Homozygous Animals Research Articles

Disruption of insulin receptor substrate 2 (IRS2) causes non-obese type 2 diabetes with β-cell dysfunction in the golden (Syrian) hamster

Because of the advent of genome-editing technology, gene knockout (KO) hamsters have become attractive research models for diverse diseases in humans. This study established a new KO model of diabetes by disrupting the insulin receptor substrate-2 (Irs2) gene in the golden (Syrian) hamster. Homozygous KO animals were born alive but with delayed postnatal growth until adulthood. They showed hyperglycemia, high HbA1c, and impaired glucose tolerance. However, they normally responded to insulin stimulation, unlike Irs2 KO mice, an obese type 2 diabetes (T2D) model. Consistent with this, Irs2 KO hamsters did not increase serum insulin levels upon glucose administration and showed β-cell hypoplasia in their pancreas. Thus, our Irs2 KO hamster provide a unique T2D animal model that is distinct from the obese T2D models. This model may contribute to a better understanding of the pathophysiology of human non-obese T2D with β-cell dysfunction, the most common type of T2D in East Asian countries, including Japan.

Role of albumin in the metabolism and excretion of ochratoxin A.

Ochratoxin A (OTA) is known to be strongly bound to serum albumin, but it remains unknown how albumin affects its metabolism and kinetics. To close this gap, we used a mouse model, where heterozygous albumin deletion reduces serum albumin to concentrations similar to hypoalbuminemic patients and completely eliminates albumin by a homozygous knockout. OTA and its potential metabolites (OTα, 4-OH-OTA, 7'-OH-OTA, OTHQ, OP-OTA, OTB-GSH, OTB-NAC, OTB) were time-dependently analyzed in plasma, bile, and urine by LC-MS/MS and were compared to previously published hepatotoxicity and nephrotoxicity data. Homozygous albumin deletion strongly accelerated plasma clearance as well as biliary and urinary excretion of the parent compound and its hydroxylation products. Decreasing albumin in mice by the heterozygous and even more by the homozygous knockout leads to an increase in the parent compound in urine which corresponded to increased nephrotoxicity. The role of albumin in OTA-induced hepatotoxicity is more complex, since heterozygous but not homozygous nor wild-type mice showed a strong biliary increase in the toxic open lactone OP-OTA. Correspondingly, OTA-induced hepatotoxicity was higher in heterozygous than in wild-type and homozygous animals. We present evidence that albumin-mediated retention of OTA in hepatocytes is required for formation of the toxic OP-OTA, while complete albumin elimination leads to rapid biliary clearance of OTA from hepatocytes with less formation of OP-OTA. In conclusion, albumin has a strong influence on metabolism and toxicity of OTA. In hypoalbuminemia, the parent OTA is associated with increased nephrotoxicity and the open lactone with increased hepatotoxicity.

Molecular detection and association of 12.1kb deletion within the high mobility AT-hook 2 gene in the Netherlands dwarf rabbit (Oryctolagus Cuniculus).

Rabbits are mainly bred for human consumption and medical research. However, it has been recently showed that several rabbit breeds are also kept as pets for human leisure. The Netherlands dwarf rabbit is currently in the immense interest of many Vietnamese customers due to its personality and miniature stature. However, 12.1kb deletion from position 44,709,089 to 44,721,236bp in the high mobility AT-hook 2 (HMGA2) gene on chromosome 4 was identified as the structural variant causing dwarfism and altered craniofacial development in this breed. It has been documented that HMGA2 plays an important role in regulating growth and individuals with genotype HMGA2 del/del are fatal several days after birth. Despite the economically high value of the Netherlands dwarf rabbit, there has been no study on the genetic survey of lethal alleles in this breed in Vietnam. The aim of this study is to develop a fast and reliable method to screen the frequency of lethal alleles of HMGA2 in the South of Vietnam. Rabbit saliva was collected, and DNA extraction was followed. Multiplex polymerase chain reaction (PCR) with three primers was optimized and performed to detect the presence of 12.1kb deletion within the HMGA2 sequence. Our data showed that the 12.1kb deletion in the Netherlands dwarf rabbit population was detected by our optimized multiplex PCR. In 100 rabbit animals, 34 and 16 individuals were homozygous wild type (+/+) and homozygous mutant (del/del), respectively, while 50 rabbits were heterozygous. The frequency of HMGA2 lethal allele carrier was 66% (66/100 individuals). Our results indicated that we successfully developed a fast, accurate multiplex PCR to detect carrier individuals. Verification of the genotypes was followed by sequencing. We recommend implementing our multiplex PCR procedure in genetic selection for carrier and homozygous wild-type animals in the mating scheme to prevent the lethality of the rabbit offspring. Additionally, awareness should be raised among rabbit breeders to monitor the genetic makeup of the Netherlands dwarf rabbit populations. However, due to the limitation of the sample size, more samples should be taken in future studies to obtain the genetic frequency of the HMGA2 lethal allele more accurately.

Investigating the Impact of Hydrocephalus on Epiplexus Cells of the Choroid Plexus

Hydrocephalus is characterized by accumulation of cerebrospinal fluid (CSF) in the brain, resulting from over-production, under-absorption, or blockage of the flow of CSF. Current treatments are limited to surgically placed shunts that often require follow-up procedures. The goal of this research group is to understand the mechanisms of hydrocephalus with the aim of finding effective pharmacological treatments. The animal model used exhibits a mutation in the transmembrane 67 (Tmem67) gene. Homozygous recessive animals present with ventriculomegaly at birth and severe hydrocephalus by postnatal day 15 (P15). Experiments in this study compare tissue obtained from wildtype animals and homozygous mutant animals. The choroid plexus (CP) is the main producer of CSF, making it a target structure in hydrocephalus research. It is a branched tissue that includes a stroma with fenestrated capillaries, wrapped by a tightly regulated barrier epithelium. Choroid plexus epithelial cells (CPe) are multiciliated cells that strictly regulate the osmotic balance of CSF. Epiplexus cells are poorly understood immune cells localized on the apical (CSF-facing) side of the CPe. These cells are believed to share functional characteristics with microglia and macrophages. Microglia are the innate immune cells of the central nervous system; a specialized type of glial cell that interact with neurons, and aid in the structure and immunosurveillance of the brain. Macrophages originate from monocytes and reside in most tissues of the body, responding to cellular signals of injury and infection. Macrophages can be found free-roaming in tissues or associated to epithelial cells, known as border-associated macrophages (BAMs). Due to their association with the CPe, epiplexus cells are considered BAMs. Beyond their apparent similarities to other immune cells, little else is known about the properties and functions of epiplexus cells or their interaction with the CPe or CSF, nor if they play a role in the development of hydrocephalus. This study furthers the understanding of epiplexus cells, specifically how they impact, or are impacted by hydrocephalus. Rat pups (P15) were terminally anesthetized, perfused with 1x PBS, and fixed with 4% PFA. In the lateral ventricle CP, we have visualized CPe-associated cells with morphology consistent with epiplexus cells. These cells are labeled with microglial/macrophage/epiplexus cell markers: allograft inflammatory factor1/ionized calcium-binding adaptor molecule 1 (Iba1), macrosialin (CD68), and mannose receptor type C-1 lectin (CD206). Iba1 is thought to play a role in restructuring the actin cytoskeleton in cells of monocyte lineage (macrophages and microglia), resulting in morphological differences across levels of activation. CD68 is a transmembrane protein commonly expressed in macrophages and monocytes, supposedly related to the oxidized low-density lipoprotein (OxLDL) pathway, which is involved in exacerbating oxidative stress. CD206 is a transmembrane protein that may aid in the roles of detecting pathogens, removing proteins, and sending waste through lymphatic system. Confocal analysis combining cell morphology and immunohistochemical labeling will be used to quantify CPe-associated epiplexus cells in wildtype and hydrocephalic animals. The results will be substantiated using quantitative real-time PCR. Funding: United States Department of Defense Congressionally Directed Medical Research Program Award; Hydrocephalus Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hox gene–specific cellular targeting using split intein Trojan exons

The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in Drosophila to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.

Impairments of cerebellar structure and function in a zebrafish KO of neuropsychiatric risk gene znf536

Genetic variants in ZNF536 contribute to the risk for neuropsychiatric disorders such as schizophrenia, autism, and others. The role of this putative transcriptional repressor in brain development and function is, however, largely unknown. We generated znf536 knockout (KO) zebrafish and studied their behavior, brain anatomy, and brain function. Larval KO zebrafish showed a reduced ability to compete for food, resulting in decreased total body length and size. This phenotype can be rescued by segregating the homozygous KO larvae from their wild-type and heterozygous siblings, enabling studies of adult homozygous KO animals. In adult KO zebrafish, we observed significant reductions in anxiety-like behavior and social interaction. These znf536 KO zebrafish have decreased cerebellar volume, corresponding to decreased populations of specific neuronal cells, especially in the valvular cerebelli (Va). Finally, using a Tg[mbp:mgfp] line, we identified a previously undetected myelin structure located bilaterally within the Va, which also displayed a reduction in volume and disorganization in KO zebrafish. These findings indicate an important role for ZNF536 in brain development and implicate the cerebellum in the pathophysiology of neuropsychiatric disorders.

Genotyping KIF1C (c.608G>A) Mutant Reveals a Wide Distribution of Progressive Ataxia in German Charolais Cattle

Bovine progressive ataxia in Charolais cattle was first described in the 1970s; then, cases were reported in Charolais worldwide. A homozygous loss-of-function mutation within the KIF1C gene (c.608G>A) was found to be responsible for this neurodegenerative disease. The aim of this study was to determine whether the mutated KIF1C allele segregates in the German Charolais population and whether the estimated breeding values for growth and muscle conformation are associated with the mutated genotypes. Genetic test results of the KIF1C:c.608G>A variant were available for 1315 Charolais cattle from 35 herds located in Germany. In addition, 324 samples from eight other beef cattle breeds were tested for the mutated KIF1C allele. We were able to demonstrate that the KIF1C mutation is common, with a frequency of 11.75% in the German Charolais population. All but two of the eight (2/8 = 25%) homozygous mutated individuals showed clinical signs consistent with progressive ataxia. The estimated breeding values of muscle conformation in 200- and 365-day-old animals indicated a significant superiority for homozygous mutated animals when compared either with heterozygous or homozygous wild-type genotypes; this was also the case for heterozygous genotypes in comparison with homozygous wild-type genotypes. For the estimated breeding values of daily weight gain in 200- and 365-day-old animals, the significant differences between homozygous mutated and heterozygous or wild-type genotypes were in favour of the homozygous mutant animals. There were no differences in the estimated maternal breeding values among all three KIF1C genotypes. For the first time, two German Angus cattle carrying the KIF1C mutation heterozygous were detected. The breeders’ survey highlighted that increased awareness would facilitate increased conviction among breeders of the need for genetic testing in order to eliminate the lethal KIF1C allele.

Large scale sequence-based screen for recessive variants allows for identification and monitoring of rare deleterious variants in pigs.

Most deleterious variants are recessive and segregate at relatively low frequency. Therefore, high sample sizes are required to identify these variants. In this study we report a large-scale sequence based genome-wide association study (GWAS) in pigs, with a total of 120,000 Large White and 80,000 Synthetic breed animals imputed to sequence using a reference population of approximately 1,100 whole genome sequenced pigs. We imputed over 20 million variants with high accuracies (R2>0.9) even for low frequency variants (1-5% minor allele frequency). This sequence-based analysis revealed a total of 14 additive and 9 non-additive significant quantitative trait loci (QTLs) for growth rate and backfat thickness. With the non-additive (recessive) model, we identified a deleterious missense SNP in the CDHR2 gene reducing growth rate and backfat in homozygous Large White animals. For the Synthetic breed, we revealed a QTL on chromosome 15 with a frameshift variant in the OBSL1 gene. This QTL has a major impact on both growth rate and backfat, resembling human 3M-syndrome 2 which is related to the same gene. With the additive model, we confirmed known QTLs on chromosomes 1 and 5 for both breeds, including variants in the MC4R and CCND2 genes. On chromosome 1, we disentangled a complex QTL region with multiple variants affecting both traits, harboring 4 independent QTLs in the span of 5 Mb. Together we present a large scale sequence-based association study that provides a key resource to scan for novel variants at high resolution for breeding and to further reduce the frequency of deleterious alleles at an early stage in the breeding program.

Fused in sarcoma regulates glutamate signaling and oxidative stress response

Mutations in fused in sarcoma (fust-1) are linked to ALS. However, how these ALS causative mutations alter physiological processes and lead to the onset of ALS remains largely unknown. By obtaining humanized fust-1 ALS mutations via CRISPR-CAS9, we generated a C. elegans ALS model. Homozygous fust-1 ALS mutant and fust-1 deletion animals are viable in C. elegans. This allows us to better characterize the molecular mechanisms of fust-1-dependent responses. We found FUST-1 plays a role in regulating superoxide dismutase, glutamate signaling, and oxidative stress. FUST-1 suppresses SOD-1 and VGLUT/EAT-4 in the nervous system. FUST-1 also regulates synaptic AMPA-type glutamate receptor GLR-1. We found that fust-1 ALS mutations act as loss-of-function in SOD-1 and VGLUT/EAT-4 phenotypes, whereas the fust-1 ALS mutations act as gain-of-function in redox homeostasis and the microbe-induced oxidative stress response. We hypothesized that FUST-1 is a link between glutamate signaling and SOD-1. Our results may provide new insights into the human ALS alleles and their roles in pathological mechanisms that lead to ALS.

Role of the Glycine Receptor β Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and β subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRβ, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRβ that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRβ under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRβ was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRβ enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2β complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.

A nematode model to evaluate microdeletion phenotype expression.

Microdeletion syndromes are genetic diseases caused by multilocus chromosomal deletions too small to be detected by karyotyping. They are typified by complex pleiotropic developmental phenotypes that depend both on the extent of the deletion and variations in genetic background. Microdeletion alleles cause a wide array of consequences involving multiple pathways. How simultaneous haploinsufficiency of numerous adjacent genes leads to complex and variable pleiotropic phenotypes is not well understood. CRISPR/Cas9 genome editing has been shown to induce microdeletion-like alleles at a meaningful rate. Here, we describe a microdeletion allele in Caenorhabditis elegans recovered during a CRISPR/Cas9 genome editing experiment. We mapped the allele to chromosome V, balanced it with a reciprocal translocation crossover suppressor, and precisely defined the breakpoint junction. The allele simultaneously removes 32 protein-coding genes, yet animals homozygous for this mutation are viable as adults. Homozygous animals display a complex phenotype including maternal effect lethality, producing polynucleated embryos that grow into uterine tumors, vulva morphogenesis defects, body wall distensions, uncoordinated movement, and a shortened life span typified by death by bursting. Our work provides an opportunity to explore the complexity and penetrance of microdeletion phenotypes in a simple genetic model system.

DDB2-associated incidence of squamous cell carcinoma in Haflingers: risk minimization by genotyping

SCC (squamous cell carinomas) are among the most common eye neoplasms in horses. In recent studies Haflinger horses with a homozygous genotype for a missense variant in the DDB2 gene (damage specific DNA binding protein 2) had a significant increased risk of developing ocular SCC. The aims of this study were to determine the frequency of the SCC-associated risk allele in the DDB2 gene in Swiss and Austrian Haflinger populations and to validate the previously described phenotypic correlation. For this purpose, Haflingers presented at various horse clinics in Switzerland (n = 21, including 11 SCC cases), privately kept Haflingers (n = 52, including 1 SCC case), and Haflingers from a stud farm in the Austrian Tyrol (n = 53) were recruited. The individual DDB2 genotype of the animals was determined using a polymerase chain ceaction (PCR) test using hair follicle or whole blood samples. Of the 12 horses suffering from SCC, nine had ocular SCC and three had non-ocular SCC. Six of the nine Haflingers with ocular SCC and one of the three Haflingers with non-ocular SCC were homozygous for the DDB2 variant. Of the 113 clinically normal animals, 7/113 were homozygous (6 %) and 42/113 were heterozygous (37 %), which corresponds to an allele frequency of 24,8 % in the control cohort. The risk of ocular SCC occurring in Haflingers is significantly increased with the homozygous DDB2 genotype. However, not all animals with SCC carry this gene variant and not all DDB2 homozygous animals develop SCC, which can be explained by the multifactorial genesis of the disease. Due to the high frequency of the undesirable allele, we recommend taking the individual DDB2 genotype of breeding animals into account in order to avoid homozygous offspring with a greatly increased SCC risk by excluding high-risk matings.

SAT275 Generation And Characterization Of A Humanized CYP21A2 Mouse Model For Congenital Adrenal Hyperplasia

Abstract Disclosure: A. Huebner: None. S. Thirumalasetty: None. T. Schubert: None. R. Naumann: None. I. Reichardt: None. M. Rohm: None. D. Landgraf: None. F. Gembardt: None. M.F. Hartmann: None. S.A. Wudy: None. M. Peitzsch: None. N. Reisch: None. K. Koehler, PhD: None. 21-hydroxylase deficiency (21OHD) is the most common form of congenital adrenal hyperplasia (CAH) and is caused by mutations in the CYP21A2 gene. 21OHD causes a wide array of clinical symptoms that result from gluco- and mineralocorticoid deficiency and adrenal androgen excess. Treatment firstly aims to substitute lacking steroid hormones, and secondly to restore negative feedback towards CRH and pituitary ACTH secretion to diminish adrenal androgen overproduction. In most cases supra-physiological glucocorticoid doses are necessary which may cause short stature, obesity, hypertension, and cardiovascular and metabolic co-morbidity with reduced quality of life. Hence, current steroid substitution regimens have significant limitations, so novel therapeutic strategies are required. In recent years new therapeutic approaches have emerged including new non-glucocorticoid substances interfering with the HPA axis to minimize adrenal androgen production and to lower external glucocorticoid substitution to physiological levels. However, valuable in-vivo models for pre-clinical testing of such drugs are lacking. Here we present the first viable and humanized mouse model in which the mouse gene Cyp21a1 is replaced by the human orthologue CYP21A2 in which the human point mutation R484Q is integrated. Twenty-weeks-old homozygous mice show marked adrenal hyperplasia, enhanced expression of the CYP21A2 gene and a weak increase of Cyp11a1 and Cyp11b2 gene expression. Tandem mass spectrometry measurements of the mice plasma at 20 weeks show decreased corticosterone and 11-deoxycorticosterone levels in the presence of increased ACTH levels in both male and female homozygous animals. Progesterone levels in homozygous mice are significantly higher (p<0.01) than in wildtype mice. We also observed increased aldosterone levels in female mutants whereas blood pressure does not differ between wildtype and mutant mice strains. Histologically, mutants exhibit adrenocortical hyperplasia. While mutant male mice are fertile with normal appearing testes, females are infertile, remain in the diestrus phase and present with a reduced number of ovarian follicles. In parallel, a second mouse strain bearing the I173N mutation was developed. This mutation is frequent in human patients causing simple virilizing or rarely salt wasting CAH. Homozygous mice require dexamethasone treatment during pregnancy and until weaning but are viable without treatment afterwards. Preliminary results show adrenal hyperplasia and alteration in steroidogenic gene expression and steroid profiles. In conclusion, we demonstrate that the humanized mutant CYP21A2 mice may represent an excellent animal CAH model to test novel treatment strategies for CAH patients. We believe that this model(s) will facilitate the transition from basic research into clinical application. Presentation: Saturday, June 17, 2023

Prenylation is essential for the enrichment of cone phosphodiesterase-6 (PDE6) in outer segments and efficient cone phototransduction.

Phosphodiesterase-6 (PDE6) is the key phototransduction effector enzyme residing in the outer segment (OS) of photoreceptors. Cone PDE6 is a tetrameric protein consisting of two inhibitory subunits (γ') and two catalytic subunits (α'). The catalytic subunit of cone PDE6 contains a C-terminus prenylation motif. Deletion of PDE6α' C-terminal prenylation motif is linked to achromatopsia (ACHM), a type of color blindness in humans. However, mechanisms behind the disease and roles for lipidation of cone PDE6 in vision are unknown. In this study, we generated two knock-in mouse models expressing mutant variants of cone PDE6α' lacking the prenylation motif (PDE6α'∆C). We find that the C-terminal prenylation motif is the primary determinant for the association of cone PDE6 protein with membranes. Cones from PDE6α'∆C homozygous mice are less sensitive to light, and their response to light is delayed, whereas cone function in heterozygous PDE6α'∆C/+ mice is unaffected. Surprisingly, the expression level and assembly of cone PDE6 protein were unaltered in the absence of prenylation. Unprenylated assembled cone PDE6 in PDE6α'∆C homozygous animals is mislocalized and enriched in the cone inner segment and synaptic terminal. Interestingly, the disk density and the overall length of cone OS in PDE6α'∆C homozygous mutants are altered, highlighting a novel structural role for PDE6 in maintaining cone OS length and morphology. The survival of cones in the ACHM model generated in this study bodes well for gene therapy as a treatment option for restoring vision in patients with similar mutations in the PDE6C gene.

β-Casein A1 and A2: Effects of polymorphism on the cheese-making process

Of late, "A2 milk" has gained prominence in the dairy sector due to its potential implications in human health. Consequently, the frequency of A2 homozygous animals has considerably increased in many countries. To elucidate the potential implications that beta casein (β-CN) A1 and A2 may have on cheese-making traits, it is fundamental to investigate the relationships between the genetic polymorphisms and cheese-making traits at the dairy plant level. Thus, the aim of the present study was to evaluate the relevance of the β-CN A1/A2 polymorphism on detailed protein profile and cheese-making process in bulk milk. Based on the β-CN genotype of individual cows, 5 milk pools diverging for presence of the 2 β-CN variants were obtained: (1) 100% A1; (2) 75% A1 and 25% A2; (3) 50% A1 and 50% A2; (4) 25% A1 and 75% A2; and (5) 100% A2. For each cheese-making day (n = 6), 25 L of milk (divided into 5 pools, 5 L each) were processed, for a total of 30 cheese-making processes. Cheese yield, curd nutrient recovery, whey composition, and cheese composition were assessed. For every cheese-making process, detailed milk protein fractions were determined through reversed-phase HPLC. Data were analyzed by fitting a mixed model, which included the fixed effects of the 5 different pools, the protein and fat content as a covariate, and the random effect of the cheese-making sessions. Results showed that the percentage of κ-CN significantly decreased up to 2% when the proportion of β-CN A2 in the pool was ≥25%. An increase in the relative content of β-CN A2 (≥50% of total milk processed) was also associated with a significantly lower cheese yield both 1 and 48 h after cheese production, whereas no effects were observed after 7 d of ripening. Concordantly, recovery of nutrients reflected a more efficient process when the inclusion of β-CN A2 was ≤75%. Finally, no differences in the final cheese composition obtained by the different β-CN pools were observed.

Genetic characteristics of Poltavske sriblo rabbits by myostatin and progesterone receptor gene and selection indices

Background. Rabbit breeding is a branch of animal husbandry that produces cheap and high-quality meat products in a short period of time. Productive, maternal, reproductive and technological characteristics determine the economic efficiency of rabbit breeding under the conditions of full realization of the genetic potential of animals and a good fodder base. The main factor in the development of rabbit breeding is the increase in the number of animals, which is supported by improved maintenance, veterinary support and breeding practices. Materials and Methods. The experiment was conducted on a population of rabbits (200 heads) of the Poltavske sriblo breed. After weaning at 45 days of age, the young were separated by sex and kept 3–4 animals in a cage. 3-month-old males were placed in individual cages until they reached the age of breeding use – 150–160 days. Rabbits were fed granulated compound feed: concentrated fodder, grass meal, feed additives of animal origin, minerals and premixes. Blood was taken from the ear vein. DNA was isolated using a standard commercial kit “DNA-sorb B” with some modifications. Wright’s F-statistic, including several F-coefficients of inbreeding, was used to assess the genetic differentiation of populations. Testing of the population according to the studied genes for Hardy–Weinberg genetic equilibrium was carried out using the Pearson χ2 test. To determine Poltavske sriblo rabbits’ breeding value, indices were calculated based on economic and beneficial traits, including genetic and economic values of individual traits. As a genetic parameter, the heredity coefficient of rabbits was used, and the economic one was the cost of the products produced. Results. The results of the peculiarities of Poltavske sriblo rabbits genetic structure analysis by the distribution of allelic variants of the myostatin gene and progesterone receptor in the context of genealogical lines are presented. It was found that the grea­test frequency of the C allele by the myostatin gene was in the descendants of the male Cooper (0.551). The frequency of the T allele in this sample of animals was 0.449, respectively. For Snowball’s line rabbits, higher values of the G allele by progesterone receptor gene (0.488) were noted due to the advantage of homozygous animals. The highest value of the effective number of alleles by the myostatin gene was characteristic of Cooper’s rabbit line (0.500), and the lowest – of Bach’s lines (0.215) and Barry’s lines (0.230). According to the progesterone receptor gene, the highest values of Ne were for the Cooper (0.500), Snowball (0.507), and Mini (0.511) rabbits, and the lowest values were for Fox (0.307). The influence of the rabbit genotype on the manifestation of economic and beneficial traits – average daily growth and fertility – has also been established. The use of the breeding value evaluation of the Poltavske sriblo breed rabbits according to the selection and genetic index according to the productivity of daughters, made it possible to single out the main males among the group, as fertilizers. Male fertilizers included Snowball, Long, and Mini. Conclusions. The obtained data can be used for selective and breeding practices in rabbit breeding with the aim of selecting breeders for the improvement of the meat productivity and reproductive ability of rabbits.

Heterozygosity for Crohn’s Disease Risk Allele of Atg16L1 Protects from Salmonella Infection

Abstract Autophagy is an evolutionarily conserved pathway for the formation and trafficking of lipid vesicles in response to stress, including inflammation and microbial infection. A missense variant of the essential autophagy gene Atg16L1 (rs2241880, T300A) is carried by a majority of the human population and is linked to Crohn’s Disease (CD). Previous studies found that T300A enhances proteolytic cleavage and degradation of Atg16L1, leading to dysregulated cytokine expression and impaired control of microbial infection in knock-in mice. However, these studies have focused on homozygous animals while the plurality of the human population is heterozygous for the allele. Here we demonstrate that heterozygosity for T300A protects mice from Salmonella infection. We find the variant allele results in altered complex formation independent of cleavage, allowing for enhanced inflammasome activation but impaired conjugation of Atg8 to single membranes (CASM). In vivo, one copy of T300A is sufficient to elevate the production of inflammatory cytokines, which is required for protection from Salmonella infection. Meanwhile, mice deficient in CASM genes are susceptible to Salmonella infection and this pathway is shown to be impaired in homozygous but not heterozygous mice. Finally, we demonstrate that heterozygosity in human macrophages also results in elevated cytokine expression, while maintaining functional non-canonical autophagy. These results clarify the altered autophagy function of the CD risk allele Atg16L1T300A and suggest it can be beneficial to heterozygous carriers, providing a potential explanation for its prevalence in the human population. NIH R01 AI121244 F31 HL149238

Association of functional sequence variants of the myosin heavy chain 3 gene with muscle collagen content in pigs.

This study examined the association between functional sequence variants (FSVs) of myosin heavy chain 3 (MYH3) genotypes and collagen content in a Landrace and Jeju native pig (JNP) crossbred population. Four muscles (Musculus longissimus dorsi, Musculus semimembranosus, Musculus triceps brachii, and Musculus biceps femoris) were used for the analysis of meat collagen content, and the same animals were genotyped for the FSVs of the MYH3 gene by using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Three FSVs of MYH3 genotypes were identified and had genotype frequencies of 0.358, 0.551, and 0.091 for QQ, Qq, and qq, respectively. QQ animals for the FSVs of the MYH3 genotypes showed higher collagen content in their M. longissimus dorsi (p < 0.001), M. semimembranosus (p < 0.001), M. triceps brachii (p < 0.001), and M. biceps femoris (p < 0.001) than qq homozygous animals. After the validation of this result in other independent populations, the FSVs of MYH3 genotypes can be a valuable genetic marker for improving collagen content in porcine muscles and can also be applied to increase the amount of collagen for biomedical purposes.

Evaluating the potential impact of selection for the A2 milk allele on inbreeding and performance in Australian Holstein cattle

Selection decisions are generally based on estimated breeding values (EBV) for a combination of traits that are polygenic (e.g. milk production). However, in some cases, there is additional intense selection for a single allele, or SNP, for a perceived benefit, such as selection for polled or A2 milk. Using a dataset where the A2 mutation was imputed using a reference population with whole genome sequence, we tested the hypothesis that intense selection in Australian Holstein cattle for the A2 allele in the β-casein gene may have resulted in increased inbreeding. We also estimated the average difference in performance between animals homozygous for the A1 or A2 allele for a range of traits. Using high-density genotypes we compared differences in genome-wide and regional inbreeding between Holstein cows homozygous for either the A1 or A2 β-casein alleles i.e. A1/A1 or A2/A2. This study shows that between the years 2000 to 2017, the frequency of the A2/A2 genotype increased by 20% in Holstein cows (from 32% to 52%). Our results suggest that selection for homozygosity at the β-casein A2 allele has increased inbreeding both across the genome and on chromosome 6 in A2/A2 Holstein cows. Animals that were A2/A2 were twice as likely to have a run of homozygosity of at least 1Mb long across the β-casein locus compared to animals that were A1/A1. Cows that are homozygous for the A2 allele had an average protein yield EBV advantage of 0.24 genetic standard deviations (SD) compared to A1/A1 homozygous cows. In contrast, A2/A2 homozygous animals were on average 0.2 genetic SD inferior on fertility EBV. As a result, the difference in the overall economic index (that includes traits contributing to profitability) there was only a small advantage of 0.05 SD for A2/A2 cows compared to A1/A1 cows. However, strong selection for the A2 allele has likely led to a higher level of regional and overall inbreeding which in the long term could harm genetic progress for some or all economic traits. Therefore, applying approaches that mitigate rapid inbreeding while selecting for preferred alleles and quantitative traits may be desirable.

Genomic regions underlying positive selection in local, Alpine cattle breeds.

We used genome-wide SNP data from 18 local cattle breeds from six countries of the Alpine region to characterize population structure and identify genomic regions underlying positive selection. The geographically close breeds Evolèner, Eringer, Valdostana Pezzata Nera, and Valdostana Castana were found to differ from all other Alpine breeds. In addition, three breeds, Simmental, and Original Braunvieh from Switzerland and Pinzgauer from Austria built three separate clusters. Of the 18 breeds studied, the intra-alpine Swiss breed Evolèner had the highest average inbreeding based on runs of homozygosity (FROH ) and the highest average genomic relationship within the breed. In contrast, Slovenian Cika cattle had the lowest average genomic inbreeding and the lowest average genomic relationship within the breed. We found selection signatures on chromosome 6 near known genes such as KIT and LCORL explaining variation in coat color and body size in cattle. The most prominent selection signatures were similar regardless of marker density and the breeds in the data set. In addition, using available high-density SNP data from 14 of the breeds we identified 47 genome regions as ROH islands. The proportion of homozygous animals was higher in all studied animals of local breeds than in Holstein and Brown Swiss cattle, the two most important commercial breeds in the Alpine region. We report ROH islands near genes related to thermoregulation, coat color, production, and stature. The results of this study serve as a basis for the search for causal variants underlying adaptation to the alpine environment and other specific characteristics selected during the evolution of local Alpine cattle breeds.