Bovine Leukemia Virus molecular detection and associated factors among dairy herd workers in Antioquia, Colombia.

The Bovine Leukemia Virus (BLV) affects mainly cattle, is transmitted by exposure to contaminated biological fluids, and generates lymphomas in 5 % of infected animals. The zoonotic potential of BLV has been studied, and it is currently unknown if it circulates in human workers on dairy herds in Antioquia. Objective: To determine the frequency of BLV detection, the genotypes of the virus, and the factors associated with its detection in workers for dairy herds in Antioquia, Colombia. Through a cross-sectional study in 51 dairy herds, 164 adults were recruited. A peripheral blood sample was collected from each participant for molecular detection of the BLV env and tax genes, and associated factors were explored through bivariate and multivariate mixed Poisson model analyses. The analysis showed that 82 % (134/164) of the participants were men, with an average age of 40. Using qPCR, the constitutive gene GAPDH was amplified to evaluate the presence of amplification inhibitors in the DNA samples. Using nested PCR, the amplification of the env viral gene was obtained in 13 % (22/164) of the total samples analyzed, while all the samples tested negative for tax. The amplicons of the env gene were sequenced, and the identity compatible with BLV was verified by BLAST analysis (NCBI). Using molecular phylogeny analysis, based on maximum likelihood and haplotype network analysis, it was identified that BLV genotype 1 is present in the evaluated population. 16 % (26/164) of the participants reported having ever had an accident with surgical material during work with cattle; this variable was associated with BLV positivity even after adjusting for other variables (PRa =2.70, 95 % CI= 1.01- 7.21). Considering that other studies have reported the circulation of BLV genotype 1 in cattle from this same region and the present report in humans from dairy herds, the results suggest a possible zoonotic transmission of BLV genotype 1 in Antioquia, reinforcing the need to continue investigating to determine the potential role of this virus as an etiological agent of disease in livestock farmers in the department.

Phylogenetic analysis of the partial sequences of the env and tax BLV genes reveals the presence of genotypes 1 and 3 in dairy herds of Antioquia, Colombia.

Bovine leukemia virus (BLV) is a retrovirus that primarily infects dairy cows. Although few studies have also used the tax gene, phylogenetic studies of BLV use mostly the env gene. The aim of this work was to establish the circulating genotypes of BLV in specialized dairy cattle from Antioquia, Colombia. Twenty blood samples from Holstein Friesian cows were collected, and their DNA was isolated. A PCR was performed for a partial region of the env and tax genes. A phylogenetic analysis was carried out using the maximum likelihood and Bayesian methods for both genes. Nineteen sequences were identified as genotype 1 by env and tax genes. Only one sequence was clustered with genotype 3 and had the highest proportion of different nucleotide sites compared to other strains. Four amino acid substitutions in the 134 amino acid residue fragment of the Env protein were identified in the Colombian sequences, and three new amino acid substitutions were reported in the 296 amino acid residue fragment of the Tax protein. R43K (Z finger), A185T (Activation domain), and L105F changes were identified in the genotype 3 sample. This genotype has been reported in the United States, Japan, Korea, and Mexico, but so far, not in Colombia. The country has a high rate of imported live animals, semen, and embryos, especially from the United States. Although it is necessary to evaluate samples from other regions of the country, the current results indicate the presence of two BLV genotypes in specialized dairy herds.

Genome-wide association study for reproduction traits in Colombian Creole Blanco Orejinegro cattle.

The profitability of the beef cattle production system relies heavily on reproductive traits. Unfortunately, certain traits, such as age at first calving (AFC), calving interval (CI), and gestation length (GL), can pose challenges in traditional breeding programs because of their low heritability (0.01-0.12) and sex-limited characteristics. Another important aspect is the conservation of the genetic resources of animals adapted to the Colombian regions, which implies the preservation and rational use of the creole breeds in the country market. Therefore, this study aimed to identify genomic regions in the creole cattle breed Blanco Orejinegro (BON) that influence the reproductive traits in females. The dataset comprised 439 animals and 118,116 single-nucleotide polymorphisms' (SNPs) markers. The GS3 program was used to identify the SNP effects employing the BAYES Cπ methodology. The number of SNPs with effect for AFC was 25, 1527 for CI, and 23 for GL. Some of the genes found associated with reproductive and growth traits as well as immune response and environmental adaptation ECE1, EPH, EPHB2, SMARCAL1, IGFBP5, IGFBP2, FCGRT, EGFR, MUL1, PINK1, STPG1, CNGB1, TGFB1, OXTR, IL22RA1, MYOM3, OXTR, CNR2, HIVEP3, CTPS1, CXCL8, FCGRT, MREG, TMEM169, PECR, and MC1R. Our results evidenced a high contribution of the genetic architecture of the Colombian creole cattle breed Blanco Orejinegro that may impact should be included in implementing genetic improvement and conservation programs.

Bovine leukosis virus, bovine viral diarrhea, and bovine neosporosis seroprevalence in specialized dairy herds in Antioquia-Colombia.

Enzootic bovine leukosis (EBL) is a chronic infectious disease caused by the bovine leukosis virus (BLV), a Deltaretrovirus. Bovine viral diarrhea (BVD) is an infectious disease caused by a pestivirus. Bovine neosporosis is caused by the obligate intracellular parasite Neospora caninum (Nc). These pathogens can have horizontal (postnatal) or vertical (transplacental) transmissions and affect the productive and reproductive performance of infected bovines. This work aimed to detect BLV, BVD, and Nc seroprevalence in specialized dairy cattle from the north, east, and Aburrá Valley regions of the Department of Antioquia, the highest in milk production regions in Antioquia. A total of 599 blood samples, obtained from 53 specialized dairy cattle herds, were evaluated by the ELISA test. The results revealed a seroprevalence of 41.13% for BLV (242/599), 28.48% (163/599) for Nc, and 22.7% (132/599) for BVD. Regarding the regional seroprevalence evaluation, BLV was found in 47.02% of the samples from the east, 36.87% from the north, and 46.02% from the Aburrá Valley. Nc was found in 31.03% of the samples from the east, 24.26% from the north, and 36.63% from Aburrá Valley. BVD was found in 21.62% of the samples from the east, 25.03% from the north region, and 10.39% of the samples from the Aburrá Valley. It is highlighted by these results that the north region, with the highest milk production in Antioquia, had the lowest BLV and Nc seroprevalences but the highest seroprevalence of BVD. BLV has increased in Antioquia in recent years, and as an immunosuppressive infection, opportunities for other pathogens are created by it. A significant statistical difference was found in the average prevalence of the pathogens according to the municipality, cattle breed, and region of origin of the sample. The seroprevalence of these pathogens in specialized dairy herds in Antioquia can be classified as medium-low. However, it is recommended that biosecurity practices should be maximized to avoid the spread of these pathogens due to the variability detected in the region, municipality, breed group, and herd age. The rapid and efficient diagnosis of these three pathogens through reliable methodologies will allow for the control of dissemination in dairy herds.

Bovine leukemia virus detection in humans: A systematic review and meta-analysis.

To review the available studies on the frequency of detection of the bovine leukemia virus in human samples, a systematic review with meta-analysis of the scientific literature was carried out, including papers published in English, Spanish, and Portuguese in 5 multidisciplinary databases. We collected information from different populations following a detailed and reproducible search protocol in which two researchers verified the inclusion and exclusion criteria. We identified 759 articles, of which only 33 met the inclusion criteria. Analyzed studies reported that the presence of the virus was measured in human samples, such as paraffin-embedded breast tissue and peripheral blood from 10,398 individuals, through serological and molecular techniques. An overall virus frequency of 27% (Ranging between 17 and 37%) was observed, with a high-frequency data heterogeneity between studies. The presence of this virus in different human biological samples suggests the need to investigate further its transmission route to humans and its potential role in developing and progressing diseases.

Genetic-quantitative uni- and bi-trait analysis for growth traits in the Colombian creole breed Blanco Orejinegro (BON).

The Blanco Orejinegro (BON) is a Colombian creole cattle breed that is not genetically well characterized for growth traits. The aim of this work was to estimate genetic parameters for birth weight (BW), weaning weight (WW), yearling weight (YW), daily weight gain between birth and weaning (DWG), time to reach 120 kg of live weight (T120), and time to reach 60% of adult weight (T60%), and establish the selection criteria for growth traits in the BON population of Colombia. Genealogical and phenotypic information for BW, WW, YW, DWG, T120, and T60% traits of BON animals from 14 Colombian herds were used. These traits were analyzed with the AIREML method in a uni- and bi-trait animal model including the maternal effect for BW, WW, DWG, and T120. The direct heritability estimates values were 0.22 ± 0.059 (BW), 0.20 ± 0.057 (WW), 0.20 ± 0.153 (YW), 0.17 ± 0.07 (DWG), 0.26 (T120), and 0.44 ± 0.03 (T60%). The maternal heritability estimates values were 0.14 ± 0.040 (BW), 0.15 ± 0.039 (WW), 0.25 ± 0.06 (DWG), and 0.16 (T120). The direct genetic correlations were high ( >|0.60|) among all the traits, except between T60% with BW, WW, YW, and DWG (ranged from - 0.02 to - 0.51), all in a favorable direction. The results showed that there is genetic variation in the growth traits associated with the additive genetic effect, and they might respond to selection processes. Furthermore, genetic gains would improve through selection, especially for YW and T60% when WW is used as criterion.

Frequency of genotypic markers for genetic disorders, colour, polledness, and major genes in Blanco Orejinegro cattle.

The purpose of this work was to establish DNA marker frequencies for genetic disorders, colour, horned/polled trait, and major genes of importance for productive and reproductive traits in Blanco Orejinegro (BON) cattle. The Blanco Orejinegro breed is a Colombian creole breed characterized by a white hair coat on black skin with black ears, black hair on the middle part of the legs, and absence of horns. We genotyped 420 animals of Colombia, 70 with the GGP-Bovine 150 K chip and 350 with the GGP-Bovine 50 K chip. The markers were associated with 50 genetic diseases, 52 major gene variants related to productive traits, and 12 variants related to coat coloration, presence of horns, and adaptation, selected from the information contained in the chips. Genotype frequencies were estimated using the R statistical program. Genetic disorder annotations were derived using the Online Mendelian Inheritance in Animals tool (OMIA) and the average inbreeding coefficient (F) (n = 7799) using the MTDFNRM program. Carriers were found for 16 of the genetic disorders evaluated but with low frequencies (0.24 to 2.46%); no homozygous animals were found for the disorders. Carriers were associated with disorders such as bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), syndactyly, and epidermolysis bullosa (EB). The F was 4.41%. Concerning the genes associated with colour (TYR, MC1R, and PMEL), alleles related to black pigmentation, the absence of horns (polledness), and slick coat (an adaptive trait) were highly frequent (> 81.90%). Markers associated with milk production and quality, yellow fat, and fertility showed variable frequencies, indicating selection potential. Allele frequency of genetic disorders in BON cattle was low, suggesting few genetic disorder problems, with syndactyly being the most frequent condition. The markers associated with colour and polledness were almost fixed, with a frequency at or near 100%. Production and reproduction markers showed variability for selection.

Antibody seropositivity for Neospora caninum in Blanco Orejinegro cattle in Colombia and factors associated with the infection.

Neosporosis is a parasitic disease that causes abortions and economic losses in bovine production systems, but no studies have been found concerning its effect on the Creole cattle breed, Blanco Orejinegro (BON). The aim of this research was to establish the serological status of Colombian BON cattle against Neospora caninum and to determine the factors associated with seropositivity. Blood samples were taken from 363 animals from 13 herds in six states of Colombia, and analyzed by indirect ELISA using a commercial test. Information on sex, herd, generation group, and state was recorded. A survey was carried out with 26 questions related to productive, reproductive, and health factors per herd. A logistic regression analysis was performed and the ORs for significantly associated variables were estimated using the R software. General seropositivity of 73.5% (95% CI 68.6-77.9%) was obtained, and sex, age group, and herd were the variables significantly associated with seropositivity (p < 0.05). For the sex variable, seroprevalence levels of 79.6% (95% CI 74.3-84.1%) were recorded for females and 54.5% (95% CI 43.6-65.1%) for males. Herd seroprevalence varied between 58.3 and 95.8%, and the last generation showed the lowest positivity (51.2%, 95% CI 42.1-60.2%). The inadequate disposal of fetuses was a risk factor, while carrying out serological tests to new animals that enter the herd, the use of new gloves and palpation utensils for each animal, supplementation, and stabling were stated as protective factors. No effect of positivity was found in the last calving interval. The implementation of bovine neosporosis control programs to support breeding and conservation programs of the BON breed in Colombia is recommended.

Effect of the energy level of the diet of Holstein cows in early and mid-lactation on the sanitary and hygienic quality of milk / Efecto del nivel energético de la dieta de vacas Holstein en lactancia temprana y media sobre la calidad higiénica y sanitaria de la leche

ABSTRACT During the first weeks of lactation, the energy balance of dairy cows is generally negative, allowing for decompensation in a variety of tissues and systems, especially the immune system. The energy levels of a cow's diet during the first third of lactation, in specialized dairying, will modulate the immune system functionality of the mammary glands. The aim of this study was to assess the sanitary quality of milk through the somatic cell score (SCS), the colony-forming units (CFU), and the isolated bacteria of Holstein cows with different energy levels in the diet. Thus, 24 Holstein cows were studied during their first third of lactation, after the lactation peak (50 to 100 lactation days). They were assigned into 4 treatments, taking into account the energy requirements of each animal: Isoenergetic (ISO), Hypoenergetic (HYPO), Hyper energetic (HYPER), and Isoenergetic plus sunflower oil supplementation (OIL). Milk samples were taken for the somatic cell count (SCC) and CFUs, and isolated pathogens in milk were identified by microbiological culture. The diet had a significant effect on SCS with a value p of 0.0331, but not on the CFUs (p< 0.5141). 11 pathogens were identified and in 33.9% of samples, microorganisms were not isolated. The Isoenergetic diet favors the presence of some microorganisms and the increase of SCS.

RESUMEN En las primeras semanas de lactancia, generalmente, el balance energético de la vaca lechera es negativo, determinando una descompensación en diferentes tejidos y sistemas, en especial, el inmunológico. Los niveles de energía en la dieta de vacas, en primer tercio de lactancia en lechería especializada, modularán la funcionalidad del sistema inmune en glándula mamaria. El objetivo del presente trabajo fue evaluar la calidad sanitaria de la leche, a través de la medición de células somáticas (SCS), las unidades formadoras de colonias (UFC) y las bacterias aisladas de vacas Holstein, con diferentes niveles de energía en la dieta. Se usaron 24 vacas Holstein en el primer tercio de gestación, posterior al pico de lactancia (entre 50 y 100 días de lactancia), dispuestas en 4 tratamientos, de acuerdo con el cubrimiento de los requerimientos energéticos del animal: isoenergético (ISO), hipoenergético (HIPO), hiperenergetico (HIPER) e isoenergético más suplementación con aceite de girasol (ACEITE). Se tomaron muestras de leche para el recuento de células somáticas (RCS) y UFC y se identificaron patógenos en leche, por cultivos microbiológicos. La dieta tuvo un efecto significativo sobre el SCS, con un valor p de 0.0331 y no sobre las UFC (p<0.5141). Se identificaron 11 patógenos y en 33,96% de las muestras no se aislaron microorganismos. La dieta isoenergética favorece la presencia de algunos microorganismos y el aumento del SCS.

Diversity analysis, runs of homozygosity and genomic inbreeding reveal recent selection in Blanco Orejinegro cattle.

Blanco Orejinegro (BON) cattle have 500 years of adaptation to the Colombian tropic, but little is known about their genetic history. Our aim was to estimate levels of linkage disequilibrium (LD), effective population size (Ne), genomic inbreeding for runs of homozygosity (FROH ), genomic relation matrix (FGRM ), excess of homozygotes (FHOM ) and pedigree information (FPEDCOMP ) and to characterize the runs of homozygosity (ROH), searching for selection signatures. A total of 419 BON animals were genotyped, 70 with a 150K chip and 349 with a 50K chip. Next, an imputation to 50K was performed, and, after editing, databases of 40K were obtained. The PLINK v1.90 and R programs were used to estimate LD, ROH, FROH and FHOM . The SNeP v1.1 program was used to obtain Ne, and PreGSf90 was used to elaborate the scaled G matrix. The MTDFNRM program was used to estimate FPEDCOMP . The LD mean as r2 at 1 Mb was 0.21 (r2  > 0.30 at a distance of 96.72kb), and Ne was 123 ± 1. A total of 7,652 homozygous segments were obtained, with a mean of 18.35 ± 0.55 ROH/animal. Most of the genome was covered by long ROHs (ROH>8 Mb  = 4.86%), indicating significant recent inbreeding. The average inbreeding coefficient for FPEDCOM , FGRM , FHOM and FROH was 4.41%, 4.18%, 5.58% and 6.78%, respectively. The highest correlation was observed between FHOM and FROH (0.95). ROH hotspots/islands were defined using the extreme values of a box plot that was generated, and correspond to QTLs related to milk yield (55.11%), external appearance (13.47%), production (13.30%), reproduction (8.15%), health (5.24%) and meat carcass (4.74%).

Comparison of three methodologies for the genetic study of lactation persistency in Holstein cattle from Antioquia.

Persistency is the rate of decrease after milk production peak, mathematical models such as Wood's can be used to estimate it for describing the lactation curve and its rate of descent; random regression models are also useful, as they describe the genetic lactation curve for each animal. The objective of this study was to compare Best Linear Unbiased Prediction (BLUP), marker-assisted BLUP (MBLUP) model and random regression model (RRM) to estimate genetic parameters and breeding values for the lactation persistency curve. 4,658 test day measurements were available for 733 individuals, from which lactation curves were described to calculate persistency, estimating genetic parameters and values for this trait through BLUP and MBLUP. A similar process was done for RRM, where persistency was estimated from the genetic lactation curve. The heritability obtained using RRM was 0.51, greater than that obtained by BLUP (0.29) and MBLUP (0.21). The reliability of the genetic value for persistency in bulls was greater when RRM was used, but there was no correlation between the genetic values of different models. The highest heritability for persistency and the more reliable genetic values for bulls were achieved under the RRM, it allows positioning this methodology as an important tool for genetic evaluation of persistency.

The energy level of the diet in Holstein cows in the first third of lactation alters the expression of immune genes in the mammary gland.

Our goal was to define the effect of the energy intake in Holstein cows' diet in the first third lactation on gene expression profile of immune system in mammary gland through RNA-seq. Twelve Holstein cows were used in the first third of lactation, arranged in four treatments or diets: (a) hypocaloric (HYPO, 85% of the net energy of lactation (NEl) requirements), (b) isocaloric (ISO, 100% of the NEl requirements, control diet), (c) hypercaloric (HYPER, 115% of the NEl requirements) and (d) isocaloric plus sunflower oil supplementation (OIL, 106% of the NEl requirements). A biopsy of mammary gland tissue was carried out after 25 days per diet, from which the RNA was extracted and sequenced using the Illumina HiSeqTM 2,500 sequencer. The analysis of reads obtained from the sequencing in the QIAGEN® CLC Genomics Workbench 10.0 bioinformatics software was performed. From 27,368 genes annotated in the reference genome, 17,429 genes expressed in the evaluated treatments were identified. Moreover, 1,743 differentially expressed genes (DEGs) were found, of which 15 DEGs were found in the ISO vs. OIL comparison, 1,196 DEGs in the ISO vs. HYPO comparison and 532 DEGs in the ISO vs. HYPER comparison. Thus, of the 1,743 DEGs, 401 correspond to genes involved in the functioning of the immune system, encompassing 23% of the total number of DEGs involved in the analysis, and 13.6% of the total number of genes involved in the functioning of the immune system. The energy intake in Holstein cows' diet has impact in the expression of immune genes CXCL13, TRDC among others, present in the regulation of immune system processes. This immune system altered might increase the somatic cells score and therefore reach some diseases. It is recommended to measure the energy intake according to the animals' energy requirements and to cover them the closest to the 100%.

Association of BoLA DRB3 gene polymorphisms with BoHV-1 infection and zootechnical traits.

BACKGROUND: The dairy sector is one of the leading in agricultural production sectors in the world and the bovine herpesvirus 1 (BoHV-1) is an important pathogen that causes great losses in most production systems. Moreover, BoLA DRB3 immunological gene presents different alleles related to protection against many pathogens. METHODS: Serological diagnosis was carried out to determine the BoHV-1 infection and through PCR-RFLP 506 Holstein cows from several municipalities of Antioquia were genotyped for BoLA DRB3.2 gene polymorphisms. RESULTS: Alleles 8, 16, 22, and 24 were the most common out of the 42 alleles found. By indirect ELISA technique, a 58.7% prevalence of BoHV-1 infection in this population was diagnosed and Odd ratios for found alleles were calculated by logistic regression; the only significant association was held for allele 37, which showed that it effects confers susceptibility to infection. On the other hand, by using generalized linear models, a significant association between BoLA DRB3.2 gene and milk and fat yield in primiparous and services per conception in multiparous was found, with the most favorable alleles being 11 and 28 in primiparous and 22 and 28 in multiparous; allele 37 was unfavorable only in primiparous. CONCLUSION: BoLA DRB3.2 gene polymorphisms have shown high variability and significant effects on Holstein cattle and their performance in production systems in Antioquia are at both sanitary or health and productive levels.

Aislamiento de una cepa genotipo 3 del virus de la hepatitis E obtenida de heces humanas / Cell culture isolation of hepatitis e virus genotype 3 strain obtained from human feces

ABSTRACT Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/µL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/µL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

RESUMEN El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/µL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/µL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV.

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality.

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 µM Resveratrol (CR, C- respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C-VR, CRVR) or without (C-V-, CRV-) 0.5 µM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in CRVR group compared with CRV- group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but CRV- and CRVR groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C-VR) restored GSH content (P < 0.05) to the level found in the CR group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 µM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.

Ascorbic acid-cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl-ß-cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC-CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC-CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap-frozen for gene expression analysis by RT-qPCR. Besides, in vitro-produced zygotes were cultured with 100 µM of AC-CD and blastocysts were as well snap-frozen for gene expression analysis. A group without AC-CD (control- ) and other with CD (control+ ) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC-CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC-CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC-CD, while in blastocysts derived from zygote cultured with AC-CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC-CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC-CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.

Resveratrol supplementation promotes recovery of lower oxidative metabolism after vitrification and warming of in vitro-produced bovine embryos.

Although vitrification is the current method of choice for oocyte and embryo cryopreservation, it may have detrimental effects on reduction-oxidation status and mitochondrial activity. The aim of this study was to evaluate the effect of supplementing invitro culture (IVC) media and/or vitrification solutions with the antioxidant resveratrol on active mitochondria, mitochondrial superoxide production and lipid peroxidation. Abattoir-derived oocytes were matured and fertilised invitro using standard procedures. Following IVF (21h later), zygotes were cultured in IVC medium supplemented with 0 or 0.5µM resveratrol. On Day 7, blastocysts were vitrified using the Cryotech Vitrification Kit (Cryo Tech Laboratory) with or without 0.5µM resveratrol. After warming, active mitochondria, mitochondrial superoxide production and lipid peroxidation were evaluated using Mito Tracker Green FM, MitoSOX Red and BODIPY581/591 C11 staining respectively. The vitrification-warming process significantly increased active mitochondria and mitochondrial superoxide production in bovine embryos (P<0.05, ANOVA). The addition of 0.5µM resveratrol to the IVC medium or vitrification solutions significantly attenuated the increase in active mitochondria (P<0.05), but not in mitochondrial superoxide production, whereas embryos cultured and vitrified with resveratrol showed the highest values for both parameters (P<0.05). Regarding lipid peroxidation, no significant differences were detected between treatments. In conclusion, resveratrol supplementation of IVC medium or vitrification solutions contributes to recovery of an embryo's 'quieter' state (i.e. lower oxidative metabolism) after vitrification. However, supplementation of both solutions with resveratrol seemed to have a pro-oxidant effect.

Efecto del aceite esencial de orégano (Lippia origanoides) sobre metabolitos sanguíneos en pollos de engorde / Effect of oregano essential oil (Lippia origanoides) on blood metabolites in broiler chickens / Efeito do óleo essencial de orégano (Lippia origanoides) sobre metabolitos sanguíneos em frangos de corte

Resumen Objetivo: evaluar parámetros sanguíneos en pollos de engorde de la línea genética Cobb500, luego de la administración de aceite esencial de orégano (Lippia origanoides) (AEO). Materiales y métodos: se utilizaron 200 pollos de línea genética Cobb 500 y se realizaron mediciones los días 14, 28 y 42. Los animales fueron aleatorizados a una de dos dietas: dieta comercial con antibiótico y sin este. A esta última se adicionaron diferentes concentraciones de AEO (75 ppm, 100 ppm o 200 ppm AEO). Se realizó un diseño estadístico de bloques al azar en un arreglo de parcelas dividas. Resultados: los pollos del grupo D5 (200 ppm) presentaron mayores valores en glucosa, fosfatasa y fósforo que los pollos alimentados con antibiótico (D2) a lo largo del experimento. Conclusión: la adición de 200 ppm de AEO en el alimento de pollos de engorde de la línea genética Cobb 500 induce una mejora en metabolitos sanguíneos. Este trabajo permitió evaluar las variables metabólicas de pollos que consumieron AEO.

Abstract Objective: To evaluate blood parameters in broiler chickens of the Cobb 500 genetic line, after the administration of oregano essential oil (Lippia origanoides) (OEO). Materials and methods: 200 chickens of the Cobb 500 genetic line were used, and measurements were taken on days 14, 28, and 42. The animals were randomly assigned to one of two diets: commercial diet with and without antibiotic supplementation. To the antibiotic-free diet different concentrations of OEO were added (75 ppm, 100 ppm, or 200 ppm). A statistical randomized block design was used in an array of divided plots. Results: Chickens from group D5 (200 ppm of OEO) had higher values in glucose, phosphatase, and phosphorus than chickens fed with antibiotic supplementation (D2) throughout the experiment. Conclusion: The addition of 200 ppm of OEO in the food of broiler chickens of the Cobb 500 genetic line induces an improvement in blood metabolites. This work allowed evaluating metabolic variables of chickens that consumed OEO.

Resumo Objetivo: avaliar parâmetros sanguíneos em frangos de corte da linha genética Cobb500, após administração de azeite essencial de orégano (Lippia origanoides) (AEO). Materiais e métodos: Foram utilizados 200 frangos de linha genética Cobb 500 e foram realizadas medições nos dias 14, 28 e 42. Os animais foram submetidos aleatoriamente a uma de duas dietas: dieta comercial com antibiótico e sem este. A esta última foram adicionadas diferentes concentrações de AEO (75 ppm, 100 ppm ou 200 ppm AEO). Se realizou um desenho estatístico de blocos aleatórios em uma matriz de parcelas dividas. Resultados: os frangos do grupo D5 (200 ppm) apresentaram maiores valores em glicose, fosfatase e fósforo que os frangos alimentados com antibiótico (D2) ao longo do experimento. Conclusão: a adição de 200 ppm de AEO no alimento de frangos de corte da linha genética Cobb 500 induz a uma melhora em metabolitos sanguíneos. Este trabalho permitiu avaliar as variáveis metabólicas de frangos que consumiram AEO.

Presence of bovine leukemia virus genotypes 1 and 3 in antioquia, colombia / Presencia de los genotipos 1 y 3 del virus de la leucosis bovina en antioquia, colombia

SUMMARY Bovine leukemia virus (BLV) is an immunosuppressant retrovirus that primarily affects dairy livestock, its target cells are B lymphocytes in which it integrates its genome infecting cattle for life. It is important to identify the distribution of the BLV in the region and to reconstruct its evolutionary history through phylogenetic trees, for the province of Antioquia this is the first report of the BLV genotypes. The aim of this study was to identify the genotype of BLV circulating in dairy cattle of different regions of the province of Antioquia, Colombia. DNA was extracted from 8 Holstein cows. Nested PCR was performed to amplify a fragment of 444 pb of the env gene. The env viral gene codes for surface protein gp51, gene is highly conserved and it is used for phylogenetic analysis. Obtained amplicons were sequenced, manually aligned in MEGA V7 program, and compared to 53 viral env gene sequences registered in GenBank. Phylogenetic analysis was performed by Maximum Likelihood and Bayesian methods. Two circulating genotypes were found: the most common genotype was 1, found in seven samples; they grouped with sequences from EE. UU, Argentina and Japan; only one sample was classified as genotype 3 and was grouped with samples from EE. UU and Japan. At least two genotypes (1 and 3) of BLV are circulating in Antioquia; however, more cattle and herds should be evaluated to elucidate the diversity and distribution of BLV in Colombia.

RESUMEN El virus de la leucosis bovina (BLV) es un retrovirus inmuno-supresor que afecta, principalmente, al ganado lechero; sus células diana son los linfocitos B, en los cuales, integra su genoma, infectando al ganado de por vida. Es importante identificar la distribución del BLV en la región y reconstruir su historia evolutiva, a través de árboles filogenéticos; para el departamento de Antioquia, este es el primer reporte de los genotipos del BLV. El objetivo de este estudio fue identificar el genotipo de BLV que circula en ganado lechero de diferentes regiones del departamento de Antioquia, Colombia. Se extrajo ADN de 8 vacas Holstein. Se realizó una PCR anidada, para amplificar un fragmento del gen env de 444 pb. El gen env viral codifica la proteína de superficie gp51, altamente conservado y es usado en análisis filogenéticos. Los amplicones obtenidos se secuenciaron, se alinearon manualmente en el programa MEGA V7 y se compararon con 53 secuencias del gen env viral, registradas en GenBank. El análisis filogenético, se realizó por métodos de Máxima Verosimilitud y Bayesianos. Se encontraron dos genotipos circulantes: el genotipo más común fue 1, hallado en siete muestras, agrupadas con secuencias de EE. UU, Argentina y Japón; solo una muestra se clasificó como genotipo 3 y se agrupó con muestras de EE. UU y Japón. Al menos dos genotipos (1 y 3) de BLV están circulando en Antioquia; sin embargo, se deben evaluar más bovinos y hatos para elucidar la diversidad y la distribución de BLV en Colombia.

Microorganismos aislados en cultivo bacteriológico de muestras de leche de vacas holstein clínicamente sanas / Microorganisms isolated in bacteriological culture from milk samples from clinically healthy holstein cows / Microrganismos isolados em cultivo bacteriológico de amostras do leite de vacas holandesas clinicamente saudáveis

Resumen El objetivo del presente estudio fue determinar la frecuencia de aislamiento de microorganismos de tipo infeccioso y ambiental en leche de un grupo vacas clínicamente sanas en dos diferentes tipos de ordeño. Para ello se utilizaron muestras de leche de 289 vacas ubicadas en hatos de cuatro municipios del Norte del departamento de Antioquia en diferentes sistemas de ordeño (manual y mecánico), a cada muestra de leche se le hizo cultivo bacteriológico para determinar la presencia de microorganismos. De 289 muestras de leche evaluadas, 193 (66,78%) fueron positivas para aislamiento de cualquier tipo de patógeno (ocho diferentes patógenosaislados), de las cuales 81 (28,02%) muestras provenían de ordeño manual y 112 (38,75%) pertenecían a ordeño mecánico, encontrando un mayor porcentaje de aislamiento de patógenos bacterianos en muestras de leche provenientes de sistema de ordeño mecánico (p=0,0236). El patógeno de mayor aislamiento fue el Arcanobacterium haemolyticum (antes clasificado como Corinebacterium sp), microorganismo que hace parte de la flora saprófita del animal, con presencia de manera individual en el 20,14% de las muestras analizadas y en confección con otros patógenos en 0,7% de las muestras. El microorganismo que siempre se reporta como mayor productor de mastitis subclínica en bovinos es el Streptococcus agalactiae, un patógeno infeccioso, que en el presente estudio se aisló del 12,50% de las muestras de leche. Se determinó el Odds ratio (OR), entre aislamiento de Streptococcus agalactiae y Score de Células somáticas (SCS) que fue estadísticamente significativo, indicando que cuando está presente este patógeno el SCS aumenta y los animales son más susceptibles a padecer mastitis.

Abstract A microbiological analysis was performed to determine the frequency of isolation of microorganisms of infectious and environmental type in milk from a group of clinically healthy cows in two different types of milking (manual and mechanical). To each sample of milk was made bacteriological culture to determine the presence of microorganisms. Of 289 milk samples evaluated, 193 (66.78%) were positive for isolation of any type of pathogen, of which 81 (28.02%) samples came from manual milking and 112 (38.75%) belonged to mechanical milking, finding a higher percentage of isolation of bacterial pathogens in milks coming from mechanical milking system (p = 0.0236). The most isolated pathogen was the Arcanobacterium haemolyticum, A microorganism that forms part of the saprophytic flora of the animal, with an individual presence in 20.14% and in coinfection with other pathogens in 0.7% of the samples. The most common microorganism of subclinical mastitis in cattle is Streptococcus agalactiae, which in the present study was isolated from 12.50% of milk samples. The odds ratio (OR) between the isolation of Streptococcus agalactiae and the Somatic Cell Score (SCS) was determined, which was statistically significant, indicating that when this pathogen is present the SCS increases and the animals are more susceptible to mastitis.

Resumo O objetivo deste estudo foi determinar a frequência de isoLamento de microorganismos de tipo infecciosos e ambientaL no Leite de um grupo de vacas cLinicamente saudáveL em dois diferentes tipos de ordenha. Para este fim utiLizaram-se amostras do Leite de 289 vacas LocaLizadas em rebanhos de quatro municipaLidades do Norte de Antioquia com dois tipos diferentes de ordenha (manuaL e mecânica), a cada amostra do Leite foi feito um cuLtivo bacterioLógico para determinar a presença de microorganismos. De 289 amostras do Leite avaLiadas, 193 (66,78%) foram positivas para o isoLamento de quaLquer tipo de agente patogênico (oito diferentes agentes patogênicos isoLados), das quais 81 (28,02%) amostras eram de ordenha manuaL e 112 (38, 75%) foram de ordenha mecânica, encontrando uma maior percentagem de isoLamento dos agentes patogênicos bacterianos nos Leites provenientes do sistema de ordenha mecânica (p = 0,0236). O agente patogênico mais isoLado foi Arcanobacterium haemolyticum (Corinebacterium sp), microrganismo que faz parte da flora saprófita do animaL, presentes individuaLmente em 20,14% das amostras anaLisadas, e em reLação com outros agentes patogênicos o 0,7% das amostras. O microrganismo que sempre se reporta como o maior produtor de mastite subcLínica em gado é o Streptococcus agalactiae, um agente patogênico infeccioso, que neste estudo foi isoLado no 12,50% das amostras do Leite. Foi determinado o Odds ratio (OR) entre o isoLamento de Streptococcus agalactiae e Pontuação das céLuLas somáticas (SCS), e foi estatisticamente significativo, indicando que quando o agente patogênico está presente, o SCS aumenta e os animais são mais susceptíveis à mastite.