Bioinformatic identification of Single Nucleotide Polymorphisms (SNPs) in keratin-associated protein genes in alpacas (Vicugna pacos) / Identificación bioinformática de Polimorfismos de Nucleótido Simple (PNSs) en genes de proteínas asociadas a queratinas en alpacas (Vicugna pacos)

Abstract This study aims to identify single nucleotide polymorphisms (SNPs) within KRTAP genes in alpacas (Vicugna pacos), which play a fundamental role in defining their physico-mechanical properties and potentially the quality of alpaca fiber, the primary product of their breeding. Thirty-four KRTAP genes, as annotated in the reference genome VicPac3.1, were investigated. Utilizing the reference genome, along with nine additional genomes and reads from 300 reduced representation DNA libraries of alpacas, SNPs were identified. Minor allele frequency (MAF) and genotyping rates were computed using PLINK software, while Illumina Scores were determined for each SNP using Illumina Design Studio software. Markers meeting the criteria of MAF ≥ 0.05, genotyping rate > 45%, and Illumina Score ≥ 0.6 per SNP were selected. A total of 67 SNPs were identified within intronic, exonic, and untranslated regions of KRTAP genes. Among these, 35 SNPs were incorporated into the 76K Alpaca SNP microarray, with 32 SNPs subsequently validated in a population of 936 alpacas. In conclusion, our findings delineate SNPs within KRTAPs that hold potential utility in genome-wide association studies, thereby facilitating the integration of modern breeding technologies into alpaca breeding programs.

Resumen Este estudio tiene como objetivo identificar polimorfismos de nucleótido simple (PNSs) dentro de los genes KRTAP en alpacas (Vicugna pacos), que juegan un papel fundamental en la definición de sus propiedades físico-mecánicas y la calidad de la fibra de alpaca, producto principal de su crianza. Se investigaron treinta y cuatro genes KRTAP, tal como están anotados en el genoma de referencia VicPac3.1. Utilizando el genoma de referencia, junto con nueve genomas adicionales y lecturas de 300 bibliotecas de representación reducida de ADN de alpacas, se identificaron los PNSs. La frecuencia de los alelos menores (MAF) y las tasas de genotipificación se calcularon utilizando el software PLINK, mientras que los Illumina Score se determinaron para cada PNS utilizando el software Illumina Design Studio. Se seleccionaron marcadores que cumplían con los criterios de MAF ≥ 0.05, tasa de genotipado > 45% e Illumina Score ≥ 0.6 por PNS. Se identificaron un total de 67 PNSs dentro de regiones intrónicas, exónicas y/o no traducidas de genes KRTAP. Entre estos, se incorporaron 35 PNSs al microarray 76K Alpaca SNP, y posteriormente se validaron 32 PNSs en una población de 936 alpacas. En conclusión, nuestros hallazgos identificaron los PNSs dentro de los KRTAP que tienen una utilidad potencial en estudios de asociación de todo el genoma, facilitando así la integración de tecnologías de reproducción modernas en los programas de reproducción de alpacas.

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library / 中国实验血液学杂志

OBJECTIVE@#To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.@*METHODS@#We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA.@*RESULTS@#A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies.@*CONCLUSION@#A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

Exportación y sanitación internacional en empresas productoras de prendas de alpaca de Huancayo y Lima / Exportation and international sanitation in companies producing alpaca garments in Huancayo and Lima

El Perú se considera un país potencia en cuanto a la exportación textil de la alpaca, cuya fibra natural es muy fina, ligera y con alto valor a nivel mundial. Para el año 2019, la exportación total de fibra de alpaca en Perú llegó a un total de 23 países, incluyendo mercados en Europa, Asia y Norteamérica. La tendencia en el comercio exterior incluyendo las empresas textiles de alpaca, se ha dirigido a garantizar el cuidado del medio ambiente y la inocuidad de los productos respecto a la salud humana. Se realizó un estudio desde el paradigma post-positivista, con enfoque cualitativo y el método hermenéutico, para interpretar la situación de exportación y sanitación internacional en empresas peruanas productoras de prendas de alpaca en Huancayo y Lima. Se obtuvo información a partir de cuatro informantes voluntarios de cada empresa acerca de aspectos relacionados con exportación de las prendas de alpaca: competitividad, promoción, proveedores, producción, calidad de producto, valor agregado, calidad de servicio, mercados destino, demanda y herramientas de inteligencia comercial, entre otros. Asimismo, se indagó sobre la concepción y cumplimiento del Reglamento Sanitario Internacional. Las empresas evaluadas, emplean excelente materia prima, garantizan un control de calidad de sus productos ofreciendo textiles competitivos que se distribuyen en diversos mercados internacionales, emplean herramientas de comercialización que favorecen la identificación de regulaciones arancelarias y la capacitación de su personal. No obstante, muestran carencias en el cumplimiento del RSI, requiriendo adoptar medidas que garanticen la salud pública al comercializar sus productos(AU)

Peru is considered a powerful country in terms of alpaca textile exports, whose natural fiber is very fine, light and highly valued worldwide. For the year 2019, the total export of alpaca fiber in Peru reached a total of 23 countries, including markets in Europe, Asia and North America. The trend in foreign trade, including alpaca textile companies, has been aimed at guaranteeing care for the environment and the safety of products with respect to human health. A study was carried out from the post-positivist paradigm, with a qualitative approach and the hermeneutical method, to interpret the export situation and international sanitation in Peruvian companies that produce alpaca garments in Huancayo and Lima. Information was obtained from four volunteer informants from each company about aspects related to the export of alpaca garments: competitiveness, promotion, suppliers, production, product quality, added value, service quality, destination markets, demand and marketing tools. commercial intelligence, among others. Likewise, the conception and compliance with the International Health Regulations were inquired about. The evaluated companies use excellent raw material, guarantee quality control of their products by offering competitive textiles that are distributed in various international markets, use marketing tools that favor the identification of tariff regulations and the training of their personnel. However, they show deficiencies in compliance with the RSI, requiring the adoption of measures that guarantee public health when marketing their products(AU)

Polimorfismos de nucleótido simple (PNSs) del gen MC1R en alpacas negras y marrones / Single nucleotide polymorphisms (SNPs) of the MC1R gene in black and brown alpacas

Resumen En alpacas los fenotipos del color de vellón tienen diferentes terminologías que induce a una confusión dentro del color marrón y sus tonalidades, el que requiere de una mejor descripción y cuantificación. En consecuencia los objetivos del estudio fueron cuantificar el color de fibra e identificar los PNSs informativos del gen MC1R (receptor 1 de melanocortina) en alpacas marrones y negras. Un fenotipo vicuña (n=14) y cuatro fenotipos de alpacas (n=79), marrón claro, marrón oscuro, marrón-negro y negro fueron evaluados por colorimetría. El vellón de vicuña mostró mayor luminosidad (47.74) e intensidad de color (24.33) respecto a las alpacas marrones. Los valores obtenidos de CIE L*a*b* (luminosidad e intensidad) sugieren valores bajos en alpacas eumelánicas y altos en alpacas feomelánicas. En vicuña y alpaca la secuencia codificante del gen MC1R tiene un solo exón de 954 pb, las vicuñas no mostraron la deleción (c.224_227del). Sin embargo, esta deleción se ha observado en los tres fenotipos de alpaca (marrón claro, marrón oscuro y negro), al igual que los cinco PNSs no sinónimos que ya fueron descritos en otras poblaciones, c.82A>G, c.259G>A, c.376G>A, c.587T>C, c.901C>T (p.T28A, p.M87V, p.G126S, p.F196S y p.R301C). Para las dos especies, se identificaron un total de ocho haplotipos definidos por los cinco PNSs. No se observaron asociaciones entre los fenotipos de color y los PNSs: c.259G>A, c.376G>A y c.901C>T (p>0.05), probablemente debido a la influencia de otros genes como el ASIP en la expresión del color. Nuestros resultados, así como los estudios previos evidenciaron regiones altamente conservadas en la secuencia codificante del gen MC1R.

Abstract In alpacas color fleece phenotypes have different terminologies that induces confusion within the brown color and its shades, it requires a better description and quantification. Consequently, the aims of the study were to quantify the color of fiber and identify the informational SNPs in the MC1R gene (melanocortin 1 receptor) in brown and black alpacas. A vicuña phenotype (n=14) and four alpaca phenotypes (n=79), light brown, dark brown, brown-black and black were evaluated by colorimetry. The vicuña fleece showed greater lightness (47.74) and color intensity (24.33) compared to brown alpacas. The CIE L*a*b* values (lightness and intensity) suggest low values in eumelanic alpacas and high in pheomelanic alpacas. In vicuña and alpaca, the coding sequence of the MC1R gene has a single exon of 954 bp, in vicuñas the deletion (c.224_227del) was not observed. However, this deletion was observed in three alpaca phenotypes (light brown, dark brown and black), as well as the five non-synonymous SNPs described in other populations, c.82A>G, c.259G>A, c.376G>A, c.587T>C, c.901C>T (p.T28A, p.M87V, p.G126S, p.F196S, and p.R301C). Eight haplotypes defined by the five SNPs were identified in both species. The associations between color phenotypes and SNPs were not observed (p>0.05), probably due to the influence of other genes such as ASIP on color expression. Our results as well as previous studies showed highly conserved regions in the coding sequence of the MC1R gene.

First characterization of preantral follicles in the ovaries of adult alpaca / Primera caracterización de folículos preantrales de ovario de alpaca adulta

It is a fact that while even basic reproductive information on alpacas is unavailable, the normal ovarian reserve of this species in comparison to other species is also unidentified. In this study, the ovarian preantral follicles in healthy adult alpacas were characterized in order to establish a general model to in vitro studies. Ten ovaries were collected from five adult alpacas. The ovarian cortex samples were fixed with paraformaldehyde and histological analysis was done. Normal and degenerated follicles percentages were determined. The normal follicles were measured and classified in primordial, transitional, primary and secondary stages. Most of the preantral follicles present in the ovarian cortex of alpacas were primordial and transitional stages; primary (6.10%) and secondary (0.37%) follicles were rarely found. The primary and secondary follicles were larger in diameter when compared with the primordial and transitional follicles. The largest oocyte diameter was recorded in the secondary follicles (P < 0.05). This study serves to establish a biological model for future reproduction studies in Alpacas or as possible biological model for studies of folliculogenesis in humans(AU)

Es un hecho que, si bien no se dispone de información reproductiva básica sobre las alpacas, la reserva ovárica normal de esta especie en comparación con otras especies tampoco está identificada. En este estudio, se caracterizaron los folículos preantrales ováricos en alpacas adultas sanas. Se recogieron diez ovarios de cinco alpacas adultas. Las muestras de la corteza ovárica se fijaron con paraformaldehído y se realizó un análisis histológico. Se determinaron los porcentajes de folículos normales y degenerados. Los folículos normales se midieron y clasificaron en estadios: primordiales, de transición, primarios y secundarios. La mayoría de los folículos preantrales presentes en la corteza ovárica de las alpacas eran estadios primordiales y de transición; Raras veces se encontraron folículos primarios (6.10%) y secundarios (0.37%). Los folículos primarios y secundarios tenían un diámetro mayor en comparación con los folículos primordiales y de transición. El mayor diámetro de ovocitos se registró en los folículos secundarios (P <0.05). Este estudio sirve para establecer un modelo biológico para futuros estudios de reproducción en alpacas o como posible modelo biológico para estudios de foliculogénesis en humanos(AU)

Identificación bioinformática de Polimorfismos de Nucleótido Simple (PNSs) en genes candidatos para las características de la fibra en alpacas (Vicugna pacos) / Bioinformatic identification of Single Nucleotide Polymorphisms (SNPs) in candidate genes for fiber characteristics in alpacas (Vicugna pacos)

El objetivo fue identificar y predecir la ubicación de polimorfismos de nucleótido simple (PNSs) en genes relacionados al crecimiento de la fibra. Se realizó el estudio con un total de 31 genes de queratina (KRT9, KRT12, KRT13, KRT14, KRT16, KRT18, KRT20, KRT25, KRT1, KRT3, KRT5, KRT6a, KRT6b, KRT6c, KRT7, KRT8, KRT71, KRT80, KRT31, KRT32, KIRT40, KRT81, KRT82, KRT10, KRT15, KRT17, KRT19, KRT2, KRT4, KRT79 y KRT83) asociados con las características de lana, fibra y pelo en ovinos, cabras y humanos respectivamente, cuyas secuencias fueron encontradas en la base de datos del National Center for Biotechnology Information (NCBI). Mediante el uso de bases de datos y herramientas bioinformáticas como el Conserved Domains Database, Spling, y MegaBlast se logró ubicar secuencias únicas para cada gen. Estas secuencias fueron comparadas con los genomas de referencia Vicugna_pacos-2.0.2 y Vi_pacos_V1.0. Se identificaron 48 PNSs ubicados en las regiones intrónicas y exónicas de 22 genes. No se localizaron PNSs en o alrededor de los genes KRT10, KRT15, KRT17, KRT19, KRT2, KRT4, KRT6b, KRT6c y KRT79. El análisis comparativo entre las cuatro especies estudiadas permitió observar que los genes KRT81, KRT6b y KRT6c no están presentes en los genomas de referencia de alpaca, los genes KRT31, KRT14, KRT81, KRT83, KRT6b y KRT6c no están presentes en el genoma de referencia de ovino y los genes KRT31, KRT13, KRT81, KRT83, KRT6b y KRT6c no están presentes en el genoma de referencia de cabra.

The objective was to identify and predict the location of single nucleotide polymorphisms (SNPs) in genes related to fiber growth. The study was carried out with 31 keratin genes (KRT9, KRT12, KRT13, KRT14, KRT16, KRT18, KRT20, KRT25, KRT1, KRT3, KRT5, KRT6a, KRT6b, KRT6c, KRT7, KRT8, KRT71, KRT80, KRT31, KRT32, KIRT40, KRT81, KRT82, KRT10, KRT15, KRT17, KRT19, KRT2, KRT4, KRT79 y KRT83) associated with wool, fiber and hair characteristics in sheep, goat and human, respectively. These gene sequences were retrieved from the National Center for Biotechnology Information (NCBI) database. Using databases and bioinformatics tools such as the Conserved Domains database, Spling and Megablast, unique sequences for each gene were identified. These sequences were compared to the reference genomes: Vicugna_pacos-2.0.2 and Vi_pacos_V1.0 to identify single nucleotide polymorphisms (SNPs). In this manner, 48 SNPs were identified and localized in both intronic and exonic regions of 22 genes. We did not identify SNPs for KRT10, KRT15, KRT17, KRT19, KRT2, KRT4, KRT6b, KRT6c and KRT79. Comparative analysis among the four species studied allow to identify that sequences for KRT81, KRT6b and KRT6c genes are not present in the alpaca reference genomes. Similarly, genes KRT31, KRT14, KRT81, KRT83, KRT6b and KRT6c are not present in the ovine reference genome and, genes KRT31, KRT13, KRT81, KRT83, KRT6b and KRT6c are not present in the goat reference genome.

Aspectos patológicos da intoxicação por aceturato de diminazeno em camelídeos sul-americanos / Pathological aspects of diminazene aceturate toxicosis in american camelids

Aceturato de diminazeno é um fármaco quimioterápico sintético comumente usado na medicina veterinária para o tratamento de doenças causadas por parasitos hematozoários. Entretanto, seu uso pode levar a efeitos colaterais, como alterações neurológicas graves e morte. A criação de camelídeos é uma atividade recente no Brasil, fazendo-se necessário conhecer mais sobre as doenças que acometem essas espécies. De dez camelídeos (seis lhamas e quatro alpacas) da propriedade, seis tiveram sinais clínicos e, destes, apenas uma lhama com manifestações leves recuperou-se. Os sinais clínicos incluíam apatia, andar cambaleante, fraqueza, sialorreia, cabeça baixa e pendida lateralmente, dificuldade em levantar e dispneia, observados a partir de 18 horas após o uso do medicamento. À necropsia e ao exame histopatológico foram observadas alterações de encefalopatia hemorrágica bilateral e simétrica, mais graves em tronco encefálico e tálamo. Este trabalho descreve as principais lesões observadas em um surto de intoxicação por diminazeno em alpacas (Lama pacos) e lhamas (Lama glama) e alerta criadores e veterinários sobre o risco de intoxicação por aceturato de diminazeno em camelídeos sul americanos.(AU)

Diminazene aceturate is a synthetic chemotherapeutic drug commonly used in veterinary medicine for the treatment of diseases caused by hematozoan parasites. However, side effects as severe neurological disorders and death can occur. The raising of american camelids is a recent activity in Brazil, requiring knowledge about diseases that affect these species, in order to avoid misguided conducts. In a herd of ten camelids (six llamas and four alpacas) six showed clinical signs and five died; only a llama with mild signs recovered. The clinical signs included apathy, difficulty to stand up, staggering gait, weakness, down head and drooping the head laterally, dyspnea and drooling of saliva, observed from 18 hours after use of the drug. At necropsy and histopathological examination was found bilateral and symmetrical hemorrhagic encephalopathy, more severe in brainstem and thalamus. This paper describes the main lesions observed in an outbreak of diminazene aceturate poisoning in alpacas (Lama pacos) and llamas (Lama glama) and alert breeders and veterinarians about the risk of poisoning by this drug in american camelids.(AU)

Estudio morfológico de testículo de híbridos de alpaca (Lama pacos L.1758) y llama (Lama glama L. 1758) / Morphological study of testis of hybrid alpaca (Lama pacos L. 1758) and llama (Lama glama L. 1758)

En el presente estudio se ha realizado una descripción histológica básica de la estructura de testículos de machos híbridos resultantes del cruce de llama (Lama glama L.1758) y alpaca (Lama pacos L.1758), a los que se llama guarizos o huarizos. Como la fisiología reproductiva de estos híbridos es desconocida, nos pareció importante y necesario realizar estudios histológicos básicos para predecir si tienen capacidad reproductiva, ya que como híbridos se les considera estériles. Para ello se han analizado los testículos izquierdos de 10 híbridos de edades comprendidas entre 2 y 5 años, fijándolos en solución Bouin e incluyéndolos en parafina. Se han estudiado al microscopio óptico cortes histológicos de 5 micras de grosor, conteniendo en todos los casos túbulos seminíferos, para estudiar el epitelio germinal. En algunos casos se incluía el epidídimo, para determinar si su histología es normal y compatible con una posible funcionalidad. Estas secciones fueron teñidas con hematoxilina­eosina (HE) y/o con HE-PAS. El estudio de estas secciones permitió observar en los túbulos seminíferos todos los tipos celulares esperados: células de Sertoli, células de Leydig, espermatogonias y espermatocitos, todos ellos de aspecto normal. En el caso de las células de Sertoli, algunas de ellas presentaban un alto grado de vacuolización, lo que también se considera normal por la gran presencia de gotas lipídicas. En otras ocasiones en ciertas células de Sertoli se detectaron burbujas o huecos esféricos vacíos, que en la literatura se considera que corresponde a los espacios que ocupaban células germinales ya desaparecidas. La constitución histológica del epidídimo resultó normal. Por lo tanto, el estudio histológico de los testículos de estos híbridos permite concluir que la estructura de los túbulos seminíferos es aparentemente normal y que parecen formar espermatocitos también normales, lo que podría sugerir la posibilidad de que estos híbridos tengan cierto grado de fertilidad...

In the present study was carried out a description of the basic histological structure of testes in male hybrids of crossings of llama (Lama glama L.1758) and alpaca (Lama pacos L.1758), which is called guarizos or huarizos. As the reproductive physiology of these hybrids is unknown, it seemed important and basic histological studies are needed to predict if they have reproductive capacity because they are considered as sterile hybrids.To do this we have analyzed the left testes of 10 hybrid aged 2 and 5 years, fixing them in Bouin solution and including them in paraffin. Were studied by light microscopy histological sections of 5 microns in thickness, containing in all cases seminiferous tubules to study the germinal epithelium. In some cases, including the epididymis, to determine if the histology is normal and compatible with a possible functionality. These sections were stained with hematoxylin-eosin (HE) and / or HE-PAS. The study of these sections allowed the seminiferous tubules observed in all cell types expected: Sertoli cells, Leydig cells, spermatogonia and spermatocytes, all of them look normal. In the case of Sertoli cells, some of which exhibited a high degree of vacuolization, which is also considered normal by the presence of large lipid droplets. At other times in some Sertoli cells were detected hollow spherical bubbles or voids, in the literature is considered to correspond to the spaces occupied by germ cells and missing. The constitution of the epididymis was histologically normal.Therefore, histologic examination of the testes of these hybrids can be concluded that the structure of seminiferous tubules with apparently normal and spermatocytes also appear to be normal, which could suggest the possibility that these hybrids have some degree of fertility...

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

EXPRESSION OF TRANSFORMING GROWTH FACTOR-?1 AND ITS SIGNAL TRANSDUCER SMAD2 AND SMAD4 IN ALPACA TESTIS / 解剖学报

Objective To study the expression and localization of TGF?1 and its signal transducer Smad2 and Smad4 which play important roles during testicular development and spermatogenesis in alpaca testis. Methods The whole testes were obtained from alpacas aged 24 months(n=3) at the Scientific Research Base of Shanxi Agriculture University.The protein expressions of TGF-?1、smad2、smad4 in alpaca testes were examined by Western blotting and SABC. Results The distributions of(TGF-?1)、Smad2 and Smad4 in the testes of alpacas aged 24 months were demonstrated.Conclusion Our findings show that TGF-?1、Smad2 and Smad4 are involved in the regulation of spermatogenesis,and provide direct evidences for the moleculer mechnism of TGF-?1 action in the alpaca testis during spermatogenesis.