Skin Cortisol and Acoustic Activity: Potential Tools to Evaluate Stress and Welfare in Captive Cetaceans.

As people's focus broadens from animals on farms to zoos and aquaria, the field of welfare science and the public's concern for animal welfare continue to grow. In captive animals, stress and its causes are topics of interest in welfare issues, and the identification of an objective method that can be used to assess animals' stress as a physiological state is essential. Both behavioral and physiological parameters can be used as indicators in order to assess animal stress quantitatively. To validate this approach, acoustic activity and the sloughed scrape skin cortisol concentration were used to evaluate the animal welfare of captive beluga whales (Delphinapterus leucas). The acoustic activity (5 min at 10:00 am) of three captive D. leucas was routinely recorded by a transducer and analyzed using audio editing software. The calls were separated into three main categories: whistles, pulses, and combo calls. The sloughed scrape skin samples were collected non-invasively once a week from all three animals' fluke and/or flipper. Cortisol was extracted using a modified skin steroid extraction technique, and detected via commercially available enzyme immunoassays. The results showed that the cortisol concentration increased by varying levels when the whales encountered the same event. In addition, the number and distribution of the calls changed along with the events. This indicated that the changes in the cortisol concentration and acoustic behavior may have reflected the fluctuations in the environment and body condition. Therefore, the scrape cortisol measurement and acoustic recordings could be used to monitor stress levels in captive beluga whales. We recommend that aquaria consider incorporating skin scrape cortisol and acoustic activity monitoring into their standards for animal welfare.

Effects of Para-Toluenesulfonamide on Canine Melanoma Xenotransplants in a BALB/c Nude Mouse Model.

The pharmacological pathway of para-toluenesulfonamide (PTS) restricts the kinase activity of the mammalian target of rapamycin, potentially leading to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical effect on tumorigenesis. We aimed to examine the antitumor effect of PTS or PTS combined with cisplatin on canine melanoma implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. The mice were randomly divided into four groups: control, cisplatin, PTS, and PTS combined with cisplatin. Mice treated with PTS or PTS combined with cisplatin had retarded tumor growth and increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase phosphorylation, decreased inflammatory cytokine levels, reduced inflammation-related factors, enhanced anti-inflammation-related factors, and inhibition of metastasis-related factors. Mice treated with PTS combined with cisplatin exhibited significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with those treated with cisplatin or PTS alone. PTS or PTS combined with cisplatin could retard canine melanoma growth and inhibit tumorigenesis. PTS and cisplatin were found to have an obvious synergistic tumor-inhibiting effect on canine melanoma. PTS alone and PTS combined with cisplatin may be antitumor agents for canine melanoma treatment.

Analysis of persistent organochlorine pesticides in shellfish and their risk assessment from aquafarms in Taiwan.

In Taiwan, freshwater clams (Corbicula fluminea) and hard clams (Meretrix lusoria) are the most frequently raised shellfish in land-based pond aquaculture, but research on the accumulation of organochlorine pesticides (OCPs) in these shellfish is limited. We detected the levels of 14 OCPs in 62 shellfish from Taiwanese aquafarms by performing gas chromatography-tandem mass spectrometry. OCP residues were detected in 4.84% of the samples including readings of 0.04 mg/kg chlordane (in a freshwater clam), 0.03 mg/g p,p'-DDE (in a freshwater clam), and 0.02 mg/g p,p'-DDE (in a hard clam). However, the associated estimated daily intake values were less than the acceptable daily intake levels of chlordane and p,p'-DDE Therefore, the consumption of these shellfish presents no immediate health risks. Our findings contribute to food safety and serve as a reference for OCP screenings for aquatic shellfish.

Analytical Detection of Sulfonamides and Organophosphorus Insecticide Residues in Fish in Taiwan.

Exposure to residues of antibiotics (e.g., sulfonamides) and insecticides (e.g., organophosphorus insecticides) in aquacultured food can adversely affect humans and animals and thus affect public health globally. Here, using a validated method, we examined the levels of residues of 12 sulfonamides as well as 18 organophosphorus insecticides in aquacultured fish in Taiwan. A total of 52 fish samples (i.e., 20 tilapia, 16 milk fish, and 16 perch samples) were obtained from Taiwanese aquafarms from June 2018 to October 2019. We detected 0.02 and 0.03 mg/kg of sulfamethazine (a sulfonamide) in one tilapia and one milk fish, respectively, and 0.02, 0.05, and 0.03 mg/kg of chlorpyrifos (an organophosphorus insecticide) in one tilapia, one milk fish, and one perch, respectively; thus, among the samples, 3.85% and 5.77% contained sulfonamides and organophosphorus insecticide residues, respectively. Furthermore, we assessed human health risk based on the estimated daily intakes (EDIs) of these residues: EDIs of sulfonamide and organophosphorus insecticide residues were <1.0% of the acceptable daily intake recommended by the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives. The risk of exposure to sulfonamide and organophosphorus insecticide residue by consuming aquacultured fish in Taiwan was thus negligible, signifying no immediate health risk related to the consumption of fish. Our findings can constitute a reference in efforts geared toward ensuring food safety and monitoring veterinary drug and insecticide residue levels in aquacultured organisms. Residue levels in fish must be continually monitored to further determine possible effects of these residues on human health.

Analysis of Pollution of Phthalates in Pork and Chicken in Taiwan Using Liquid Chromatography-Tandem Mass Spectrometry and Assessment of Health Risk.

Phthalates are widely used plasticizers that can cause endocrine disruption, mutagenicity, and carcinogenic effects and can contaminate food through various pathways. Investigations are scanty on phthalate pollution of livestock and poultry meat and their dietary exposure to humans. The present study assessed residual levels of phthalates in unpackaged pork (30 samples) and unpackaged chicken (30 samples) and their relevance to meat consumption and health risks in the Taiwanese population. Phthalate quantity was assessed by liquid chromatography-tandem mass spectrometry; the materials included diisononyl phthalate, diisodecyl phthalate, benzyl butyl phthalate, di-2-ethylhexyl phthalate (DEHP), and di-n-butyl phthalate. The Taiwan Food and Drug Administration (TFDA) has established values of tolerable daily intake (TDI) for the five phthalates. The major compound detected was DEHP, which ranged from 0.62 to 0.80 mg/kg in two pork samples, and 0.42-0.45 mg/kg in three chicken samples. Collectively, 8.33% of the phthalate-residue-containing samples tested positive for DEHP. The concentrations of DEHP were lower than the screening value of 1.0 mg/kg, as defined by the TFDA. Health risk was calculated as the estimated daily intake (DI) for any likely adverse effects; the DI of DEHP residues was <1% of the TDI value. The estimated risk was insignificant and considered to be safe, indicating that there is no risk to the health of Taiwanese population due to meat consumption. However, it is suggested that a phthalate monitoring program in meat should be instituted for any possible effects in future on human health.

Phthalates and organophosphorus insecticide residues in shrimp determined by liquid/gas chromatography-Tandem mass spectrometry and a health risk assessment.

Shrimps are the most widely and increasingly cultured crustaceans in land-based ponds in Taiwan. However, few studies have investigated the phthalate contamination of and insecticide residues in shrimp. In this study, we applied a validated method to analyze the phthalate and 18 insecticides residues in shrimp. A total of 46 samples of whiteleg, grass, or giant river shrimp were collected from aquafarms and production areas in Taiwan. We detected 0.02-0.70 mg/kg of di-2-ethylhexyl phthalate (DEHP) in three shrimps; 0.02-0.03 mg/kg of chlorpyrifos in three shrimps, and 0.03 mg/kg of trichlorfon in one shrimp, indicating that 6.52% and 8.70% of the samples contained phthalate and insecticide residues, respectively. Furthermore, the assessed risk was negligible and indicated no immediate health risk associated with shrimp consumption. Continual monitoring of the residues in shrimps is critical for further assessment of possible effects on human health.

Sound exposure-induced cytokine gene transcript profile changes in captive bottlenose dolphin (Tursiops truncatus) blood identified by a probe-based qRT-PCR.

Cetacean health may be potentially affected by anthropogenic sound. We have initiated investigations on the effect of low-frequency underwater sound on immunological gene transcript profiles of captive bottlenose dolphins (Tursiops truncatus) using a probe-based quantitative gene expression assay. Six immunologic genes (IL-2Rα, -4, -10, -12, TNFα and IFNγ) were selected for analysis using two validated housekeeping genes (PGK1 and HPRT1) as reference genes. Twenty-four blood samples from six clinically healthy individuals and six blood samples from individuals after sound exposures were available. The gene transcript profile of sound-exposed dolphins was consistent with a stress-induced TH2 shift profile as compared to controls. This study may lead to better understanding of the effects of anthropogenic sound on immune responses of cetaceans.

Benefits of Intraluminal Agarose Stents during End-to-End Intestinal Anastomosis in New Zealand White Rabbits.

In the present study, we evaluated the utility of an intraluminal agarose stent (IAS) for end-to-end intestinal anastomoses in rabbits. Female New Zealand white rabbits (n = 14) underwent conventional sutured anastomosis (CSA) with or without an IAS. IAS were used to maintain the luminal diameter for more rapid and accurate suturing, and then was squeezed transluminally to crush it into fragments, which passed through the intestines and were eliminated. The rabbits were euthanized on postoperative day 21. At necropsy, the anastomoses were assessed for adhesion formation, stenosis, and bursting pressure and were examined histologically for collagen content and blood vessel formation. Anastamosis surgery took less time in the IAS group (15.0 ± 2.6 min) than in the CSA-only group (30.1 ± 7.9 min). Only 1 postoperative death occurred (in the CSA group), and postmortem examination revealed evidence of anastomotic leakage. Adhesion formation and stenosis did not differ between groups, but bursting pressure, collagen content, and blood vessel formation were all significantly increased in the IAS group. IAS may decrease the operative time by maintaining a clear surgical field at the anastomotic site. In addition, the use of IAS promotes rapid healing and maintains the luminal diameter during end-to-end intestinal anastomosis.

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Development of a sedation protocol using orally administered tiletamine-zolazepam-acepromazine in free-roaming dogs.

OBJECTIVE: To investigate the sedative effects in dogs of tiletamine-zolazepam-acepromazine (TZA) or ketamine-flunitrazepam (KF) administered orally and to evaluate the effectiveness of encapsulated TZA for capturing free-roaming dogs. STUDY DESIGN: Experimental study followed by a field trial. ANIMALS: Six research dogs and 27 free-roaming dogs. METHODS: In a pilot study, six research dogs were administered liquid TZA (20 mg kg-1 tiletamine-zolazepam and 2 mg kg-1 acepromazine) or liquid KF (50 mg kg-1 ketamine and 2 mg kg-1 flunitrazepam) orally: treatment 1, forcefully squirting liquid medication into the mouth; treatment 2, encapsulating liquid medication for administration in canned food; treatment 3, administering liquid medication mixed with gravy. Sedation was scored. A follow-up field trial attempted capture of 27 free-roaming dogs. RESULTS: In the pilot study, the median time (range) to lateral recumbency (% dogs) after TZA administration was: treatment 1, 47.5 (35-80) minutes (67%); treatment 2, 30 (15-65) minutes (83%); and treatment 3, 75 (45-110) minutes (100%). No dogs in KF treatment 2 or 3 achieved lateral recumbency. Based on these results, 20 free-roaming dogs were offered encapsulated TZA in canned food: TZ (20 mg kg-1) and acepromazine (2 mg kg-1). Of these, no further drugs to four dogs (one dog captured), 10 dogs were administered a second dose within 30 minutes (five dogs captured) and six dogs were administered TZ (5 mg kg-1) and xylazine (1.1-2.2 mg kg-1) intramuscularly by blow dart (six dogs captured). Seven dogs were initially offered twice the TZA dose (five dogs captured). In total, 63% free-roaming dogs were captured after administration of encapsulated TZA in canned food. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of encapsulated TZA in canned dog food can aid in the capture of free-roaming dogs, but additional drugs may be required. The sedation onset time and medication palatability influenced the capture rate.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality.

The current study aimed to investigate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen was collected from Duroc boars and was allocated to 32 treatment groups for cryopreservation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI) was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b) dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference observed among these three treatments. The farrowing rates after AI with post-thawed semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa.

Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.

Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan.

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our laboratory recently confirmed the first case of encephalitozoonosis in a rabbit in Taiwan; the prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being infected. This study surveys the seropositivity of E. cuniculi using carbon immunoassay (CIA) and enzyme-linked immunosorbent assay (ELISA). Serological examination of 171 rabbits using CIA and ELISA showed that 63.2% (108/171) and 67.8% (116/171) were seropositive against E. cuniculi, respectively. Thirteen of the 14 rabbits (92.9%) with neurological symptoms were seropositive. Except for gender, health status and location had a significant effect on E. cuniculi seropositivity (p<0.05). Adult rabbits aged older than 4 months exhibited significantly higher seropositivity for E. cuniculi than young rabbits (p<0.05). In conclusion, this study shows that E. cuniculi is present and widespread among healthy rabbits in Taiwan. Therefore, the fields of veterinary and human medicine in Taiwan should be aware of this zoonotic issue and the resulting public health concern of encephalitozoonosis.

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor.

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.