in plasma and urine of cats

Resveratrol has generated interest in cats due to reported health benefits. Cats have low activity of β‐glucuronidase, and we hypothesized they could not form two common resveratrol metabolites, resveratrol‐3‐O‐glucuronide and resveratrol‐4′‐O‐glucuronide. Resveratrol, 3 mg/cat/day, was given orally to intact male (n = 5) and female cats (n = 5) for 4 weeks. A control group (8 intact males) was used for comparison. Plasma and urine were collected weekly and analysed using high‐pressure liquid chromatography coupled with tandem mass spectrometry. Resveratrol and resveratrol‐3‐O‐sulphate, but no glucuronide metabolites, were detected in plasma and urine. Median (range 10–90th percentile) plasma resveratrol for control and treatment groups was 0.46 ng/ml (0.02–1.74 ng/ml) and 0.96 ng/ml (0.65–3.21 ng/ml). Median (range) plasma resveratrol‐3‐O‐sulphate for control and treatment groups was 6.32 ng/ml (2.55–10.29 ng/ml) and 11.45 ng/ml (1.47–53.29 ng/ml). Plasma resveratrol differed from control in week 4, while plasma resveratrol‐3‐O‐sulphate was different in all weeks (p < 0.05). Median (range) urine resveratrol for control and treatment groups was 0.28 ng/ml (0.05–1.59 ng/ml) and 19.98 ng/ml (8.44–87.54 ng/ml). Median (range) urine resveratrol‐3‐O‐sulphate for control and treatment groups was 26.71 ng/ml (10.50–75.58 ng/ml) and 108.69 ng/ml (11.83–231.05 ng/ml). All time points for urine resveratrol and resveratrol‐3‐O‐sulphate were significantly different from control (p < 0.05), except for weeks 1, 3 and 4 for resveratrol. The results support our hypothesis that cats are unlikely able to glucuronidate resveratrol, most likely due to a reduction in the activity of β‐glucuronidase.     

Thiamine deficiency affects glucose transport and β‐oxidation in rats

Thiamine is recognized as a cofactor for many enzymes involved in intermediary metabolism responsible for energy production. Animal model of thiamine deficiency (TD) included direct evaluation of glucose uptake by estimation of ³H‐deoxyglucose transport across red blood cells membranes and β‐oxidation of fatty acids in isolated leucocytes. Feeding of animals with the thiamine‐deficient diet (0.018 mg/kg diet) for 30 days resulted in disturbances in energy production. The thiamine intake was limited not only by vitamin B₁ deficiency in the diet, but also by time‐dependent drop of feed consumption by rats fed this diet. At the end of experiment, diet consumption in this group of rats was 52% lower than in the control group. This was accompanied by low glucose uptake by erythrocytes of rats suffering vitamin B₁ deficiency for longer time. At the end of experimental period, glucose uptake was over 2 times lower in TD erythrocytes than in control RBC. Such drop of energy production was not compensated by delivery of energy from fatty acid degradation. In leucocytes from TD rats, the β‐oxidation was also suppressed. Observed significant decrease of serum insulin from 2.25 ± 0.25 ng/ml (day 0) to 1.94 ± 0.17 ng/ml (day 30) might have significant impact on observed energy production disorders. The results from this study indicate that the thiamine deficiency significantly reduces feed intake and causes modest abnormalities in glucose and fatty acid utilization.     

Marijuana Poisoning

The plant Cannabis sativa has been used for centuries for the effects of its psychoactive resins. The term “marijuana” typically refers to tobacco-like preparations of the leaves and flowers. The plant contains more than 400 chemicals but the cannabinoid δ-9-tetrahydrocannabinol (THC) is the major psychoactive constituent. “Hashish” is the resin extracted from the tops of flowering plants and generally has a much higher THC concentration. Marijuana is the most commonly used illicit drug in the United States. Currently, several states have passed legislation to decriminalize possession of small amounts of marijuana for both medical and personal use and several other states have similar legislation under consideration. The most common form of marijuana use in humans is inhalation of the smoke of marijuana cigarettes, followed by ingestion. In animals, although secondhand smoke inhalation is possible, the most common source of exposure is through ingestion of the owner's marijuana supply. The minimum lethal oral dose for dogs for THC is more than 3 g/kg. Although the drug has a high margin of safety, deaths have been seen after ingestion of food products containing the more concentrated medical-grade THC butter. There are two specific cannabinoid receptors in humans and dogs, CB₁ (primarily in central nervous system) and CB₂ (peripheral tissues). In animals, following oral ingestion, clinical effects begin within 60 minutes. All of the neuropharmacologic mechanisms by which cannabinoids produce psychoactive effects have not been identified. However, CB₁ activity is believed to be responsible for the majority of cannabinoid clinical effects. Highly lipid soluble, THC is distributed in fat, liver, brain, and renal tissue. Fifteen percent of THC is excreted into the urine and the rest is eliminated in the feces through biliary excretion. Clinical signs of canine intoxication include depression, hypersalivation, mydriasis, hypermetria, vomiting, urinary incontinence, tremors, hypothermia, and bradycardia. Higher dosages may additionally cause nystagmus, agitation, tachypnea, tachycardia, ataxia, hyperexcitability, and seizures. Treatment of marijuana ingestion in animals is largely supportive. Vital signs including temperature and heart rate and rhythm must be continually monitored. Stomach content and urine can be tested for cannabinoids. Gas chromatography and mass spectrometry can be utilized for THC detection but usually may take several days and are not practical for initiation of therapy. Human urine drug-screening tests can be unreliable for confirmation of marijuana toxicosis in dogs owing to the interference of a large number of the metabolites in canine urine. False negatives may also arise if testing occurs too recently following THC ingestion. Thus, the use of human urine drug-screening tests in dogs remains controversial. No specific antidote presently exists for THC poisoning. Sedation with benzodiazepines may be necessary if dogs are severely agitated. Intravenous fluids may be employed to counter prolonged vomiting and to help control body temperature. Recently, the use of intralipid therapy to bind the highly lipophilic THC has been utilized to help reduce clinical signs. The majority of dogs experiencing intoxication after marijuana ingestion recover completely without sequellae. Differential diagnoses of canine THC toxicosis include human pharmaceuticals with central nervous system stimulatory effects, drugs with central nervous system depressant effects, macrolide parasiticides, xylitol, and hallucinogenic mushrooms.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Radioimmunoassay of parathyroid hormone in cats

A radioimmunoassay for measurement of midmolecule parathyroid hormone (PTH) concentration in serum from dogs was validated for use on serum from cats. The assay detected an increase in serum concentration of PTH after IV infusion of Na2 EDTA in healthy cats. Infusion of calcium chloride caused a decrease in measured PTH. Accuracy of the assay was demonstrated by quantitative recovery of a feline parathyroid gland extract added to pooled feline sera. Mean interassay and intra-assay coefficients of variation were 0.13 and 0.07, respectively. Sensitivity of the assay was 0.1 ng of PTH/ml. The median PTH concentration measured in 40 adult cats was 3.5 ng/ml, with a range of 1.16 to 11.0 ng/ml.     

Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia

Hip dysplasia (HD) is one of the most important bone and joint diseases in dogs. Making the radiographic diagnosis is sometime possible when the disease has markedly progressed. Chondroitin sulfate (CS) and hyaluronan (HA) are the most important cartilage biomolecules that are elevated in the serum taken from dogs with osteoarthritis. The serum CS and HA can be detected by an ELISA technique, with using monoclonal antibodies against CS epitope 3B3 and WF6 and the HA chain as the primary antibodies. The aim of this study was to compare the levels of serum CS (both epitopes) and HA in non-HD and HD dogs. All 123 dogs were categorized into 2 groups. The non-HD group was composed of 98 healthy dogs, while the HD group was comprised of 25 HD dogs. Blood samples were collected for analyzing the serum CS and HA levels with using the ELISA technique. The results showed that the average serum level of the CS epitope WF6 in the HD group (2,594 ± 3,036.10 ng/ml) was significantly higher than that in the non-HD group (465 ± 208.97 ng/ml) (p < 0.01) while the epitope 3B3 in the HD group (105 ± 100.05 ng/ml) was significantly lower than that in the non-HD group (136 ± 142.03 ng/ml) (p < 0.05). The amount of serum HA in the HD group (134.74 ± 59.71 ng/ml) was lower than that in the non HD group (245.45 ± 97.84 ng/ml) (p < 0.05). The results indicate that the serum CS and HA levels might be used as biomarkers for osteoarthritis in HD dogs.     

Effects of cannabidiol without delta-9-tetrahydrocannabinol on canine atopic dermatitis: A retrospective assessment of 8 cases

Objective We aimed to examine the effects of cannabidiol (CBD)-containing hemp oil without delta-9-tetrahydrocannabinol (THC) as a supplemental treatment for canine atopic dermatitis (CAD), as well as its adverse effects, and effects on concurrent drug use in dogs.Animal In this retrospective case series, 8 dogs with CAD were diagnosed by veterinary dermatologists certified by the Japanese Society of Veterinary Dermatology.Procedure The medical records of dogs supplemented with CBD-containing hemp oil were evaluated with respect to signalment, physical examination, plasma C-reactive protein concentrations, pharmacologic management, the CAD Extent and Severity Index (4th iteration), and the Pruritus Visual Analog Scale.Results Overall, CBD, used as a supplement in combination with other drugs, was well-tolerated over a wide dose range and decreased the occurrence of pruritus in dogs with CAD when ingested twice a day.Conclusion This study provides the first report of supplementation with CBD without THC that was effective in controlling pruritic behavior in dogs with CAD.Clinical relevance Further controlled studies are required to investigate the dose range, efficacy, and safety.     

Alpha‐Linolenic Acid Supplementation in Tris Extender Can Improve Frozen–Thawed Bull Semen Quality

The study was conducted to evaluate the effects of α ‐linolenic acid (ALA) on frozen–thawed quality and fatty acid composition of bull sperm. For that, twenty‐four ejaculates obtained from three bulls were diluted in a Tris extender containing 0 (control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was incubated at 37°C for 15 min, to allow absorption of ALA by sperm cell membrane. The sample was chilled for 2 h, packed into 0.25‐ml straws and frozen in liquid nitrogen for 24 h. Subsequently, straws were thawed and evaluated for total sperm motility (computer‐assisted semen analysis), membrane functional integrity (hypo‐osmotic swelling test), viability (eosin‐nigrosin), fatty acid composition (gas chromatography) and lipid peroxidation (thiobarbituric acid‐reactive substances (TBARS)). A higher (p < 0.05) percentage of total sperm motility was observed in ALA groups 5 ng/ml (47.74 ± 07) and 10 ng/ml (44.90 ± 0.7) in comparison with control (34.53 ± 3.0), 3 ng/ml (34.40 ± 2.6) and 15 ng/ml (34.60 ± 2.9). Still, the 5 ng/ml ALA group presented a higher (p < 0.05) percentage of viable sperms (74.13 ± 0.8) and sperms with intact membrane (74.46 ± 09) than all other experimental groups. ALA concentration and lipid peroxidation in post‐thawed sperm was higher in all treated groups when compared to the control group. As such, the addition of 5 ng/ml of ALA to Tris extender improved quality of frozen–thawed bull spermatozoa.     

The effects of ketoconazole and cimetidine on the pharmacokinetics of oral tramadol in greyhound dogs

Tramadol is administered to dogs for analgesia but has variability in its extent of absorption, which may hinder its efficacy. Additionally, the active opioid metabolite (M1) occurs in low concentrations. The purpose of this study was to determine if administration of oral tramadol with suspected metabolism inhibitors (ketoconazole, cimetidine) would lead to improved bioavailability of tramadol and M1. Six healthy Greyhounds were included. They were administered tramadol orally and intravenously, M1 intravenously, oral tramadol with oral ketoconazole and oral tramadol with oral cimetidine. Oral tramadol bioavailability was low (2.6%). Ketoconazole and cimetidine significantly increased tramadol bioavailability to 18.2% and 20.3%, respectively. The mean maximum plasma concentration of tramadol alone was 22.9 ng/ml, and increased to 109.9 and 143.2 ng/ml with ketoconazole and cimetidine, respectively. However, measured tramadol plasma concentrations were below the minimum concentration considered effective in humans (228 μg/ml). In all treatment groups, measured M1 concentrations (<7 μg/ml) were below concentrations associated with efficacy in humans. To conclude, tramadol and M1 concentrations were low and variable in dogs after oral dosing of tramadol, even in combination with cimetidine or ketoconazole, but effective concentrations in dogs have not been defined.     

Prolactin,Androstenedione and IGF1 Serum Concentrations During Induced Follicular Growth by eCG Administration in the Bitch

The oestrus cycle in the domestic bitch, a monoestrous species, differs considerably from that of other veterinary domestic animals species. In the bitch the combined use of eCG and hCG is effective to induce oestrus predictably and safely (Stornelli et al., Theriogenology, 78, 2012 and 1056). Although several studies were done to describe the hormonal changes during the canine oestrus cycle, to our knowledge none was done to describe the hormonal changes during induced follicular growth after the administration of eCG. The aim of this work was to study prolactin (PRL), insulin‐like growth factor (IGF1) and androstenedione (ANDR) serum concentrations during follicular growth induced by a single dose of eCG administered to late anoestrous bitches. PRL and ANDR concentrations were lower before than after eCG TRT (before eCG vs pro‐oestrus, oestrus and dioestrus; 4.3 ± 1.8 ng/ml vs 6.5 ± 1.6 ng/ml, p < 0.05; 0.08 ± 0.2 ng/ml vs 0.42 ± 0.16 ng/ml, p < 0.05). Conversely, IGF1 concentrations were similar before and after eCG TRT (286.0 ng/ml ±32.2, p > 0.53). Additionally, PRL concentrations were similar before oestrus compared to during oestrus and dioestrus (6.9 ± 1.7 ng/ml, p > 0.19). Furthermore, IGF1 concentrations were higher before and during oestrus compared to first day of dioestrus (286.1 ± 29.8vs 200.4 ± 29.2 ng/ml, p < 0.01). On the contrary, ANDR concentrations were lower before and during oestrus compared to first day of diestrum (0.35 ± 0.17 ng/ml and 0.38 ± 0.15 vs 0.68 ± 0.17 ng/ml, p < 0.05). These results show that treatment with a single injection of 50 IU/kg of eCG in late anoestrous bitches successfully induced changes in follicular growth which were paralleled with changes in PRL, IGF1 and ANDR serum concentration similar to those occurring during a normally occurring oestrous cycle. In addition, our results suggest that IGF1 in the bitch could play an important role in ovarian folliculogenesis.     

The effect of tributyrin supplementation to milk replacer on plasma glucagon‐like peptide 2 concentrations in pre‐weaning calves

The objective of this study was to evaluate the effect of tributyrin (TB) supplementation to milk replacer (MR) on performance, health, and blood concentrations of metabolite and glucagon‐like peptide (GLP‐2) in pre‐weaning calves. Twenty Holstein heifer calves were raised on an intensified nursing program using MR supplemented with either palm oil (CON) or TB (TB) at 0.3% (as fed basis) for 7 weeks starting 1 week after birth. Calves were fed a calf starter and kleingrass from the beginning of the study. Blood samples were obtained weekly to measure blood glucose, serum β‐hydroxybutyric acid (BHBA), insulin‐like growth factor 1 (IGF‐1), and plasma GLP‐2 concentrations. Starter DMI and metabolizable energy (ME) intake were lower in TB calves at 46, 47, from 49 to 55 days after birth compared with the CON calves. However, any growth parameters were not affected by TB treatment. Blood glucose, serum BHBA, and IGF‐1 concentrations were not affected by TB supplementation. On the other hand, mean plasma GLP‐2 concentration among whole experimental period was higher for TB (0.60 ng/ml) compared with CON (0.41 ng/ml). In conclusion, feeding MR supplemented with TB increases plasma GLP‐2 concentration, which might counterbalance the growth performance of TB calves despite the decreased ME intake.     

Role of histamine in heartworm extract-induced shock in dogs

OBJECTIVE: To determine whether heartworm (HW) extract-induced shock in dogs is consistent with anaphylactic shock by examining the role of histamine. ANIMALS: 6 mixed-breed dogs (3 without and 3 with HW infections) and 4 specific pathogen-free (SPF) Beagles. PROCEDURE: Four experiments were performed as follows: 1) 6 mixed-breed dogs were treated IV with 2 ml of HW extract, and plasma histamine concentrations were determined; 2) 4 SPF dogs were treated IV with 2 ml of HW extract and examined for shock; 3) sera from 6 dogs of experiment 1 and from 4 SPF dogs of experiment 2 that were obtained before HW extract treatment were tested for heterologous passive cutaneous anaphylaxis (PCA), using rabbits during a sensitization period of 48 to 72 hours; and 4) mast cell degranulation by HW extract was tested, using rat mesentery and canine cultured mast cells. RESULTS: Experiment 1: 6 dogs developed shock, and plasma histamine concentrations increased significantly from 0.3 +/- 0.2 (mean +/- SD) ng/ml before HW extract treatment to 44.6 +/- 68.9 ng/ml at the onset of shock; experiment 2: all SPF dogs developed shock and had an increase in plasma histamine concentrations; experiment 3: sera from mixed-breed dogs without HW infection and from SPF dogs had negative PCA reactions; experiment 4: HW extract degranulated rat mesentery mast cells and released histamine directly from canine mast cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that an unknown mast cell-degranulating substances contained in HW extract may degranulate mast cells directly, consequently releasing histamine that may participate in the onset of shock in HW extract-induced shock in dogs.     

Serum hydrocortisone (cortisol) values in normal and adrenopathic dogs as determined by radioimmunoassay

Using a specific radioimmunoassay, serum hydrocortisone values were measured in dogs. Between 9:00 AM and 10:00 AM, the base-line hydrocortisone value for 56 clinically normal dogs ranged from 6.0 to 28.5 ng/ml, with a mean value of 17.8 +/- 1.32 ng/ml (mean +/- SEM). Marked differences due to age, sex, body weight, or breed were not observed in the hydrocortisone values. In 11 dogs with definitive hyperadrenocorticism, serum hydrocortisone values were from 32 to 148 ng/ml. In 2 dogs with hyperadrenocorticism, the values were 4.5 and 3.1 ng/ml. The estimation of serum hydrocortisone values by radioimmunoassay is simple and precise, and can be utilized to aid in the diagnosis of adrenopathy in dogs.