Genome-wide association study of copy number variations with shank traits in a F2 crossbred chicken population.

Copy number variations (CNVs) are large-scale changes in the DNA sequence that can affect the genetic structure and phenotype of an organism. The purpose of this study was to investigate the existing CNVs and their associations with the shank diameter (ShD) and shank length (ShL) traits using data from an F2 crossbred chicken population. To carry out the study, 312 chickens were genotyped using the Illumina 60k SNP Beadchip. The shank traits of the birds were measured from day 1 to 12 weeks of age. penncnv and cnvruler tools were used to find copy numbers and regions with copy number changes (CNVR), respectively. The CNVRanger package was used to perform a genome-wide association study between shank traits and CNVs. Gene ontology research in CNVRs was carried out using the david database. In this investigation, 966 CNVs and 606 regions with copy number changes were discovered. The copy number states and variations were randomly distributed along the length of the autosomal chromosomes. Weeks 1-4, 9 and 12 of growth revealed a significant association of copy number variations with shank traits, false discovery rate (FDR-corrected p-value < 0.01), and the majority of CNVs that were statistically significant were found on chromosomes 1-3. These CNV segments are nearby genes such as KCNJ12, FGF6 and MYF5, which are fundamental to growth and development. In addition, gene set analyses revealed terms related to muscle physiology, regulation of cellular processes and potassium channels.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Genetic variants in the TAC1 transcriptional regulatory region affect on trainability and excitability levels in Belgian Malinois dogs.

BACKGROUND: Trainability in dogs is affected by learning aptitude and memory capacity. While this trait has a heritable basis in canines, the specific genetic loci responsible for it remain unknown. Our previous results suggested that the BDNF, CCK and TAC1 genes are associated with learning and memory in canines. Experimental validation is crucial to confirm the effects of these candidate genes on trainability. Understanding the genetic foundation of this trait would offer insight into the inheritance pattern of complex behavioural characteristics. OBJECTIVE: This study aimed to assess the genetic variations within candidate genes and explore their potential associations with behavioural phenotypes in dogs. METHODS: The behavioural characteristics of 123 male Belgian Malinois dogs were assessed using a customised questionnaire. Target regions of candidate genes were screened for genetic variation by single-strand conformational polymorphism (SSCP). Following that, SSCP banding patterns were sequenced, and putative transcription factor binding sites were predicted using bioinformatics tools. Quantitative association analysis between identified genetic variants and behavioural trait scores was performed using the general linear model (GLM). RESULTS: Sequencing the coding and flanking regions revealed three mutations (c.-89C>T, c.-162G>C and c.*33T>A) in the dog TAC1 gene. Bioinformatics analysis predicted two single nucleotide polymorphisms (SNPs) were located within the putative TAC1 promoter and could disrupt transcription factor binding sites. Statistical tests revealed that the c.-89C>T was significantly associated with excitability (p < 0.01), while the c.-162G>C was significantly associated with trainability level (p < 0.05). CONCLUSION: In summary, we identified two regulatory SNPs in the 5'-UTR promoter region of the TAC1 gene that are associated with excitability and trainability in Belgian Malinois dogs. These genetic variations have the potential to alter the binding sites of transcription factors NRF1 and OTX1, consequently influencing TAC1 expression and related behavioural characteristics. Our findings implicate TAC1 polymorphisms as candidates influencing breed-specific behavioural characteristics in canines. Further studies on diverse breeds of dogs are necessary to validate these SNPs' effects.

The effect of peroxisome proliferator-activated receptor gamma (PPARγ) as a mediator of dietary fatty acids and thiazolidinedione in pulmonary arterial hypertension induced by cold stress of broilers.

The objective of this experiment was to evaluate the effects of dietary fish oil and pioglitazone as peroxisome proliferators-activated receptor gamma (PPARγ) activating ligands on the reduction of cold-induced ascites in broiler chickens. A total of 480 one-day-old (Ross 308) male chicks were randomly allocated to four treatment groups with eight replicates of 15 birds each. The following treatments were used: 1) ambient temperature (negative control), with basal diet; 2) cold-induced ascites (positive control), with basal diet; 3) cold-induced ascites, with basal diet +10 mg/kg/day pioglitazone and 4) cold-induced ascites, with basal diet +1% of fish oil. When compared with the positive control, body weight gain was higher (P ≤ 0.05) for broilers fed diets containing fish oil and pioglitazone at 28, 42, and 0-42 d. Broilers under cold-induced ascites had the highest blood pressure at 21 and 42 d, while fish oil and pioglitazone treatment reduced the blood pressure (P ≤ 0.05). Red blood cells, white blood cells, hematocrit, erythrocyte osmotic fragility, bursa of Fabricius and spleen weights were improved (P ≤ 0.05) for chickens fed fish oil diets and pioglitazone compared to the cold-induced ascites (positive control). Exposure to cold temperature resulted in an increase in plasma T3 and T3/T4 ratio and decline in plasma T4 (P ≤ 0.05). In conclusion, PPARγ agonist pioglitazone and fish oil as source of omega-3 polyunsaturated fatty acid could be used as a strategy to reduce the negative effects of pulmonary arterial hypertension and ascites in broiler chickens.

Investigating the expression of fertility-regulating LncRNAs in multiparous and uniparous Shal ewe's ovaries.

Sheep is the primary source of animal protein in Iran. Birth type is one of the significant features that determine total meat output. Little is known about how long non-coding RNAs (LncRNAs) affect litter size. The purpose of this research is to investigate the DE-LncRNAs in ovarian tissue between multiparous and uniparous Shal ewes. Through bioinformatics analyses, LncRNAs with variable expression levels between ewes were discovered. Target genes were annotated using the DAVID database, and STRING and Cytoscape software were used to evaluate their interactions. The expression levels of 148 LncRNAs were different in the multiparous and uniparous ewe groups (false discovery rate (FDR) < 0.05). Eight biological process terms, nine cellular component terms, 10 molecular function terms, and 38 KEGG pathways were significant (FDR < 0.05) in the GO analysis. One of the most significant processes impacting fertility is mitogen-activated protein kinase (MAPK) signaling pathway, followed by oocyte meiosis, gonadotropin-releasing hormone signaling pathway, progesterone-mediated oocyte maturation, oxytocin signaling pathway, and cAMP signaling pathway. ENSOARG00000025710, ENSOARG00000025667, ENSOARG00000026034, and ENSOARG00000026632 are LncRNAs that may affect litter size and fertility. The most crucial hub genes include MAPK1, BRD2, GAK, RAP1B, FGF2, RAP1B, and RAP1B. We hope that this study will encourage researchers to further investigate the effect of LncRNAs on fertility.

Peroxisome proliferator-activated receptor gamma (PPARγ) activation: a potential treatment for ascites syndrome in broiler chickens.

Ascites (serous fluid accumulation in the abdominal cavity) has been observed worldwide in fast growing broilers. Pulmonary vascular remodeling is an important pathological feature of broiler ascites syndrome. Peroxisome proliferators-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) are expressed in pulmonary vascular endothelial cells and vascular smooth muscle cells (VSMC) where they participate in the regulation of normal pulmonary vascular function. The objective of the present study was to investigate the effects of omega-3 fatty acids (found in fish oil) and pioglitazone (PIO) as natural and synthetic PPARγ ligands supplementation on PPARγ and PGC-1α expression in the prevention of pulmonary arterial hypertension (PAH) syndrome in broiler chickens. The experiment was conducted with 4 treatment groups: 1) negative control, normal temperature conditions with basal diet; 2) positive control, low-temperature conditions with basal diet; 3) positive control + 10 mg PIO/kg of weight/d and 4) positive control + 1% FO. Each treatment had 5 replicates. Ascites heart index (RV/TV) was significantly (P < 0.05) reduced in chickens receiving FO (0.20) and PIO (0.21) compared to the positive control group (0.26). The addition of PIO in broilers under cold-induced ascites significantly increased the expression of PPARγ (9.44) and PGC-1α (5.81) genes in lung tissue compared to the negative control group (1.03, P < 0.05). Proliferative indexes of VSMC in pulmonary arteries such as PMT, PIT, and percentage wall thickness were significantly elevated in positive control group, indicating that pulmonary vascular remodeling occurred following VSMC proliferation in ascites. The vessel internal diameter was increased in FO and PIO groups. Based on these results, activation and expression of PPARγ and PGC-1α genes as a critical regulator of pulmonary artery smooth muscle cell using ligands, especially PIO, can be effective in reducing the incidence of PAH in broiler chickens.

Trade-off between feed efficiency and immunity in specialized high-performing chickens.

Based on resource allocation theory, ignoring importance of immunity, and focus on growth and feed efficiency (FE) traits in breeding plans may lead to serious weakness in immune system performance. However, in poultry the adverse effects of selection for FE on the immune system are unclear. Therefore, an experiment was conducted to study the trade-off between FE and immunity using a total of 180 high-performing specialized male chickens from a commercial broiler line which were selected over 30 generations for growth (body weight gain, BWG) and FE (residual feed intake, RFI). Birds were reared for 42 d and 5 FE-related traits of the birds in the last week were considered including daily feed intake (DFI), feed conversion ratio (FCR), residual feed intake (RFI), residual BW gain (RG), and residual intake and gain (RIG). For all 180 chickens, immune system performance including humoral immune response, cell-mediated immunity (CMI), and the activity of lysozyme enzyme (L. activity) as innate immunity was measured. After ascending sort of each FE records, 10% of higher records (H-FE: N = 18) and 10% of lower records (L-FE: N = 18) were determined, and immunity between L-FE and H-FE groups were compared. Moreover, L-BWG and H-BWG were analyzed because BWG is one of components in the FE formula. Performance of the immune system was not statistically different for CMI in none of the studied FE groups. Moreover, high and low groups for DFI and BWG were not different regarding the immunity of the birds. Antibody titers against Newcastle disease virus (NDV) were different between low and high groups of FCR, RG, and RIG. Likewise, SRBC-derived antibodies were significantly different between RFI groups. Rather than humoral immunity, RIG had adversely effect on the innate immunity. Results of the present study showed that although RIG is a more appropriate indicator for FE, choosing for high RIG can weaken the performance of the both humoral and innate immune systems, while RFI had fewer adverse effects.

Gene expression pattern and molecular mechanisms involved in Shal and Sangsari sheep fertility using RNA-Seq.

Ovulation rate and litter size are the main reproductive traits with high economic value in the sheep breeding industry. In this study, three Shal ewes (multiparous) and three Sangsari ewes (uniparous) at the age of 5 were used. The live weight was between 45 and 50 kg at an extremely body condition score of 3. These breeds are marked seasonal reproduction activity and are often bred in semi-closed breeding systems. Total RNAs were extracted from the ovarian tissues, and RNA sequencing was carried out. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was then used to annotate genes, and the string database and the Cytoscape software were used to investigate their interactions. Then path-act network analysis and gene-act network analysis were investigated. The results indicated that 19,932 genes were differentially expressed. The 5968 differentially expressed genes were identified in Shal ewe's ovarian tissue compared to Sangsari ewes (FDR < 0.05), of which 2921 genes were up-regulated and 3047 genes were down-regulated. Bioinformatics analysis exhibited that most of the biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways associated with significant DEGs (Differentially Expressed Genes) in the two studied breeds are associated with oocyte maturation and metabolism. MAPK signalling pathways and Ubiquitin-mediated proteolysis are the most important biological pathways associated with reproductive and fertility traits in the Shal breed. AKT3, MAPK8, MAPK9 and RELA genes are also important genes related to the fertility of multiparous sheep. Analysis of ovarian RNA-seq data identified that most of the differentially expressed genes were involved in various reproductive processes including folliculogenesis, ovulation, ovarian and embryonic development. The MAPK signalling pathway had the most interaction with other pathways, and the AKT3 gene could be a powerful candidate gene in the reproduction and fertility of Shal sheep. These results could pave the way for future efforts to address sheep prolificacy barriers.

Identification of differentially expressed long noncoding RNAs in the ovarian tissue of Shal and Sangsari ewes using RNA-seq.

BACKGROUND: The ovary has an important role in reproductive function. Animal reproduction is dominated by numerous coding genes and noncoding elements. Although long noncoding RNAs (LncRNAs) are important in biological activity, little is known about their role in the ovary and fertility. METHODS: Three adult Shal ewes and three adult Sangsari ewes were used in this investigation. LncRNAs in ovarian tissue from two breeds were identified using bioinformatics analyses, and then target genes of LncRNAs were discovered. Target genes were annotated using the DAVID database, and their interactions were examined using the STRING database and Cytoscape software. The expression levels of seven LncRNAs with their target genes were assessed by real-time PCR to confirm the RNA-seq. RESULTS: Among all the identified LncRNAs, 124 LncRNAs were detected with different expression levels between the two breeds (FDR < 0.05). According to the DAVID database, target genes were discovered to be engaged in one biological process, one cellular component, and 21 KEGG pathways (FDR < 0.05). The PES1, RPS9, EF-1, Plectin, SURF6, CYC1, PRKACA MAPK1, ITGB2 and BRD2 genes were some of the most crucial target genes (hub genes) in the ovary. CONCLUSION: These results could pave the way for future efforts to address sheep prolificacy barriers.

An association of CEP78, MEF2C, VPS13A and ARRDC3 genes with survivability to heat stress in an F2 chicken population.

Heat stress is a serious problem in the poultry industry. An effective tool for improving heat tolerance can be genomic selection based on single nucleotide polymorphisms. This study was performed to identify genomic regions controlling survivability to heat stress in a population of F2 chickens that accidentally experienced acute heat stress, using Illumina 60K Chicken SNP Bead Chip. After quality control in markers, 47,730 SNPs remained for genome-wide association study (GWAS). The GWAS results indicated that markers Gga_rs16111480 (p = 8.503e-08), GGaluGA354375 (p = 5.99e-07) and Gga_rs14748694 (p = 7.085e-07) located on Z chromosome showed significant association with heat stress tolerance trait. The Gga_rs16111480 marker was located inside the CEP78 gene. The marker GGaluGA354375 was located inside the LOC101752071 gene and next to the MEF2C gene. The Gga_rs14748694 marker was adjacent to LOC101752071 and MEF2C genes. Moreover, the SNP maker of Gga_rs16111480 was located on 243 kb downstream of the VPS13A gene, and the GGaluGA354375 and Gga_rs14748694 SNPs were located on 947 kb and 888 kb downstream of the ARRDC3 gene, respectively. The results of this study suggest that apart from the gene LOC101752071, which its function was unknown, each of the two MEF2C and CEP78 genes were found to be closely related to heat stress resistance in bird.

Application of Metabolomics to Identify Hepatic Biomarkers of Foie Gras Qualities in Duck.

Foie gras is a traditional dish in France that contains 50 to 60% of lipids. The high-fat content of the liver improves the organoleptic qualities of foie gras and reduces its technological yield at cooking (TY). As the valorization of the liver as foie gras products is strongly influenced by the TY, classifying the foie gras in their potential technological quality before cooking them is the main challenge for producers. Therefore, the current study aimed to identify hepatic biomarkers of foie gras qualities like liver weight (LW) and TY. A group of 120 male mule ducks was reared and overfed for 6-12 days, and their livers were sampled and analyzed by proton nuclear magnetic resonance (1H-NMR). Eighteen biomarkers of foie gras qualities were identified, nine for LW and TY, five specific to LW, and four specific to TY. All biomarkers were strongly negatively correlated to the liver weights and positively correlated to the technological yield, except for the lactate and the threonine, and also for the creatine that was negatively correlated to foie gras technological quality. As a result, in heavy livers, the liver metabolism was oriented through a reduction of carbohydrate and amino acid metabolisms, and the plasma membrane could be damaged, which may explain the low technological yield of these livers. The detected biomarkers have been strongly discussed with the metabolism of the liver in nonalcoholic steatohepatitis.

Identification of Plasmatic Biomarkers of Foie Gras Qualities in Duck by Metabolomics.

The foie gras is an emblematic product of French gastronomy composed of waterfowl fatty liver. The organoleptic qualities of this product depend on the liver characteristics such as liver weight (LW) and technological yield (TY) at cooking. One of the main issues for producers is to classify the foie gras with high or low technological quality before cooking them. Thus the study aims at identifying biomarkers of these characteristics with non-invasive biomarkers in duck. 1H-NMR (nuclear magnetic resonance of the proton) analyses were performed on plasma of male mule ducks at different time points during the overfeeding period to obtain a large range of liver characteristics so as to identify plasmatic biomarkers of foie gras. We used two methods, one based on bucket data from the 1H-NMR spectra and another one based on the fingerprints of several metabolites. PLS analyses and Linear models were performed to identify biomarkers. We identified 18 biomarkers of liver weight and 15 biomarkers of technological yield. As these two quality parameters were strongly correlated (-0.82), 13 biomarkers were common. The lactate was the most important biomarker, the other were mainly amino acids. Contrary to the amino acids, the lactate increased with the liver weight and decreased with the technological yield. We also identified 5 biomarkers specific to LW (3 carbohydrates: glucuronic acid, mannose, sorbitol and 2 amino acids: glutamic acid and methionine) that were negatively correlated to liver weight. It was of main interest to identify 2 biomarkers specific to the technological yield. Contrary to the isovaleric acid, the valine was negatively correlated to the technological yield.

Plant extract supplementation as a strategy for substituting dietary antibiotics in broiler chickens exposed to low ambient temperature.

The present study was conducted to investigate the effects of two plant extracts as alternatives to dietary antibiotics in broiler chickens exposed to low ambient temperature. A total of 300 one-day-old male broiler chickens were randomly assigned to four dietary treatments (5 replicate pens; 15 broiler chickens each) which consisted of starter (d 0 to 10), and grower (d 10 to 28) diets. Dietary treatments included a basal diet (negative control, NC) and three similar diets that were either supplemented with 200 mg/kg of Prosopis farcta extract (PFE), Rhus coriaria L. extract (RCE) or an antibiotic premix containing oxytetracycline (positive control, PC). In order to simulate low ambient temperature, room temperature was maintained at 32°C during the first 3 d of the trial and afterwards, the temperature was gradually reduced by approximately 1.5°C each day to 14°C on d 21. PFE and PC treatments exerted a significant effect on body weight gain at d 28. Diet PFE was effective in reducing mortality when compared with diet NC (p < 0.05). Furthermore, diet PFE caused increases in ileal  digestibility of gross energy, dry matter and organic matter when compared with diet NC (p < 0.05). Diets PFE and PC decreased coliforms, total aerobic bacteria and total anaerobic bacteria loads in the caeca when compared with diet NC (p < 0.05). Moreover, the addition of PFE to the diet improved villous height in all small intestinal segments as well as villous height:crypt depth ratio in the duodenum when compared with diet NC (p < 0.05). The results indicated that PFE is not only a valid alternative to oxytetracycline under cold stress conditions, with no antibiotic resistance, but also has the potential to increase the resistance of broiler chickens against ascites syndrome. Moreover, the addition of RCE at the concentration of 200 mg/kg to the diet was not sufficient to improve the performance of broiler chickens (similar to diet PC) but maybe more effective at higher concentrations.

WITHDRAWN:Investigating population structure and genetic diversity of some Iranian native cattle breeds using SNP markers.

Ahead of Print article withdrawn by publisher.

Differential Expression of the Alpha S1 Casein and Beta-Lactoglobulin Genes in Different Physiological Stages of the Adani Goats Mammary Glands.

BACKGROUND: Milk proteins genes have been the focus of the researches as the candidate target genes that play a decisive role when animal breeding is desired. OBJECTIVES: In the present study, the transcriptional levels of Beta-lactoglobulin (BLG) and Alpha S1 casein (CSN1S1) genes were investigated during prenatal, milking and drying times in mammary glands of the Adani goats which showed high and low breeding values. MATERIALS AND METHODS: The breeding values of the animals were estimated first by applying multi-trait random regression model. Using the biopsy gun, the mammary gland samples were taken and real-time PCR was applied to search the expression of the genes. Fixed factors of the model were the breeding value groups, sampling times and their interactions. RESULTS: The interactions were significant for both genes. At milking time, the high breeding value group exhibited more transcriptional levels for BLG and less transcriptional levels for CSN1S1 gene compared with the low breeding value group. The expression patterns of these genes were also different between the two breeding value groups. The maximum level of BLG and CSN1S1 transcriptions were found to occur at drying time. CONCLUSIONS: A difference in the gene expression was observed between the two groups which indicate the change in the nucleotide sequence for transcription factor binding sites, or miRNA binding sites, otherwise in the coding regions. Therefore, the variations in the coding and promoter regions of this gene should be investigated in the further studies.

Identification of learning and memory genes in canine; promoter investigation and determining the selective pressure.

One of the important behaviors of dogs is trainability which is affected by learning and memory genes. These kinds of the genes have not yet been identified in dogs. In the current research, these genes were found in animal models by mining the biological data and scientific literatures. The proteins of these genes were obtained from the UniProt database in dogs and humans. Not all homologous proteins perform similar functions, thus comparison of these proteins was studied in terms of protein families, domains, biological processes, molecular functions, and cellular location of metabolic pathways in Interpro, KEGG, Quick Go and Psort databases. The results showed that some of these proteins have the same performance in the rat or mouse, dog, and human. It is anticipated that the protein of these genes may be effective in learning and memory in dogs. Then, the expression pattern of the recognized genes was investigated in the dog hippocampus using the existing information in the GEO profile. The results showed that BDNF, TAC1 and CCK genes are expressed in the dog hippocampus, therefore, these genes could be strong candidates associated with learning and memory in dogs. Subsequently, due to the importance of the promoter regions in gene function, this region was investigated in the above genes. Analysis of the promoter indicated that the HNF-4 site of BDNF gene and the transcription start site of CCK gene is exposed to methylation. Phylogenetic analysis of protein sequences of these genes showed high similarity in each of these three genes among the studied species. The dN/dS ratio for BDNF, TAC1 and CCK genes indicates a purifying selection during the evolution of the genes.

Association of single nucleotide polymorphism of GHSR and TGFB2 genes with growth and body composition traits in sire and dam lines of a broiler chicken.

Growth hormone secretagogue receptor (GHSR) modulates many physiological processes by binding to its ligand, as well as transforming growth factor-beta 2 (TGFB2) regulates cell growth and development in animals and, therefore, are candidate genes for performance in chickens. In the current study, single nucleotide polymorphisms of GHSR C3286 > T and TGFB2 T(-640) > C were genotyped in sire and dam lines of a broiler chicken to evaluate the association with the growth and body composition traits. Least squares means analysis showed that the GHSR C3286 > T SNP was significantly (P < 0.01) associated with growth (DFI and ADG) and body composition traits (AFW and %AFW). In addition, the TGFB2 T(-640) > C SNP was associated with ADG (P < 0.05) and DFI and body composition traits (DW, LBW, BAKWT, %BMW, %HNDWT and %CW) (P < 0.01). Significant associations of the single nucleotide polymorphisms (SNPs) on the traits reported in the present study might be the distinct usage of codons in avian, or relating to an enhancer element and modulating the expression of the gene in chicken. The data indicated that these SNPs could be valuable genetic elements for selection of chickens for better performance in the population.

Exclusion of NEU1 and PPGB from candidate genes for a lysosomal storage disease in Japanese Black cattle.

A case of lysosomal storage disease has been reported in a calf of Japanese Black cattle. Lysosomal storage diseases are hereditary diseases caused by deficiency of lysosomal hydrolases. The clinical and pathological features and accumulated substrates of the affected animal indicated a possibility of sialidosis or galactosialidosis caused by deficiency of neuraminidase (NEU1) or protective protein for beta-galactosidase (PPGB). In the present study, we investigated nucleotide sequences of the genes encoding these two proteins to evaluate whether mutation of these genes is involved in this disease. We determined cattle genomic sequences of these two genes by using bovine EST sequences and the nucleotide sequences of all exons of these genes were compared between affected and normal animals. The results showed several nucleotide substitutions, but none of them was a functional mutation or specific to the affected animal. Furthermore, genotyping of the microsatellite markers in the vicinity of these two genes revealed no homozygosity of the chromosomal regions including these genes in the affected animal. These findings indicated that neither NEU1 nor PPGB gene is responsible for the lysosomal storage disease of Japanese Black cattle and therefore the disease is neither sialidosis nor galactosialidosis.

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene.

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.