Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

The development of nonalcoholic steatohepatitis is subjected to breeder dependent variation in guinea pigs.

Variability in disease development due to differences in strains and breeders constitutes a substantial challenge in preclinical research. However, the impact of the breeder on non-alcoholic steatohepatitis (NASH) is not yet fully elucidated. This retrospective study investigates NASH development in guinea pigs from Charles River or Envigo fed a high fat diet (20% fat, 15% sucrose, 0.35% cholesterol) for 16 or 24/25 weeks. Charles River animals displayed more severe NASH, with higher steatosis (p < 0.05 at week 16), inflammation (p < 0.05 at both week), fibrosis (p < 0.05 at week 16) and disease activity (p < 0.05 at both weeks). Accordingly, alanine and aspartate aminotransferase were increased at week 24/25 (p < 0.01). Hepatic expression of inflammatory (Ccl2, Cxcl8) and fibrotic (Pdgf, Serpine1, Col1a1) genes was also increased (p < 0.05). Differences were observed in healthy chow (4% fat, 0% sucrose, 0% cholesterol) fed animals: Envigo animals displayed higher relative liver weights (p < 0.01 at both weeks), liver cholesterol (p < 0.0001 at week 24/25) and aspartate aminotransferase (p < 0.05 at week 16), but lower levels of alkaline phosphatase (p < 0.0001 at week 24/25). These findings accentuates the importance of the breeder and its effect on NASH development and severity. Consequently, this may affect reproducibility, study comparison and limit the potential of developing novel therapies.

Estimation of genetic parameters for four Peruvian guinea pig lines.

This study aimed to estimate genetic parameters for four guinea pig lines of a crossbreeding scheme. Two paternal lines are selected for growth rate (P1) and feed conversion rate (P2), whereas two maternal lines are selected for growth rate of litter (M1) and litter size at birth (M2). The heritabilities and genetic correlations were estimated with animal linear models employing multivariate analyses with REML. The heritabilities for birth weight (BW) were 0.21±0.02 and 0.23±0.03 for P1 and P2, respectively, and for weaning weight (WW), the heritability was 0.28±0.03 for P2. The estimates for weight at 60 days of age (W60) were 0.34±0.01 and 0.47±0.04 for P1 and P2, respectively, and for partial feed conversion rate was 0.46±0.03 for P2. Heritabilities for litter weight at birth (LW) were 0.09±0.03 and 0.10±0.03 for P1 and M1, respectively. For litter weight at 10 days of age (LW10), the heritability was 0.15±0.03 for M1. Heritabilities for litter size (LS) were 0.17±0.03, 0.20±0.03 and 0.11±0.03, and for number of pups born alive (BA) were 0.09±0.03, 0.14±0.03 and 0.09±0.03 for P1, M1 and M2, respectively. Similarly, high genetic correlations were found between BW, WW and W60 and between LW, LS, LW10 and BA. The genetic correlation between BW direct and maternal was moderately negative (- 0.24 ± 0.10) for P1. These results show the genetic status for all four guinea pig lines, which is essential for the further improvement of the currently implemented breeding programme and also indicate an opportunity for genetic improvement.

Ancient DNA of Guinea Pigs (Cavia spp.) Indicates a Probable New Center of Domestication and Pathways of Global Distribution.

Guinea pigs (Cavia spp.) have a long association with humans. From as early as 10,000 years ago they were a wild food source. Later, domesticated Cavia porcellus were dispersed well beyond their native range through pre-Columbian exchange networks and, more recently, widely across the globe. Here we present 46 complete mitogenomes of archaeological guinea pigs from sites in Peru, Bolivia, Colombia, the Caribbean, Belgium and the United States to elucidate their evolutionary history, origins and paths of dispersal. Our results indicate an independent centre of domestication of Cavia in the eastern Colombian Highlands. We identify a Peruvian origin for the initial introduction of domesticated guinea pigs (Cavia porcellus) beyond South America into the Caribbean. We also demonstrate that Peru was the probable source of the earliest known guinea pigs transported, as part of the exotic pet trade, to both Europe and the southeastern United States. Finally, we identify a modern reintroduction of guinea pigs to Puerto Rico, where local inhabitants use them for food. This research demonstrates that the natural and cultural history of guinea pigs is more complex than previously known and has implications for other studies regarding regional to global-scale studies of mammal domestication, translocation, and distribution.

Back to the future: re-establishing guinea pig in vivo asthma models.

Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.

Influences of non-nutritive sweeteners on ovarian and uterine expression of T1R2 and T1R3 in peripubertal female guinea pigs.

The underlying mechanism of taste receptor type 1 subunit 2 (T1R2) and taste receptor type 1 subunit 3 (T1R3) in the hormonal and reproductive system is still elusive. A low or a high dose of sweetness equivalent to that sodium saccharin (SS, 1.5 or 7.5 mM) and rebaudioside A (RA, 0.5 or 2.5 mM) was administered to young female guinea pigs for 28 consecutive days from the age of 28 days. Our results indicated that the sweet taste receptor subunit T1R2 was markedly expressed in the ovary and uterus of guinea pigs, whereas the T1R3 protein was expressed at a lower level. We elucidated that low-dose (1.5 mM) SS increased body and ovary weight associated with elevated ovarian expression of T1R2 in guinea pigs, unlike the high-dose (7.5 mM) SS, which suppressed the ovarian expression of T1R2 and resulted in certain adverse effects on ovarian and uterine morphology. Furthermore, high-dose (2.5 mM) RA increased the number of corpus luteum and elevated uterine expression of T1R2, whereas low-dose (0.5 mM) RA induced increased secretion of serum progesterone. Therefore, our findings suggest that we should pay more attention to the potential adverse effects, including increases in ovary weight, morphology changes, and increased progesterone that result from the dose-dependent regulation of T1R2 by non-nutritive sweeteners (NNS) in the ovaries and uteri of peripubertal females.

Hearing differences in Hartley guinea pig stocks from two breeders.

Across the world, dozens of outbred Hartley guinea pig stocks are used for auditory experiments. The genetic makeup of these different stocks will differ due to differences in breeding protocols, history and genetic drift. In fact, outbred breeding protocols are not intended to produce genetically identical animals, neither across breeders, nor across time. For this reason, it is unclear how reproducible experimental results are likely to be using animals from different stocks. We evaluated the consistency of cochlear function using both clicks and tones in Hartley guinea pigs as a function of breeder (Kuiper and Charles River) and sex using archival Auditory Brain Stem Response (ABR) data and tissue from our own laboratory. Sound levels required to reach baseline threshold for click-induced ABRs were similar between male Charles River and male Kuiper guinea pig stocks. However, the median and average thresholds after exposure to high level noise were larger in the Kuiper population than in the Charles River population with corresponding threshold shifts higher in the Kuiper than in the Charles River animals. We evaluated the relationship between pure-tone thresholds and sex, age, breeder stock, left or right cochleas, weight and 5 test frequencies before and after noise exposure using a linear mixed statistical model. Across all frequencies, the effect of breeder on baseline threshold is statistically significant, with effect sizes most pronounced at the lower frequencies before exposure to noise. After noise exposure, the differences are minimal in the model, indicating that differences in threshold shift are chiefly due to differences in initial baseline hearing. However, a contingency calculation comparing response/no response at the highest speaker output at 32 kHz gave a statistically significant difference between the stocks: 28% of Kuiper cochleas responded to the highest output of the speaker as compared with 71.4% of Charles River cochleas, indicating that noise exposure induced a larger threshold shift in a greater proportion of Kuiper animals. Using our archival cochlear tissue from these studies, we confirmed the sex of each animal by PCR, then compared males and females of the Kuiper stock. Across all baseline frequencies, the effect of sex on threshold is statistically significant, with no noticeable difference after exposure. The effect sizes for baseline thresholds are most pronounced at lower frequencies. These data demonstrate that Hartley guinea pig stocks from different breeders are not uniform in their auditory characteristics, and that due to these differences, results and conclusions can differ among laboratories. Moreover, within a single stock, males and females can provide different data, confirming that male and female animals must be individually evaluated in any auditory protocol.

Hematology and Clinical Chemistry Reference Intervals for Inbred Strain 13/n Guinea Pigs (Cavia Porcellus ).

Inbred Strain 13/N Guinea Pigs are Frequently Used As Animal Models in Studies of Emerging and High-pathogenicity Viruses. To Date, Clinical Reference Intervals Have Not Been Established for Hematology and Clinical Chemistry Parameters in This Strain. We Obtained Whole-blood Samples from the Cranial Vena Cava of Healthy Strain 13/N Colony Animals for Inhouse Cbc and Clinical Chemistry Analyses. Analyte Values Were Investigated to Determine Subpopulation Differences According to Age and Sex. Glucose, Albumin, Alp, Lymphocyte Percentage, Hgb, and Mchc Decreased with Age, Whereas Neutrophil and Monocyte Percentages, Bun, Creatinine, Calcium, and Amylase Increased with Age. Total Protein and Wbc Counts Increased Over the First 300 D of Life Before Stabilizing. Across All Age Categories, Female Guinea Pigs Consistently Had Lower Rbc, Hct, Hgb, Alt, Alp, and Amylase Levels and Higher Mcv Values Than Males. These Trends Were Strongest in Adults (age, 151 Through 900 D). Most Parameters Stabilized by 300 D; Previous Studies Used 60 D or 120 D As Adult Age and 90 to 120 D As Sexual Maturity. We Recommend Age Group Definitions of 0 Through 150 D for Juveniles, 151 Through 900 D for Adults, and Older Than 900 D for Geriatric Adult Strain 13/N Guinea Pigs.

The Diabetes Susceptibility Gene SLC30A8 that Encodes the Zinc Transporter ZnT8 is a Pseudogene in Guinea Pigs Potentially Contributing to Low Guinea Pig Islet Zinc Content.

In most mammals pancreatic islet beta cells have very high zinc levels that promote the crystallization and storage of insulin. Guinea pigs are unusual amongst mammals in that their islets have very low zinc content. The selectionist theory of insulin evolution proposes that low environmental zinc led to the selection of a mutation in Guinea pig insulin that negated the requirement for zinc binding. In mice deletion of the Slc30a8 gene, that encodes the zinc transporter ZnT8, markedly reduces islet zinc content. We show here that SLC30A8 is a pseudogene in Guinea pigs. We hypothesize that inactivation of the SLC30A8 gene led to low islet zinc content that allowed for the evolution of insulin that no longer bound zinc.

Sex Genotyping of Archival Fixed and Immunolabeled Guinea Pig Cochleas.

For decades, outbred guinea pigs (GP) have been used as research models. Various past research studies using guinea pigs used measures that, unknown at the time, may be sex-dependent, but from which today, archival tissues may be all that remain. We aimed to provide a protocol for sex-typing archival guinea pig tissue, whereby past experiments could be re-evaluated for sex effects. No PCR sex-genotyping protocols existed for GP. We found that published sequence of the GP Sry gene differed from that in two separate GP stocks. We used sequences from other species to deduce PCR primers for Sry. After developing a genomic DNA extraction for archival, fixed, decalcified, immunolabeled, guinea pig cochlear half-turns, we used a multiplex assay (Y-specific Sry; X-specific Dystrophin) to assign sex to tissue as old as 3 years. This procedure should allow reevaluation of prior guinea pig studies in various research areas for the effects of sex on experimental outcomes.

Identification and characterization of a gamma-interferon-inducible lysosomal thiol reductase homolog from guinea pig (Cavia porcellus) that exhibits thiol reductase activity in vitro.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is a key enzyme in the antigen processing and presentation pathway whereby it reduces disulfide bonds at an acidic pH. In this study, a homolog of GILT from guinea pigs (designated gpGILT) was identified and characterized using bioinformatic methods and bioactivity assays. The open reading frame of gpGILT is 705bp in length and encodes 234 amino acids, with a putative molecular weight of about 25.85kDa. The structure of gpGILT is similar to those of humans and zebrafish, containing six introns and seven exons. The deduced primary structure of the gpGILT protein includes all of the typical features of other known GILT proteins, including an active-site motif, CXXC, a GILT signature sequence, CQHGX2ECX2NX4C, three potential Asn-linked glycosylation sites, and six other conserved cysteines. The predicted tertiary structures of gpGILT, human GILT, and mouse GILT are quite similar in shape and positional arrangement of the key motifs modeled on the same template. Amino acid sequence-based alignment and phylogenetic analysis showed that gpGILT is most closely related to that from the rat, with an identity of 68.40%. Additionally, the constitutive expression and immune response to lipopolysaccharide (LPS) challenge of gpGILT were tested using real-time quantitative polymerase chain reaction. A tissue-specific expression pattern in selected tissues and remarkable up-regulation of gpGILT mRNA in spleen and blood within 12h of LPS stimulation were observed, suggesting that GILT functions as an immunological surveillance-related factor in both innate and adaptive immunity. Soluble recombinant gpGILT produced in E. coli could reduce the interchain disulfide bonds of IgG in an acidic reaction system in vitro, suggesting thiol reductase activity in antigen processing. The results of this study provide a better understanding of the molecular characteristics of gpGILT and are a useful reference for further investigation of its involvement in antigen processing and immunological surveillance using the laboratory guinea pig.

Paternal intergenerational epigenetic response to heat exposure in male Wild guinea pigs.

Epigenetic modifications, of which DNA methylation is the best studied one, can convey environmental information through generations via parental germ lines. Past studies have focused on the maternal transmission of epigenetic information to the offspring of isogenic mice and rats in response to external changes, whereas heterogeneous wild mammals as well as paternal epigenetic effects have been widely neglected. In most wild mammal species, males are the dispersing sex and have to cope with differing habitats and thermal changes. As temperature is a major environmental factor we investigated if genetically heterogeneous Wild guinea pig (Cavia aperea) males can adapt epigenetically to an increase in temperature and if that response will be transmitted to the next generation(s). Five adult male guinea pigs (F0) were exposed to an increased ambient temperature for 2 months, i.e. the duration of spermatogenesis. We studied the liver (as the main thermoregulatory organ) of F0 fathers and F1 sons, and testes of F1 sons for paternal transmission of epigenetic modifications across generation(s). Reduced representation bisulphite sequencing revealed shared differentially methylated regions in annotated areas between F0 livers before and after heat treatment, and their sons' livers and testes, which indicated a general response with ecological relevance. Thus, paternal exposure to a temporally limited increased ambient temperature led to an 'immediate' and 'heritable' epigenetic response that may even be transmitted to the F2 generation. In the context of globally rising temperatures epigenetic mechanisms may become increasingly relevant for the survival of species.

The complete mitochondrial genome of the Cavia aperea.

Cavia aperea which is a Brazilian guinea pig is found in the South America. Recently the genome sequencing of C. aperea was done, but no more information of its mitochondrial had been reported. Herein, we assembled the complete mitochondrial genome sequence of C. aperea. It is a 16 835 bp long sequence with most mitogenome's characteristic structure; 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, 1 D-loop region, 1 repeat region and 3 STS regions. The GC-content of our fresh sequence is 39%. It can verify the accuracy and utility of newly determined mitogenome sequences by the phylogenetic analysis, based on whole mitogenome alignment with C. porcellus, which is the closest relative to C. aperea. We expect that using the full mitogenome we can address the taxonomic issues and study the related the evolution events.

Effects produced by different types of laser in cornea of Guinea pigs: Identification of a laser capable of producing superficial lesions without leaving scars.

PURPOSE: Climatic droplets keratopathy (CDK) is closely associated with superficial corneal erosions and lack of protective mechanisms against the harmful effects of ultraviolet radiation (UVR) during a prolonged period of time. One of the difficulties in studying the pathogenic mechanisms involved in this human disease is the lack of an experimental animal model. In this paper, a study is conducted on the effects of 4 types of lasers at various powers and time conditions on the normal guinea pig corneas in order to select only one laser condition that reversibly injures the epithelium and superficial stroma, without leaving scarring. METHODS: Damage was induced in the cornea of Guinea pigs using different powers and exposure times of 4 types of laser: argon, CO2, diode and Nd-Yag, and any injuries were evaluated by biomicroscopy (BM) and optical microscopy. Corneas from other normal animals were exposed to argon laser (350 mW, 0.3s, 50 µm of diameter), and the induced alterations were studied at different times using BM, optical coherence tomography (OCT) and transmission electron microscopy (TEM). RESULTS: Only argon laser at 350 mW, 0.3s, 50 µm of diameter produced epithelium and superficial stroma lesions. Some leukomas were observed by BM, and they disappeared by day 15. Corneal thickness measured by OCT decreased in the eyes treated with argon laser during the first week. Using TEM, different ultra structural alterations in corneal epithelium and stroma were observed during the early days, which disappeared by day 15. CONCLUSIONS: It was possible to develop reproducible corneal epithelium and anterior stroma injuries using Argon laser at 350 mW, 0.3s, 50 µm of diameter. In vivo and in vitro studies showed that injured corneas with these laser conditions did not leave irreversible microscopic or ultra structural alterations. This protocol of corneal erosion combined with exposure to UVR and partial deficiency of ascorbate in the diets of the animals for an extended period of time has been used in order to try to develop an experimental model of CDK.

Reduced noise susceptibility in littermate offspring from heterozygous animals of the German waltzing guinea pig.

The German waltzing guinea pig is a spontaneously mutated strain with severe auditory and vestibular impairment caused by a so far unknown genetic mutation. The animals are born deaf and show a circling behavior. The heterozygote animals of this guinea pig strain have functionally normal hearing and balance. However, these animals have, in earlier studies, shown an increased resistance to noise compared with normal wild-type guinea pigs. In the present study, we explored the functional hearing with auditory brainstem response thresholds before and at different time points after noise exposure. Symptom-free littermates from heterozygote couples of the German waltzing guinea pigs were exclusively used for the study, which, after the hearing test, were sent back for breeding to confirm their genotype (i.e. heterozygote or normal). The aim of this paper was to ascertain that the previously shown reduced susceptibility to noise trauma in the heterozygote animals of the German waltzing guinea pig was also evident when littermates were used as control animals. The findings are important for further analysis of the heterozygote animals of this strain and for future investigations of the underlying mechanisms behind the diverse susceptibility to exposures of loud sound.

Engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to 11ß-hydrocortisone.

11ß-Hydrocortisone (11ß-HC) is an important anti-inflammatory drug and intermediate for the synthesis of other steroids. One of the methods for the synthesis of 11ß-HC is the asymmetric reduction of cortisone catalyzed by a highly regioselective and stereoselective 11ß-hydroxysteroid dehydrogenase (11ß-HSDH). However, this process has been prohibited by the poor soluble expression of the membrane-anchoring protein 11ß-HSDH in prokaryotes. To overcome this obstacle, a mutant III-1G1 (Phe80Leu/Thr105Ser/Ala260Thr/Tyr274Stop) truncated at position 274 with improved yield of soluble protein was stepwise obtained from the 11ß-HSDH from guinea pig by random mutagenesis combining with structural complementation assay and C-terminal truncating library screening. The improved 11ß-HSDH mutant and glucose dehydrogenase (GDH) from Bacillus subtilis were coexpressed in Escherichia coli. The resulting whole-cell biocatalyst catalyzed the reduction of cortisone to 11ß-HC with 98 % conversion in 20 h, laying foundation for the development of an asymmetric reduction process for the production of 11ß-HC.