Serum metabolic profiling in pregnant Holstein cows 3 weeks prior to parturition using two-dimensional gas chromatography time-of-flight mass spectrometry.

This preliminary study explored potential serum biomarkers for predicting the onset of milk fever (MF), a bovine parturient disease with hypocalcemia. We conducted two-dimensional gas chromatography mass spectrometry-based metabolomics in 8 and 17 pregnant Holstein cows that did and did not develop MF 3 weeks later, respectively. In principal component analysis (PCA) applied to a dataset containing 1498 metabolites, serum metabolites exhibited highly similar chemical profiles between cows with and without MF. PCA with a limited dataset of metabolites containing fatty acids, which had significantly different values between the groups and/or correlation coefficients of >0.5 for the serum calcium concentration, distinguished the two groups. These suggest the possibility of developing serum biomarkers for predicting bovine MF.

Sprayed Urine Emits a Pungent Odor due to its Increased Adhesion to Vertical Objects via Urinary Proteins Rather Than to Changes in its Volatile Chemical Profile in Domestic Cats.

Spraying urine on vertical objects by raising the tail is a commonly observed functional behavior for chemical communication in Felidae species, including domestic cats (Felis silvestris catus). The sprayed urine is recognized as a chemical signal for territorial ownership of their habitats. Previous studies reported that sprayed urine emits a more pungent odor than urine excreted from a squatting position. However, little is known about how sprayed urine acts as a strong scent mark in the environment. Here, we showed that sprayed urine originates only from bladder urine without any secretions, such as anal sac secretions, but it can effectively emit volatile organic compounds (VOCs) when smeared on vertical objects due to its strong adhesion. Chemical profiles of VOCs and odor qualities were similar between fresh sprayed urine and bladder urine sampled immediately after spraying from the same individuals. Meanwhile, feline-specific proteinuria arising from excretion of a carboxylesterase that produces a precursor of cat-specific odorants resulted in reduced surface tension of the urine and increased adhesion to vertical surfaces, which kept sprayed urine on the surfaces and led to the emission of large amounts of VOCs. In conclusion, proteinuria contributes to the emission of a strong odor through its enhanced adhesion to vertical objects without other secretions containing malodorous substances. These findings improve our understanding of the mechanism of scent marking via the spraying of urine for chemical communication in cats.

Assessing the safety and suitability of using silver vine as an olfactory enrichment for cats.

Olfactory enrichment is a strategy that can improve welfare among animals managed in captivity, such as household domestic cats. Catnip (Nepeta cataria) and silver vine (Actinidia polygama) that produce iridoids are used as olfactory enrichments for cats, but little is known about the safety or the best plant resources to use that maximize positive cat responses. We report physiological effects and suitable harvest and drying methods for using silver vine as olfactory enrichment. Continuous exposure of cats to silver vine showed no hallmarks of addictive behavior, while blood indicators of stress and hepatic or renal injury showed no increase in cats stimulated with it. Drying the leaves changed the iridoid profile, enhancing the feline response. In conclusion, dried silver vine leaves are the most suitable resource for developing olfactory enrichment that maximizes feline typical response, which would not result in dependence, stress, or toxicity to the liver or kidneys in cats.

Domestic cat damage to plant leaves containing iridoids enhances chemical repellency to pests.

Catnip (Nepeta cataria) and silver vine (Actinidia polygama) produce iridoids with arthropod-repellent effects. Cats rub and roll against these plants, transferring iridoids to their fur that repels mosquitoes. Cats also lick and chew plant leaves during this response, although the benefit of this additional behavior has remained unknown. Here, we show that feline leaf damage substantially increases iridoid emission from both plants while also diversifying iridoids in silver vine. Cats show an equivalent duration of response to the complex cocktail of iridoids in damaged silver vine and to the much higher level of a single iridoid produced by damaged catnip. The more complex iridoid cocktail produced when silver vine is licked and chewed by cats increases mosquito repellency at low concentration. In conclusion, feline leaf damage contributes by releasing more mosquito-repellent iridoids. Feline olfactory and behavioral sensitivity is fine-tuned to plant-specific iridoid production for maximizing the mosquito repellency gained.

The characteristic response of domestic cats to plant iridoids allows them to gain chemical defense against mosquitoes.

Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma ß-endorphin levels in cats, while pharmacological inhibition of µ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the µ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

Detection of urinary luteinizing hormone in Japanese black cows after administration of gonadotropin-releasing hormone.

The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.

Renal expression and urinary excretion of liver-type fatty acid-binding protein in cats with renal disease.

BACKGROUND: Liver-type fatty acid-binding protein (L-FABP) is a biomarker for early detection of renal disease in humans. Liver-type fatty acid-binding protein is cytotoxic oxidation products secreted from proximal tubules under ischemia and oxidative stress. OBJECTIVE: To examine renal expression and quantify urinary excretion of L-FABP in catswith renal disease. ANIMALS: One hundred and thirty-four client-owned cats including 34 cats with serum creatinine (sCre) values >1.6 mg/dL and 10 other cats that died in clinics. METHODS: Tissue expressions of L-FABP were examined by reverse transcription polymerase chain reaction and Western blotting. Urinary L-FABP (uL-FABP) and serum L-FABP (sL-FABP) levels were determined by enzyme-linked immunosorbent assay. Anti-liver-type fatty acid-binding protein antibody immunostained renal sections. RESULTS: Feline kidneys express L-FABP. Strong L-FABP signals were observed in the lumens of proximal tubular cells in 5 cats with high uL-FABP excretion, but not in 5 cats with low uL-FABP excretion. In 9 normal cats, uL-FABP index was <1.2 µg/g urinary creatinine (uCre). High uL-FABP indexes (>10.0 µg/g uCre) were detected in 7 of 100 cats with low sCre (<1.6 mg/dL) and 18 of 44 cats with high sCre (>1.6 mg/dL). There was a weak correlation between L-FABP index and sCre, serum symmetric dimethylarginine (SDMA), or blood urea nitrogen (BUN), and these correlation coefficients were increased by analyzing only data of cats with sCre >1.6 mg/dL. There was a weak correlation between u L-FABP index and sL-FABP in all tested cats, but not in cats with high sCre. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrates correlations between L-FABP and current renal biomarkers for chronic kidney disease in cats, such as sCre and SDMA. Liver-type fatty acid-binding protein may be a potential biomarker to predict early pathophysiological events in feline kidneys.

LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples.

The histidine-containing imidazole dipeptide carnosine and its methylated analogs anserine and balenine are present at high concentrations in vertebrate tissues. Although the physiological functions of the imidazole dipeptides have not been elucidated yet, it has been suggested that they play significant biological roles in animals. Despite increasing interest, few studies have challenged the quantifications of carnosine, anserine, and balenine by a single HPLC run because they have similar retention times. In this study, we developed a method to quantify these imidazole dipeptides in meat samples using an LC-ESI-MS/MS triple-quadrupole mass spectrometer. We improved the liquid chromatographic separation of the imidazole dipeptides by applying a mix-mode column, which provides both normal phase and ion exchange separations, and developed multiple reaction-monitoring of the transitions for quantification of m/z 227 → 110 for carnosine, m/z 241 → 126 for anserine, m/z 241 → 124 for balenine, and m/z 269 → 110 for L-histidyl-L-leucine (internal standard). The established method met all pre-defined validation criteria. Intra- and inter-day accuracy and precision were ±10.0% and ≤14.8%, respectively. The ranges of quantifications were 14.7 ng/mL to 1.5 mg/mL for carnosine, 15.6 ng/mL to 1.6 mg/mL for anserine, and 15.6 ng/mL to 1.6 mg/mL for balenine. In conclusion, the validated method was successfully applied to the quantification of imidazole dipeptides in biological samples without derivatization.

Temporal changes in urinary excretion of liver-type fatty acid binding protein (L-FABP) in acute kidney injury model of domestic cats: a preliminary study.

Liver-type fatty acid-binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. It is secreted along with cytotoxic oxidation products from proximal tubular epithelial cells under conditions of ischemia and/or oxidative stress. This study examined urinary L-FABP excretion under renal ischemia in feline acute kidney injury (AKI) model. L-FABP excretion increased immediately after renal ischemia/reperfusion, despite the absence of obvious structural damage to the kidneys, in the two AKI model cats studied. L-FABP was detected in the renal tubular lumen immediately after renal ischemia/reperfusion in the two cats, but not in a sham surgery cat. These results suggested that high L-FABP excretion is a pathophysiological response associated with antioxidant defense in proximal tubules with renal ischemia and/or oxidative stress in a feline model.

GC × GC-MS-Based Volatile Profiling of Male Domestic Cat Urine and the Olfactory Abilities of Cats to Discriminate Temporal Changes and Individual Differences in Urine.

Domestic cats (Felis silvestris catus) are solitary and territorial, and mark their territories by spraying urine, which emits a strong odor produced by volatile organic compounds (VOCs). Previous studies have focused on identifications of specific VOCs, such as 3-mercpto-3-methyl-1-butanol, a cat-specific VOCs. However, little is known about how whole volatile profiles of their sprayed urine change over time or how the profiles differ among individuals. This study investigated temporal changes and individual differences of volatile profiles produced by whole VOCs in cat urine, and the ability of cats to discriminate between these scent differences. Volatile profiles of fresh and aged cat urine were analyzed by using two-dimensional gas chromatography-mass spectrometry with a VOC preconcentrator comprehensively. Volatile profiles produced by hundreds of VOCs emitted from cat urine were influenced primarily by the age of the urine, and secondarily by individuality. During habituation-dishabituation tests, subjects discriminated between fresh and 24 h-old samples of same individuals, and between odor of different individuals from 0 h-, 3 h-old, and 24 h-old samples. These results strongly suggest that cats can recognize conspecific individuals via olfaction. Since most VOCs varied among individuals but were not stable over time, their urine may contain unknown VOCs that vary among individuals, are stable over time, and act as individual recognition signals.

Temporal changes of abomasal contents and volumes in calves fed milk diluted with oral rehydration salt solution.

Several manufacturers recommend to feed mixture comprising equal amounts of oral rehydration salt (ORS) solution and milk for diarrheic calves after milk withdrawal. Such a feeding method is expected to supply more nutrients and energy compared to feeding only the ORS solution. However, little is known about the effects of feeding milk diluted with ORS solution on calves' digestive process. This study examined the abomasal contents, volumes, and emptying rates in calves fed whole milk, milk diluted by 50% with ORS solution (50% ORS-milk), and ORS solution. Ultrasonography identified curds in the milk-fed calves, but not in the 50% ORS-milk-fed or the ORS-fed calves. The abomasal fluid of the 50% ORS-milk-fed calves contained not only ß-lactoglobulin but also α-casein (CN), ß-CN, and κ-CN, which were used for curd formation and undetectable in the milk-fed calves. Abomasal pH was relatively higher in the 50% ORS-milk-fed than that in the milk-fed calves. Abomasal emptying rates were significantly faster in the ORS-fed than in the 50% ORS-milk-fed and the milk-fed calves. These data indicate that the formation of abomasal curd is inhibited in the 50% ORS-milk-fed calves due to the resultant high abomasal pH and low κ-CN concentration. The 50% ORS-milk may not provide rehydration as quickly as the ORS solution. In conclusion, we do not recommend feeding 50% ORS-milk to calves.

The Chemical Basis of Species, Sex, and Individual Recognition Using Feces in the Domestic Cat.

Scents emitted from excretions provide important information about the owner. Volatile compounds with higher levels in a species and/or sex, or that vary among individuals could be odor cues for species and/or sex, or individual recognition. However, such compounds have been identified in only a few vertebrate species. In domestic cats (Felis silvestris catus), it is known that unburied cat feces are territorial markers asserting the border of their home range, but little was known which fecal compounds are scent cues for species, sex, and individual recognition in cats. In the present study, we demonstrated the chemical basis for species, sex, and individual recognition using feces of cats. For males, major contents were fatty acids and 3-mercapto-3-methyl-1-butanol (MMB), a derivative of the unusual amino acid, felinine. MMB emission levels from feces had sex-based differences (male > female) and dynamic temporal changes during aging. Cats distinguished fecal odors with and without MMB, and different fatty acid compositions among individuals. No cat-specific compound, such as MMB, was detectable from their anal odor emitting fatty acids. We concluded that fecal MMB is a male sex recognition pheromone in cats and also provides a temporal trace of the owner. After sensing MMB, they may distinguish individual differences of conspecific feces with variable subsets of fatty acids. In contrast to scent marks, since cats can obtain species information from visual cues before sniffing conspecific anal odors, they may use their efforts to distinguish individual differences of anal odors during sniffing.

Identification of 2-phenylethanol with a rose-like odor from anal sac secretions of the small Indian mongoose (Herpestes auropunctatus).

The small Indian mongoose (Herpestes auropunctatus) is an invasive species in Okinawa and Amami-Oshima, Japan. Major strategies for their eradication have been the use of baited traps, which suffer from decreasing efficiency with declining populations and the bycatch of native animals. To address these concerns, mongoose-specific lures are required. In this study, we aimed to identify species- and/or sex-specific compounds from anal sac secretions of small Indian mongooses. Volatile compounds emitted from male and female mongoose anal sac secretions were analyzed by thermal desorption-gas chromatography-mass spectrometry. In addition to several fatty acids, 2-phenylethanol was identified as a minor compound, which is uncommon in mammalian secretions but a dominant odorant in roses. Female samples emitted higher levels of 2-phenylethanol than male samples did. These findings indicate that 2-phenylethanol is a female-specific volatile compound of anal sac secretions in small Indian mongooses, and it may be useful as an ingredient of mongoose-specific scent lures.

Olfactory discrimination of anal sac secretions in the domestic cat and the chemical profiles of the volatile compounds.

Scent emitted from anal sac secretions provides important signals for most Carnivora. Their secretions emit a variety of volatile compounds, some of which function as chemical signals with information about the scent owners. The domestic cat has a pair of anal sac glands to secrete a pungent liquid. Their anal sac secretions may give information about sex, reproductive state, and recognition of individuals. However, little is known about the volatile compounds emitted from anal sac secretions and their biological functions in cats. In this study, we examined the volatile chemical profiles of anal sac secretions in cats and their olfactory ability to discriminate intraspecific anal sac secretions. Analysis with gas chromatography-mass spectrometry showed that the major volatile compounds were short-chain free fatty acids, whose contents varied among individuals, as well as other carnivores. There was no sex difference in the volatile profiles. In temporal analyses of individual anal sac secretions performed 2 months apart, the profiles were highly conserved within individuals. Habituation-dishabituation tests showed that cats can distinguish individual differences in the odor of anal sac secretions. These results suggest that cats utilize short-chain free fatty acids emitted from anal sac secretions to obtain scent information for individual recognition rather than species or sex recognition.

LC-MS/MS quantification of felinine metabolites in tissues, fluids, and excretions from the domestic cat (Felis catus).

Domestic cat urine contains large concentrations of the unusual amino acid, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid). A felinine derivative, 3-mercapt-3-methylbutanol is a potential scent signal for letting other animals know that the scent owners are cats. 3-Methylbutanol-glutathione (MBG) is an upstream precursor of the biosynthetic pathway of felinine that may be produced in hepatocytes by conjugation of glutathione with isopentenyl pyrophosphate, an intermediate for cholesterol biosynthesis. However, little evidence exists to support the biosynthesis of MBG in the liver. This study examined the distribution of metabolites of the felinine biosynthetic pathway in multiple tissues, body fluids, and excretions of cats. MBG, the felinine precursor, 3-methylbutanol-cysteinylglycine (MBCG), felinine, and felinine N-acetyl derivative were quantified by liquid chromatography-electron spray ionization-tandem mass spectrometry. All compounds were detected in cat serum. In males, MBG and MBCG contents were significantly higher than felinine and N-acetylfelinine contents. MBG was detected in multiple tissues, including the salivary gland, heart, liver, spleen, gut, kidney, bladder, adipose tissue, and muscle. Sex differences in MBG levels were observed in the liver and other tissues. Felinine and N-acetylfelinine were also detected in those tissues. Furthermore, we detected all compounds in cat bile and fecal samples, indicating that felinine is excreted into the feces via bile from the liver. We conclude that MBG is synthesized in several tissues in a sex-dependent manner. These findings improve our understanding of felinine metabolism and function in cats.

The therapeutic efficacy of allyl isothiocyanate in cows with bovine digital dermatitis.

Bovine digital dermatitis (BDD) is the most prevalent infectious cause of lameness in cattle. Because Treponema infection is a major etiology of BDD, the most common treatment of BDD is an antibiotic. Nonetheless, dairy cows require a withdrawal period after antibiotic treatment before their milk can be marketed. To address the problem, in this study, we tested whether 3 nonantibiotic agents (used separately)-allyl isothiocyanate (AITC), sodium alginate, and calcium hydroxide-alleviate BDD lesions in dairy cows. The AITC treatment improved the BDD lesions, whereas the sodium alginate and calcium hydroxide treatments did not. Therapeutic efficacy of AITC was similar to that of lincomycin, a topical antibiotic prescribed for BDD. These results suggest that AITC is a promising nonantibiotic agent for BDD treatment in dairy cows.

Characterisation of the carboxylesterase enzyme cauxin in the seminal fluid of the cat.

The carboxylesterase cauxin is a major urinary protein in cats that is also found in seminal fluid (SF). This study investigated cauxin in feline SF including biochemical features, concentration, distribution and gene expression in epididymal tissue, and its reaction with acylglycerol substrates. Monomeric, dimeric, and/or multimeric forms of cauxin carrying N-glycosylations were detected on Western blots of feline SF but most were monomeric. Cauxin concentrations were markedly lower in SF (0.042±0.020 mg/mL) than in urine (∼0.5 mg/mL) and cauxin gene expression was 60-fold lower in the epididymis than in the kidney. Immunohistochemical examination localised cauxin within the stereocilia and cytoplasm of epithelial cells lining the caput and corpus epididymis. Cauxin-positive spermatozoa were detected in the lumen of the cauda epididymis but not in the cytoplasm of the epithelial cell lining. Using an in vitro assay, cauxin hydrolysed saturated 1-mono- but not di- and tri-acylglycerols. The results suggest that cauxin secreted from the caput and corpus epididymis acts as an esterase on lipid within feline SF.

The effect of frequent milk feeding on abomasal curd formation of Holstein calves.

In order to understand the effects of the automatic milk replacer feeding system on calf health, we examined the effect of frequent milk feeding on curd formation in the abomasum using ultrasonographic imaging. Eight male Holstein calves were divided into a milk-replacer group and a fresh milk group. Calves were fed twice a day to 12 days after birth (Period A). From 13 days, calves were fed six times a day (Period B). The abomasal fluid was taken by paracentesis. In both periods A and B, the fresh milk group formed bigger curds faster than the milk replacer group. The curd score of the milk replacer group in period B at 2 h was significant lower than those of both groups in period A. The pH in the abomasum was lower in period B than in period A. We could not identify the location of the abomasum in the milk replacer group during period B at almost time points. Our experiment suggests the possibility that frequent feeding of milk replacer causes incomplete hydrolysis of kappa-casein as well as curd formation, thus reducing the digestibility compared to the feeding of the milk replacer twice per day or frequent feeding of fresh milk.

The effect of experimental inhibition of gastric acid secretion on curd formation in abomasum and weight gain of calves.

Eight Holstein bull calves were divided into two groups; a non-treated control group and a famotidine treated group. Fresh milk was fed twice a day. The experiment was conducted between 7 and 14 days of age. During the experimental period the control group was injected with physiological saline, and the famotidine group was injected with famotidine, a histamine-H2-receptor blocker, into the jugular vein 30 minutes prior to each feeding. The control group showed maximum curd formation 2 h after feeding at both 7 and 14 days of age. Curd scores of 7-day-old and 14-day-old calves were significantly lower in the famotidine than in the control group at 2 and 4 h after feeding. Most fecal samples from the famotidine group exhibited an acidic smell. The famotidine group showed significantly lower values for both average weight gain and the rate of weight gain from 7 to 14 days of age. The inhibition of gastric acid secretion decreased curd formation in the abomasum as well as daily weight gain compared to non-treated control calves. This suggested that curd formation in the abomasum is important for the weight gain of newborn calves.

No abomasal curd formation in pre-ruminant calves after ingestion of a clotting milk replacer.

The aim of this study was to ultrasonographically evaluate curd formation in 29 pre-ruminant calves that were fed a clotting milk replacer. Abomasal curd was absent in 8/29 calves at 2 h after feeding. In these eight calves, abomasal contents were observed as an anechoic image with small echogenic spots (five calves), or as an echogenic image with an unclear outline (three calves), but there was no echogenic image with a clear outline corresponding to curd that was visualised in the other 21 calves. The curd was not observed until 7 h after feeding in the eight calves. Our analysis also indicated that the absence of curd formation in the pre-ruminant calves did not have a significant impact on the appearance, appetite and vigour of pre-ruminant calves or on their blood parameters, including serum triglyceride, blood urea nitrogen and glucose concentrations. The study provided the first evidence that the abomasum of some calves does not form curd despite ingestion of a clotting milk replacer.