Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep.

BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.

Machine learning prediction of multiple anthelmintic resistance and gastrointestinal nematode control in sheep flocks.

The high prevalence of Haemonchus contortus and its anthelmintic resistance have affected sheep production worldwide. Machine learning approaches are able to investigate the complex relationships among the factors involved in resistance. Classification trees were built to predict multidrug resistance from 36 management practices in 27 sheep flocks. Resistance to five anthelmintics was assessed using a fecal egg count reduction test (FECRT), and 20 flocks with FECRT < 80% for four or five anthelmintics were considered resistant. The data were randomly split into training (75%) and test (25%) sets, resampled 1,000 times, and the classification trees were generated for the training data. Of the 1,000 trees, 24 (2.4%) showed 100% accuracy, sensitivity, and specificity in predicting a flock as resistant or susceptible for the test data. Forage species was a split common to all 24 trees, and the most frequent trees (12/24) were split by forage species, grazing pasture area, and fecal examination. The farming system, Suffolk sheep breed, and anthelmintic choice criteria were practices highlighted in the other trees. These management practices can be used to predict the anthelmintic resistance status and guide measures for gastrointestinal nematode control in sheep flocks.

Association of ß-globin polymorphisms and tolerance to haemonchosis in ewes and lambs of different sheep breeds.

Gastrointestinal nematodes (GIN), especially Haemonchus contortus, represent a significant challenge for sheep production. Given the global concern about GIN anthelmintic resistance, alternative control methods able to reduce the dependence on these drugs are highly advisable. Since previous studies have shown that sheep carrying the Hb-A allele of ß-globin are more resistant to H. contortus, this study aimed to investigate the relationship between the different haplotypes (Hb-AA, Hb-AB and Hb-BB) and phenotypes in Santa Inês (SI), Texel (TX) and White Dorper (DO) breeds infected with H. contortus. Blood samples were collected from 180 ewes and 123 lambs of the three breeds for DNA extraction followed by qPCR using a hydrolysis probe to identify the ß-globin haplotypes. Phenotypic data, including fecal egg count (FEC), packed cell volume (PCV), FAMACHA score and body condition score for ewes and lambs, as well as weight gain for lambs, were collected. The genotypic frequencies of ß-globin for ewes and lambs were, respectively: 21.7% and 21.4% Hb-AA, 50% and 50% Hb-AB and 28.3% and 28.6% Hb-BB in SI; 0% and 0% Hb-AA, 18.6% and 9.4% Hb-AB and 81.4% and 90.6% Hb-BB in TX; and 0% and 0% Hb-AA, 13.1% and 0% Hb-AB and 86.9% and 100% Hb-BB in DO. In ewes, mean PCV differed (p<0.05) between the three haplotypes, with higher PCV in Hb-AA animals, followed by Hb-AB and Hb-BB. When considering each breed separately, SI Hb-AA ewes presented higher PCV (p<0.05), highlighting that even in a breed already considered resistant, animals with Hb-AA haplotype showed superior performance. Lambs with the Hb-AA haplotype exhibited a higher (p<0.05) mean PCV compared to those with Hb-AB and Hb-BB. The same pattern was found in SI when analyzing each breed separately. No significant association was found between ß-globin haplotypes and FEC, FAMACHA score, body condition score, or weight gain. Nevertheless, given that anemia is the major clinical sign of haemonchosis, our findings on PCV reinforce that sheep carrying the Hb-A allele of ß-globin are more tolerant to haemonchosis. This study may support the development of a valuable tool, targeting genetic selection for GIN control, reducing the dependence on anthelmintics and boosting sheep production worldwide.

Enhanced mucosal response in sheep harbouring ßA beta-globin against haemonchosis.

Association between ovine ß-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity ßA ➔ ßC switch during anaemia, but there are no studies regarding the involved local host responses. Phenotypic parameters and local responses were evaluated in sheep from two ß-globin haplotypes naturally infected with Haemonchus contortus. Morada Nova lambs were monitored at 63, 84 and 105 days of age for faecal egg counts and packed cell volume (PCV) under natural infection with H. contortus. At 210 days of age, lambs of Hb-AA and Hb-BB ß-globin haplotypes were euthanised, and the fundic region of abomasum was sampled for evaluation of microscopic lesions and relative expression of genes related to immune, mucin and lectin activities. Lambs harbouring the ßA allele presented an improved resistance/resilience against clinical haemonchosis, showing higher PCV during infection. Hb-AA animals presented increased eosinophilia in the abomasum compared to Hb-BB animals, accompanied by higher Th2 profile, mucin and lectin activity transcripts, while the inflammatory response was increased in Hb-BB animals. This is the first report to demonstrate an enhanced local response in the primary site of H. contortus infection related to ßA allele of ß-globin haplotype.

In vitro culture of parasitic stages of Haemonchus contortus.

Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco's modified Eagle's medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes' reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.

Polymorphisms in exon 11 of the mptl-1 gene and monepantel resistance in Haemonchus contortus.

Chemical treatments are the main strategy to control gastrointestinal nematodes in sheep, and the emergence of anthelmintic resistance, as consequence, results in control failures and leads to economic losses. Thus, molecular tests may constitute an excellent tool for the early detection of anthelmintic resistance-related mutations. Thus, a polymerase chain reaction (PCR)-based genotyping assay followed by polyacrylamide gel electrophoresis (PAGE) was developed to detect polymorphisms in exon 11 of the acetylcholine receptor monepantel-1 gene (mptl-1) that were previously associated with monepantel resistance through a genome-wide study in Haemonchus contortus. DNA samples recovered from individual and pooled third-stage larvae from two susceptible field-derived isolates and five (three in vivo-derived and two field-derived) resistant populations were used. New polymorphisms, including a 6-bp deletion and a 3-bp insertion, were detected in resistant individuals. These indels, confirmed using sequencing of cloned PCR products, are predicted to result in amino acid changes in transmembrane domain 2 (TMD2) of the MPTL-1 protein. The two susceptible isolates showed only the presence of the wild-type allele (100%), whereas lower frequencies of the wild-type allele were detected in monepantel-resistant populations (11.1 to 66.7%). These findings report new polymorphisms in the mptl-1 gene, validate the results obtained through genomic mapping for monepantel resistance, and provide a PCR-based assay to genotype indels located in exon 11 of mptl-1 in H. contortus.

Genome-Wide Association Study for Haemonchus contortus Resistance in Morada Nova Sheep.

Among the gastrointestinal nematodes affecting sheep, Haemonchus contortus is the most prevalent and virulent, resulting in health problems and production losses. Therefore, selecting sheep resistant to H. contortus is a suitable and sustainable strategy for controlling endoparasites in flocks. Here, 287 lambs of the native Brazilian Morada Nova hair sheep breed were subjected to two consecutive artificial infections with H. contortus and assessed for fecal egg count (FEC), packed cell volume (PCV), and live weight (LW). Forty-four animals ranked as having extreme resistance phenotypes were genotyped using the Illumina OvineSNP50v3 chip. A case−control genome-wide association study (GWAS) detected 37 significant (p < 0.001) markers in 12 ovine chromosomes in regions harboring quantitative trait loci (QTL) for FEC, Trichostrongylus spp. adults and larvae, weight, and fat; and candidate genes for immune responses, mucins, hematological parameters, homeostasis, and growth. Four single-nucleotide polymorphisms (SNP; OAR1_rs427671974, OAR2_rs419988472, OAR5_rs424070217, and OAR17_rs401006318) genotyped by qPCR followed by high-resolution melting (HRM) were associated with FEC and LW. Therefore, molecular markers detected by GWAS for H. contortus resistance in Morada Nova sheep may support animal selection programs aimed at controlling gastrointestinal nematode infections in flocks. Furthermore, genotyping of candidate genes using HRM qPCR may provide a rapid and efficient tool for animal identification.

DNA methylation may affect beef tenderness through signal transduction in Bos indicus.

BACKGROUND: Beef tenderness is a complex trait of economic importance for the beef industry. Understanding the epigenetic mechanisms underlying this trait may help improve the accuracy of breeding programs. However, little is known about epigenetic effects on Bos taurus muscle and their implications in tenderness, and no studies have been conducted in Bos indicus. RESULTS: Comparing methylation profile of Bos indicus skeletal muscle with contrasting beef tenderness at 14 days after slaughter, we identified differentially methylated cytosines and regions associated with this trait. Interestingly, muscle that became tender beef had higher levels of hypermethylation compared to the tough group. Enrichment analysis of predicted target genes suggested that differences in methylation between tender and tough beef may affect signal transduction pathways, among which G protein signaling was a key pathway. In addition, different methylation levels were found associated with expression levels of GNAS, PDE4B, EPCAM and EBF3 genes. The differentially methylated elements correlated with EBF3 and GNAS genes overlapped CpG islands and regulatory elements. GNAS, a complex imprinted gene, has a key role on G protein signaling pathways. Moreover, both G protein signaling pathway and the EBF3 gene regulate muscle homeostasis, relaxation, and muscle cell-specificity. CONCLUSIONS: We present differentially methylated loci that may be of interest to decipher the epigenetic mechanisms affecting tenderness. Supported by the previous knowledge about regulatory elements and gene function, the methylation data suggests EBF3 and GNAS as potential candidate genes and G protein signaling as potential candidate pathway associated with beef tenderness via methylation.

Investigating the benefits of targeted selective treatment according to average daily weight gain against gastrointestinal nematodes in Morada Nova lambs.

The objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, based on the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: control (CT), routine (RT, treated every 42 days), and targeted selective (TST, treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. The anthelmintic resistance of parasites was assessed by the EPG count reduction test (FECRT), RESISTA-Test©, and molecular test. For CT, RT, and TST groups, the following results were obtained: mean EPG values were 4665.1, 3063.5, and 3462.1 in the rainy season and 4475.1, 1341.7, and 2863.4 in the dry season, respectively; mean PCV values were 32.1, 33.4, and 32.3% in the rainy season and 33.9, 36.0, and 35.1% in the dry season; mean DWG readings were 0.087, 0.101, and 0.094 kg in the rainy season and 0.102, 0.113, and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1, and 76.4% in the rainy and 90.7, 12.4, and 64.8% in the dry season, respectively; in the RESISTA-Test©, the LC50 values were 0.482, 1.926, and 0.117 µg.mL-1 in the rainy and 0.437, 0.851, and 0.045 µg.mL-1 in the dry season, respectively; the frequencies of the homozygous-resistant genotype were 57.1, 71.4, and 40.0% in the rainy and 47.8, 55.9, and 41.9% in the dry season. In conclusion, TST reduced the development of resistance through refugia maintenance, without productive losses, contributing to the sustainability of sheep breeding.

Comparison of ovine ß-globin haplotype sequences and a new multiplex PCR for identification.

Deletion of pre-adult ßC-globin in sheep harboring BB haplotype of ß-globin was associated to decreased tolerance to anemia and hypoxia, and consequently, reduced resistance to Haemonchus contortus infection, which is closely related to severe anemia. Recently, a qPCR using hydrolysis probe was successfully developed for ß-globin haplotype identification, and association between resistance against H. contortus and presence of ßA allele was observed in Morada Nova sheep. Thus, this study aimed to better investigate the differences between ß-globin haplotypes and to develop a conventional multiplex PCR, as an alternative to qPCR assay for ß-globin haplotype identification. A total of 333 Morada Nova lambs had their blood collected and tested by both qPCR and new multiplex PCR, and 100 % of agreement was observed between the results. Since different primers were designed for such assay development, including different target genes, high specificity of both methods may be also highlighted. Three A haplotype samples were submitted to DNA Sanger sequencing of ß-globin gene and compared to sequences previously deposited in Genbank. One nucleotide deletion in intronic region was observed only in AA haplotype of Morada Nova animals, while in BB animals the nucleotide remained present. To the best of our knowledge, this is the first report of multiplex conventional PCR for ovine ß-globin haplotype identification. The advantages of the developed conventional PCR are reduced reagents costs (less than a half price) and wider reachability, since even labs without real time PCR thermocyclers are able to offer this assay. Therefore, it may become an important tool for sheep producers to improve genetic selection of parasite resistant animals.

Muscle allele-specific expression QTLs may affect meat quality traits in Bos indicus.

Single nucleotide polymorphisms (SNPs) located in transcript sequences showing allele-specific expression (ASE SNPs) were previously identified in the Longissimus thoracis muscle of a Nelore (Bos indicus) population consisting of 190 steers. Given that the allele-specific expression pattern may result from cis-regulatory SNPs, called allele-specific expression quantitative trait loci (aseQTLs), in this study, we searched for aseQTLs in a window of 1 Mb upstream and downstream from each ASE SNP. After this initial analysis, aiming to investigate variants with a potential regulatory role, we further screened our aseQTL data for sequence similarity with transcription factor binding sites and microRNA (miRNA) binding sites. These aseQTLs were overlapped with methylation data from reduced representation bisulfite sequencing (RRBS) obtained from 12 animals of the same population. We identified 1134 aseQTLs associated with 126 different ASE SNPs. For 215 aseQTLs, one allele potentially affected the affinity of a muscle-expressed transcription factor to its binding site. 162 aseQTLs were predicted to affect 149 miRNA binding sites, from which 114 miRNAs were expressed in muscle. Also, 16 aseQTLs were methylated in our population. Integration of aseQTL with GWAS data revealed enrichment for traits such as meat tenderness, ribeye area, and intramuscular fat . To our knowledge, this is the first report of aseQTLs identification in bovine muscle. Our findings indicate that various cis-regulatory and epigenetic mechanisms can affect multiple variants to modulate the allelic expression. Some of the potential regulatory variants described here were associated with the expression pattern of genes related to interesting phenotypes for livestock. Thus, these variants might be useful for the comprehension of the genetic control of these phenotypes.

Allele-specific expression is widespread in Bos indicus muscle and affects meat quality candidate genes.

Differences between the expression of the two alleles of a gene are known as allele-specific expression (ASE), a common event in the transcriptome of mammals. Despite ASE being a source of phenotypic variation, its occurrence and effects on genetic prediction of economically relevant traits are still unexplored in bovines. Furthermore, as ASE events are likely driven by cis-regulatory mutations, scanning them throughout the bovine genome represents a significant step to elucidate the mechanisms underlying gene expression regulation. To address this question in a Bos indicus population, we built the ASE profile of the skeletal muscle tissue of 190 Nelore steers, using RNA sequencing data and SNPs genotypes from the Illumina BovineHD BeadChip (770 K bp). After quality control, 820 SNPs showed at least one sample with ASE. These SNPs were widespread among all autosomal chromosomes, being 32.01% found in 3'UTR and 31.41% in coding regions. We observed a considerable variation of ASE profile among individuals, which highlighted the need for biological replicates in ASE studies. Functional analysis revealed that ASE genes play critical biological functions in the development and maintenance of muscle tissue. Additionally, some of these genes were previously reported as associated with beef production and quality traits in livestock, thus indicating a possible source of bias on genomic predictions for these traits.

Extreme-QTL mapping of monepantel resistance in Haemonchus contortus.

BACKGROUND: Haemonchus contortus, a gastrointestinal nematode parasite of sheep, is mainly controlled by anthelmintics; the occurrence of anthelmintic resistance leads to treatment failures and increases economic burden. Because molecular mechanisms involved in drug resistance can be elucidated by genomic studies, an extreme quantitative trait locus (X-QTL) mapping approach was used to identify co-segregation of the resistance phenotype with genetic markers to detect the genome-wide variants associated with monepantel resistance in H. contortus. METHODS: A cross between H. contortus isolates using parental susceptible (Par-S) males and monepantel resistant (Par-R) females resulted in SR progeny, while reciprocal cross resulted in RS progeny. Pools (n = 30,000) of infective larvae (L3) recovered from Par-R, and from SR and RS populations in the F3 generation, collected both before (unselected group) and 7 days after (selected group) selection with monepantel treatment in sheep hosts, were subjected to genome sequencing (Pool-Seq). Pairwise comparisons of allele frequencies between unselected and selected groups were performed for each population by Fisher's exact test (FET) and for both populations combined by a Cochran-Mantel-Haenszel (CMH) test. RESULTS: Mapping rates varied from 80.29 to 81.77% at a 90.4X mean coverage of aligned reads. After correction for multiple testing, significant (P < 0.05) changes in allele frequencies were detected by FET for 6 and 57 single nucleotide polymorphisms (SNPs) in the SR and RS populations, respectively, and by the CMH test for 124 SNPs in both populations. The significant variants located on chromosome 2 generated a selection signal in a genomic region harboring the mptl-1, deg-3 and des-2 genes, previously reported as candidates for monepantel resistance. In addition, three new variants were identified in the mptl-1 gene. CONCLUSIONS: This study expands knowledge on genome-wide molecular events underlying H. contortus resistance to monepantel. The identification of a genome region harboring major genes previously associated with monepantel resistance supports the results of the employed X-QTL approach. In addition, a deletion in exon 11 of the mptl-1 gene should be further investigated as the putative causal mutation leading to monepantel resistance.

PCR-based genotyping of SNP markers in sheep.

Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl2 and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.

Target selected treatment with levamisole to control the development of anthelmintic resistance in a sheep flock.

Levamisole phosphate, chosen based on its 100 % efficacy demonstrated by a previous fecal egg count reduction test (FECRT), was used as the exclusive anthelmintic treatment in the Embrapa Southeast Livestock sheep flock from 2009 to 2014 in a target selected treatment scheme. In the present study, the effectiveness of this nematode control scheme was evaluated after 5 years by FECRT, larval development test (LDT), and a molecular test to assess the development of levamisole resistance in Haemonchus contortus. Animals were submitted to treatments with albendazole, levamisole, closantel, ivermectin, moxidectin, and monepantel. Eggs per gram of feces (EPG) counts and fecal cultures were performed, and anthelmintic efficacy was calculated by the RESO 4.0 program. The helminths of the flock (GIN Embrapa2014) were compared to susceptible (McMaster) and resistant (Embrapa2010) H. contortus isolates in the LDT to estimate the LC50 and LC90 of levamisole and in a molecular test to evaluate the 63-bp indel in the acr8 gene associated with levamisole resistance. In the FECRT, parasites were susceptible to monepantel (99.6 %) and closantel (98.3 %), but resistant to moxidectin (93.8 %), levamisole (70.4 %), ivermectin (48.1 %), and albendazole (0 %). In the coproculture on D14, and the control group presented 80 % H. contortus and 20 % Trichostrongylus sp., while in the monepantel group L1 were observed as well as Oesophagostomum sp. L3. LDT and resistance factors provided good separation between susceptible and resistant parasites. The genotypic frequencies of the 63-bp insertion in the acr8 gene in H. contortus were 11.9, 6.7, and 0 % in GIN Embrapa2014, Embrapa2010, and McMaster isolates, respectively. After 5 years of exclusive use, the nematodes developed resistance to levamisole, detected by FECRT and by increase in LC50 and LC90 for levamisole in the LDT. The 63-bp indel was not confirmed as a molecular marker of levamisole resistance in our isolates. The target selected treatment scheme was effective to control helminths in the sheep flock for 5 years, when levamisole's inefficacy was perceived because of no change in the clinical situation of treated animals. Through this scheme, it was possible to promote reversion towards susceptibility or increase of efficacy for other chemical classes. Thus, this is a valid recommendation to control worms and to delay the development of resistance, preserving other anthelmintic classes for future use.

Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.

Chemically assisted enucleation results in higher G6PD expression in early bovine female embryos obtained by somatic cell nuclear transfer.

Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)-derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n=4) expressed XIST transcripts, most embryos showed XIST expression (75%; n=4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless, higher (p<0.05) levels of G6PD were observed in SCNT deme and in vitro-derived groups in comparison to SCNT conv. To know whether higher G6PD expression in embryos derived from SCNT chemically assisted EN indicates higher metabolism in embryos considered of superior quality or if the presence of higher reactive oxygen species (ROS) levels generated by the increased oxygen consumption triggers G6PD activation, the expression of genes related to stress response should be investigated in embryos produced by that technique.

F200Y polymorphism in the ß-tubulin gene in field isolates of Haemonchus contortus and risk factors of sheep flock management practices related to anthelmintic resistance.

Haemonchus contortus is the most prevalent and pathogenic nematode of sheep in tropical areas. The objectives of this study were to assess the frequency of the F200Y polymorphism on the ß-tubulin gene in third-stage larvae of H. contortus from 33 sheep flocks in São Paulo state, Brazil, and to associate this frequency to risk factors based on farm management practices. The resistance allele frequency varied from 9 to 74%, and the resistance genotype frequency varied from 0 to 66.7%. Resistance genotype frequencies higher than 40% were associated with multiple risk factors - new sheep farming enterprises, the absence of farm records, the use of Dorper and Suffolk breeds, rotational grazing, the lack of wetlands on farms, pasture sharing with cattle or horses, frequent incorporation of animals into the flock, semi-intensive farming systems, whole-flock treatment, failure to use the FAMACHA method, lack of the dose-and-move practice, anthelmintic rotation after each application, visual estimation of animal weight for treatment, and lack of drug combination use. It can be concluded that genotyping the F200Y polymorphism can be used to monitor the resistance in sheep flocks and the knowledge of management strategies at the farm level is important to identify drug resistance related factors.

Multidrug and multispecies resistance in sheep flocks from São Paulo state, Brazil.

The economic importance of sheep production is increasing worldwide simultaneously with the emergence of parasitic resistance. This study aimed to survey the current situation of management practices and parasite resistance in sheep flocks in São Paulo state, Brazil. A questionnaire was given to 35 sheep farmers to obtain information related to flock management practices. Of these flocks, 30 were submitted to the fecal egg count reduction test (FECRT) with at least one of the five following anthelmintics: albendazole, closantel, ivermectin, levamisole, and moxidectin, for comparison against an untreated control group. In the survey, the median number animals per flock was 301, mainly of the Santa Ines breed (in 75.8% of the flocks) and crossbred animals (in 54.5% of the flocks). The predominant farming system was semi-intensive (82.9%), using rotational grazing (80%). Selective treatment was based on FAMACHA grade (47.1%) and in clinical signs (41.2%). The most often applied anthelmintics were macrocyclic lactones (42.9-54.2% in the last three applications). Considering the anthelmintics employed in this study, 10.7% of the farms' flocks were resistant to three, 35.7% to four, and 53.6% to all five anthelmintics. The main helminth genera observed before and after treatments were Haemonchus sp. (75.8%) and Trichostrongylus sp. (19.1%), but all observed genera (Cooperia sp., Oesophagostomum sp., and Strongyloides sp.) were detected by the FECRT. Considering efficacy values less than or equal to 90% in the FECRT as resistant, 100% of flocks were resistant to albendazole and ivermectin, 96.6% to moxidectin, 92.9% to closantel, and 53.6% to levamisole. It is thus possible to conclude that multidrug resistance is widespread in sheep flocks in São Paulo state, Brazil, and this involves all prevalent helminth genera.

Polymorphism and parent-of-origin effects on gene expression of CAST, leptin and DGAT1 in cattle.

This study aimed to investigate differential allele expression (DAE) and polymorphism and parent-of-origin effects on expression of genes related to beef traits. CAST, related to meat tenderness, and DGAT1 and leptin, related to fat deposition, were evaluated. In bovine fetal tissues CAST was expressed twice as much (P < 0.05) in muscle of homozygous GG than in heterozygous AG. Leptin was expressed about one-tenth as much (P < 0.05) in heterozygous TpCm (allele T of paternal origin and allele C of maternal origin) than in homozygous CC. No DAE was observed. The evidence of polymorphism effect on expression of CAST and parent-of-origin effect on leptin contributes to a better understanding of events controlling the expression of genes of economic interest in cattle. Furthermore, if the parent-of-origin effects observed in fetal tissues are confirmed in adult tissues and associated to phenotypic variation, this parental origin criterion may be considered in marker-assisted selection of beef traits.