Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development.

We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad-mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

Cartilage-Sparing Properties of Equine Omega Complete in an Organ Culture Model of Cartilage Inflammation.

The purpose of this study was to determine anti-inflammatory and/or chondroprotective effects of Equine Omega Complete (EOC) on cartilage explants stimulated with lipopolysaccharide (LPS). Explants were aseptically prepared from the intercarpal joints of 17 market-weight pigs and placed in culture at 37°C for a total of 120 hours. For the final 96 hours, explants were conditioned with a simulated digestion extract of EOC (0, 36 or 180 µL/mL), and for the final 48 hours explants were stimulated with LPS (0 or 15µg/mL). Media was removed and replaced every 24 hours. Samples from the final 48 hours were analyzed for biomarkers of cartilage inflammation (prostaglandin E2 [PGE2] and nitric oxide [NO]) and cartilage structure (glycosaminoglycan [GAG]). At the end of the culture period cartilage explants were stained for an estimate of cell viability. Stimulation of unconditioned explants with LPS significantly increased media concentrations of PGE2, GAG and NO compared with that from unstimulated explants. LPS stimulation did not significantly affect cell viability. Both concentrations of EOC prevented significant LPS-stimulated cartilage release of GAG without impairing chondrocyte viability. No other effects of treatment were observed. These data provide evidence for a non-cytotoxic, chondroprotective effect of EOC in cartilage. This in vitro experiment supports the use of EOC in protecting against the detrimental effects of inflammation on cartilage structure.

Mechanical behaviour of healthy versus alkali-lesioned corneas by a porcine organ culture model.

BACKGROUND: Cornea is a composite tissue exhibiting nonlinear and time-dependent mechanical properties. Corneal ulcers are one of the main pathologies that affect this tissue, disrupting its structural integrity and leading to impaired functions. In this study, uniaxial tensile and stress-relaxation tests are developed to evaluate stress-strain and time-dependent mechanical behaviour of porcine corneas. RESULTS: The samples are split in two groups: some corneas are analysed in an unaltered state (healthy samples), while others are injured with alkaline solution to create an experimental ulcer (lesioned samples). Furthermore, within each group, corneas are examined in two conditions: few hours after the enucleation (fresh samples) or after 7 days in a specific culture medium for the tissue (cultured samples). Finally, another condition is added: corneas from all the groups undergo or not a cross-linking treatment. In both stress-strain and stress-relaxation tests, a weakening of the tissue is observed due to the imposed conditions (lesion, culture and treatment), represented by a lower stiffness and increased stress-relaxation. CONCLUSIONS: Alkali-induced corneal stromal melting determines changes in the mechanical response that can be related to a damage at microstructural level. The results of the present study represent the basis for the investigation of traditional and innovative corneal therapies.

Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection.

Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved.

Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

In vitro characterization and genetic diversity of Bordetella avium field strains.

Bordetella avium (BA) is a respiratory pathogen of particular importance for turkeys. Specific adherence and damage to the respiratory epithelia are crucial steps of the pathogenesis, but knowledge about the mechanisms and the variety of virulence in field strains is limited. We analysed 17 BA field strains regarding their in vitro virulence-associated properties in tracheal organ cultures (TOC) of turkey embryos, and their genetic diversity. The TOC adherence assay indicated that BA field strains differ considerably in their ability to adhere to the tracheal mucosa, while the TOC ciliostasis assay illustrated a high degree of diversity in ciliostatic effects. These two virulence-associated properties were associated with each other in the investigated strains. Three of the investigated strains displayed significantly (P > 0.05) lower in vitro virulence in comparison to other strains. Genetic diversity of BA strains was analysed by core genome multilocus sequence typing (cgMLST). We applied a cgMLST scheme comprising 2667 targets of the reference genome (77.3% of complete genome, BA strain 197N). The results showed a broad genetic diversity in BA field strains but did not demonstrate a correlation between sequence type and virulence-associated properties. The cgMLST analysis revealed that strains with less marked virulence-associated properties had a variety of mutations in the putative filamentous haemagglutinin gene. Likewise, amino acid sequence alignment indicated variations in the protein. The results from our study showed that both adherence and ciliostasis assay can be used for virulence characterization of BA. Variations in the filamentous haemagglutinin protein may be responsible for reduced virulence of BA field strains.

Prolactin induces lipid synthesis of organ-cultured pigeon crops.

The objective of this research was to examine the effects of prolactin (PRL) on the lipid synthesis of organ-cultured pigeon crops in vitro. In experiment 1, the histology, activities of enzymes, and expression of genes involved in metabolism and apoptosis of organ-cultured pigeon crops were analyzed over a 7-d culture period. The results showed that cultured crops maintained their structural integrity for up to 3 d in vitro. Beyond 3 d, caspase-3 activity and Bak1 gene expression increased with day of culture, whereas the activities of succinate dehydrogenase, Na+-K+-ATPase, Ca2+-Mg2+-ATPase, total ATPase, and gene expression of Bcl-2 and CK-19 diminished (P < 0.05). In experiment 2, the crops were cultured for 24, 36, and 48 h in medium containing 0, 25, or 50 ng/mL PRL, respectively, and the accumulation of lipid droplets, lipid content, and expression of fatty acid transportation- and lipogenesis-related genes were analyzed. The results showed that the crops with PRL supplements showed higher amounts of lipid droplets than those of the controls, and the droplets were mainly located in the basal nutritive layer in response to PRL. The efficacy of inducing lipid accumulation increased as the concentration of PRL increased. Crops with 50 ng/mL PRL incubated for 36 h displayed the maximal lipid content. Increasing the concentration of PRL from 0 to 50 ng/mL resulted in a dose-dependent increase in the expression of acetyl-CoA carboxylase, fatty acid synthase, fatty acid translocase, fatty acid binding protein 5, acyl-CoA binding protein, and peroxisome proliferator-activated receptor γ genes after incubation for 36 h (P < 0.05). Therefore, our results indicated that the organ-cultured pigeon crops maintained good viability for up to 3 d in vitro. Furthermore, PRL induced the lipid synthesis of organ-cultured pigeon crops in a dose- and time-dependent manner, which was related to the increased expression of genes involved in fatty acid transportation and lipogenesis.

Establishment and characterization of a canine keratinocyte organoid culture system.

BACKGROUND: Perturbations of epidermal and follicular homeostasis have been attributed to a variety of skin diseases affecting dogs. The availability of an in vitro system to investigate these diseases is important to understand underlying pathomechanisms. OBJECTIVES: To establish an accurate and reliable in vitro 3D system of canine keratinocyte organoids to lay the basis for studying functional defects in interfollicular epidermis (IFE) and hair follicle (HF) morphogenesis, reconstitution and differentiation that lead to alopecic and epidermal diseases. ANIMALS: Skin biopsies were obtained from freshly euthanized dogs of different breeds with no skin abnormalities. METHODS: Cells derived from microdissected IFE and HFs were seeded in Matrigel and keratinocyte organoids were grown and characterized using immunohistochemistry, RT-qPCR and RNA sequencing. RESULTS: Both organoid lines develop into a basal IFE-like cell type. Gene and protein expression analysis revealed high mRNA and protein levels of keratins 5 and 14, IFE differentiation markers and intercellular molecules. Key markers of HF stem cells were lacking. Withdrawal of growth factors resulted in upregulation of markers such as KRT16, Involucrin, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. CONCLUSION AND CLINICAL IMPORTANCE: Our 3D in vitro culture system provides the basis to explore epidermal function, to investigate the culture conditions necessary for the development of organoids with a HF signature and to address cutaneous disorders in dogs. However, for induction of HF signatures or hair growth, addition of different growth factors or co-culture with dermal papilla will be required.

Lifting the Veil on Reptile Embryology: The Veiled Chameleon (Chamaeleo calyptratus) as a Model System to Study Reptilian Development.

Living amniotes comprise three major phylogenetic lineages: mammals, birds, and non-avian reptiles. Mouse and avian embryos continue to be the primary species used in experimental settings to further our knowledge and understanding of the genetics and embryology of amniotes. In comparison, non-avian reptiles, which constitute up to 40% of all living amniotes, have played a comparatively minor role. Studies of non-avian reptiles are, however, paramount for providing insights into the evolutionary changes that occurred in the transition from reptilian-like amniote ancestors to derived mammalian and avian species. Here, we introduce the Veiled Chameleon, a squamate reptile, as a new experimental model for examining fundamental questions in development, evolution, and disease.

Development of a novel ex vivo equine corneal model.

OBJECTIVE: To develop an ex vivo equine corneal organ culture model. Specifically, to assess the equine cornea's extracellular matrix and cellularity after 7 days using two different culture techniques: either (i) immersion system or (ii) air/liquid interface system, to determine the best ex vivo equine corneal model. ANIMALS STUDIED: Fourteen healthy equine corneas of various breeds. PROCEDURES: Equine corneas with 2 mm of perilimbal sclera were freshly harvested from 7 horses undergoing humane euthanasia. One corneal-scleral ring (CSR) from each horse was randomly placed in the (i) immersion condition organ culture system (IC), with the contralateral CSR being placed in the (ii) air/liquid interface organ culture system (ALC) for 7 days. All corneas were evaluated using serial daily gross photography, histology, qPCR, and TUNEL assay. RESULTS: corneal-scleral rings placed in the IC (i) had complete loss of corneal transparency on gross photography by 7 days, showed a significant level of corneal stromal disorganization, significantly increased α-SMA levels on qPCR, and apoptosis on TUNEL assay compared to controls. The ALC (ii) had weak stromal disorganization on histopathologic examination and was not significantly different from normal equine corneal controls on all other evaluated parameters. CONCLUSIONS: The air-liquid interface organ culture system maintains the equine cornea's extracellular matrix and preserves corneal transparency, while the immersion condition results in near complete degradation of normal equine corneal architecture after 7 days in culture. The air-liquid organ culture is a viable option to maintain a healthy equine cornea in an ex vivo setting for wound healing studies.

An ovarian bioreactor for in vitro culture of the whole bovine ovary: a preliminary report.

BACKGROUND: Improved cancer therapeutics and enhanced cancer survivorship have emphasized the severe long-term side effects of chemotherapy. Specifically, studies have linked many chemotherapy agents with primary ovarian insufficiency, although an exact insult model has not yet been determined. To investigate and ultimately solve this problem, a novel device for extended study of mammalian ovaries in vitro was developed. METHODS: A bioreactor was fabricated for bovine ovarian culture that provides intravascular delivery of media to the ovary through isolation and cannulation of a main ovarian artery branch. Whole ovaries were cultured in vitro using three methods: (1) continuously supplied fresh culture media, (2) recirculated culture media, or (3) continuously supplied fresh culture media supplemented with 500 nM doxorubicin for 24 or 48 h. TUNEL assay was used to assess apoptotic cell percentages in the three groups as compared to uncultured baseline ovaries. RESULTS: The ovary culture method was shown to maintain cell viability by effectively delivering nutrient-enriched pH-balanced media at a constant flow rate. Lower apoptosis observed in ovaries cultured in continuously supplied fresh culture media illustrates that this culture device and method are the first to sustain whole bovine ovary viability for 48 h. Meanwhile, the increase in the percentage of cell apoptosis with doxorubicin treatment indicates that the device can provide an alternative model for testing chemotherapy and chemoprotection treatments to prevent primary ovarian insufficiency in cancer patients. CONCLUSIONS: An ovarian bioreactor with consistent culture media flow through an ovarian vasculature-assisted approach maintains short-term whole bovine ovary viability.

Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane.

In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm(3) cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal-Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm(3) in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and/or development. The results show that the CAM is not a suitable system for feline ovarian tissue and highlight the necessity to improve IVC systems in cats.

Establishment of a 2-week canine skin organ culture model and its pharmacological modulation by epidermal growth factor and dexamethasone.

Although canine skin models are already available as either monocellular or organotypic cultures, they only partly recapitulate normal skin morphological features and function. The objective of this study was to establish a canine serum-free skin organ culture model and verify whether dexamethasone could rescue epidermal growth factor-induced changes. The study of morphological changes as a response to pharmacological substances may indeed help to investigate skin physiology and pathology. Normal skin was obtained from five client-owned dogs subjected to surgical procedures unrelated to dermatological conditions. Two experimental designs were performed: (i) two-week viability of the skin culture; (ii) dexamethasone (DMS) inhibition of epidermal growth factor (EGF)-induced effects. Serum-free submerged organ cultures were established in Williams' E medium supplemented with penicillin-streptomycin, insulin, hydrocortisone and l-glutamine. General morphological features of skin anatomical structures were well maintained up to day 14, scattered pyknotic nuclei were visible in the epidermis from day 7. Normal keratinocyte differentiation was confirmed by cytokeratin (K) 10, K14 and loricrin immunostaining. Epidermal thickness did not decrease throughout the study. A decrease in keratinocyte proliferation was observed at day 7 and 14. Treatment with EGF induced both keratinocyte proliferation and thickening of the epidermis; both responses were counteracted by DMS. Treatment with EGF increased the length of epithelial tongues at the edge of the skin explants; this effect was further enhanced by DMS supplementation. Our findings demonstrate the potential use of a full-thickness canine skin organ culture model for the study of skin physiology and pharmacological response to exogenous compounds, especially in the field of re-epithelialisation and keratinization disorders.

Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder.

To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K(+) or carbachol (CCh) and with and without hypoxia (achieved by bubbling N2 instead of O2) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K(+)) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K(+) and CCh. However, hypoxia significantly attenuated H-65K(+)- and CCh-induced contraction. H-65K(+) and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K(+)- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K(+), hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K(+)- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder.

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus-host interactions.

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus-host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus-host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P < 0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P < 0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P < 0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV - A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.

Morphologic characterization of isolated bovine early preantral follicles during short-term individual in vitro culture.

To provide new insights in the molecular mechanism controlling preantral follicular development and to unravel the needs to support in vitro follicular development of early-stage preantral follicles (PAFs), there is a need for alternative in vitro bovine follicle culture methods. In this study, we aimed to characterize follicular dynamics using an IVC system of isolated and individually cultured bovine early PAFs during 10 days to generate individual follicle follow-up data. Preantral follicles (<50 µm) were isolated from slaughterhouse ovaries and cultured individually for 10 days. Individual follicle morphology, growth, survival, quality, and cell proliferation were evaluated in time by combining noninvasive and invasive assessment methods. The PAFs were light microscopically evaluated during culture to assess follicular dynamics, stained with neutral red to determine follicle viability, stained with 4',6-diamidino-2-phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling to evaluate cell proliferation and follicle quality, and processed for histologic evaluation to assess follicle morphology. On the basis of their morphology, follicles were subdivided in three categories, with category 1 follicles showing the best morphologic features. On Day 0, only category 1 follicles were selected, but follicle categories were reassigned on evaluation Days 1, 2, 4, 7, or 10. Although 67% of the follicles survived 10 days of IVC, the number of follicles exhibiting a normal morphology decreased significantly from Day 7 onward and the apoptotic index increased significantly from Day 10. Both category 1 and 2 follicles showed a significant increase in follicular diameter (Day 10: 21.80 ± 0.86 and 11.82 ± 0.80, respectively). This increase in follicular diameter showed to be correlated with an increase in the total cell number. In conclusion, this culture system showed to support follicular development until Day 10, although the proportion of follicles showing normal morphologic features and the follicular quality decreased after 10 days of IVC. Follicles maintaining their category 1 morphologic features over time seem to be of a better quality and show a higher developmental competence as compared to category 2 and 3 follicles.

Identification and response to metals of metallothionein in two ancient fishes: white sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens).

White sturgeon (Acipenser transmontanus) are among the most sensitive species of fishes to Cu, Cd, and Zn, but there is no information about sensitivity of lake sturgeon (Acipenser fulvescens). To begin to elucidate molecular mechanism(s) of sensitivity of sturgeons to metals a cDNA encoding metallothionein (MT) was amplified from livers of white sturgeon (WS-MT) and lake sturgeon (LS-MT), and expression in response to Cu, Cd, or Zn was characterized in liver explants from each species. The primary structure of WS-MT and LS-MT contained 20 cysteine residues, which is the same as MTs of teleost fishes. However, the primary structure of WS-MT and LS-MT contained 63 amino acids, which is longer than any MT identified in teleost fishes. Abundance of transcripts of WS-MT in explants exposed to 0.3, 3, 30, or 100 µg/L of Cu was 1.7-, 1.7-, 2.1-, and 2.6-fold less than in controls, respectively. In contrast, abundances of transcripts of WS-MT were 3.3- and 2.4-fold greater in explants exposed to 30 µg/L of Cd and 1000 µg/L of Zn, respectively. Abundance of transcripts of LS-MT was not significantly different at any concentration of Cu, Cd, or Zn. MT is hypothesized to represent a critical mechanism for detoxification of metals. Therefore, results of this study suggest that sensitivity of sturgeons to exposure to Cu, Cd, or Zn might be a result of the relatively lesser maximal response of MT to metals. The study also suggestslake sturgeon might be more sensitive than white sturgeon to metals.

Expression pattern of vascular endothelial growth factor in canine folliculogenesis and its effect on the growth and development of follicles after ovarian organ culture.

In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen-thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs.

The required sample size in vaccination-challenge experiments with infectious bronchitis virus, a meta-analysis.

For statistical, animal welfare and financial reasons the choice of the number of chickens per group in experiments is important. This estimation, together with the number of tracheal organ cultures (TOCs) that need to be examined from each chicken in order to assess protection, should be based on the difference in level of protection that one would like to be able to detect (effect size), the expected variability of the results between and within the chickens, the desired confidence level and the power of the study. To obtain data that would facilitate this estimation, a meta-analysis was performed on the data from 18 infectious bronchitis virus (IBV) vaccination-challenge experiments performed at the Dutch Animal Health Service Deventer, the Netherlands (GD) in order to determine and quantify the source of variation in the mean level of protection of different groups. For the calculations, 137 groups of chickens were subdivided into 10 clusters based on age (young or adult), vaccination (none, homologous or heterologous), challenge (IBV or mock infected) and location of vaccination (isolator at GD or in the field). The results were used to estimate the required number of chickens per group for the different clusters using 2, 5 or 10 TOCs per chicken to be able to detect effect sizes of 6.25%, 12.5%, 25% and 50% between groups of chickens with 95% confidence (P<0.05) and 80% power. The number of chickens that was required for the mentioned effect sizes varied greatly from 2 to 650. This meta-analysis provided data that allow research workers to estimate the number of chickens that should be included in each group in order to obtain reliable results based on particular combinations of infectious bronchitis vaccination and challenge strains as defined by the presented clusters.

Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification.

There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 µm in diameter) containing growing oocytes (approximately 60 µm in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (P<0.05). Higher percentages of granulosa cell-enclosed oocytes were recovered from the follicles cultured in BPL-supplemented media with 0 and 25 ng/ml FSH, and the oocytes grew to 90 µm or more in diameter. In FCS- and BFF-supplemented media, FSH increased the numbers of degenerating follicles. Next, vitrified-warmed secondary follicles were cultured in BPL-supplemented medium. One third of the follicles showed no degenerative signs, and the oocytes increased in diameter to 88.8 ± 3.1 µm after 4 weeks of culture. These results suggest that a BPL-supplemented medium supports oocyte growth in bovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.