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Due to the negative impact of Haemonchus contortus in the tropics and subtropics, the detection of serum protein profiles that occur in infected sheep is of high relevance for targeted selective treatment strategies (TST). Herein, we integrated proteomics with phenotypic traits to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper - D) and resistant (Santa Inês - S) sheep breeds. Naïve female lambs were infected with H. contortus third-stage larvae on day zero (D0), and samples were collected weekly, for 28 days. Feces were used for individual fecal egg counts (FEC) blood for packed cell volume (PCV) and serum for specific antibody quantification through ELISA. Sera was collected on D0 (-) and D21 (+), and analyzed using a LC-MS/MS based proteomics approach. FEC, PCV, and anti-H. contortus antibody levels confirmed the absence of infection on D0. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3 % and S = 24.3 %, p=0.038). From a total of 754 proteins identified, 68 differentially abundant proteins (DAPs) were noted. Phosphopyruvate hydratase (ENO3) was a DAP in all comparisons, while S+ vs D+ and S- vs D- shared the highest number of DAPs (8). Each of the four experimental groups clustered separately in a principal component analysis (PCA) of protein profile. Among the DAPs, proteins associated with the innate and adaptive immune system were detected when comparing S- vs D- and S+ vs D+. In D-, some proteins were linked to stress response to handling, sampling and heat. Focusing on the consequences of infection in each breed, in the D+ vs D- comparison, upregulated proteins were associated with inflammation control and immune response, where downregulated proteins pointed to a negative impact of infection on tissue anabolism, compromising muscle growth and fat deposition. In the S+ vs S- comparison, upregulated proteins were related to immune response, while the downregulated proteins were possibly linked to muscular development and growth, impaired by infection. Collectively, it can be concluded that ENO3 regulation emerges as a potential factor underlying the differential immune response observed between Santa Inês and Dorper sheep infected with H. contortus. In turn, detected acute phase proteins (APPs) reinforce their relation with infection, inflammation and stress conditions, whereas THEMIS-like may contribute to the immune system in Dorper. GSDMD, Guanylate-binding protein and ACAN warrant further investigation as possible biomarkers for TST strategy development., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ana Carolina S. Chagas reports financial support was provided by State of Sao Paulo Research Foundation. Andre M. Almeida reports financial support was provided by Foundation for Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lambs harboring the Hb-AA β-globin haplotype present improved cell-mediated responses and increased resistance against Haemonchus contortus infection. The aim of the present study was to compare the effect of sex and β-globin haplotypes on specific humoral responses and phenotypes of resistance during H. contortus infection in Morada Nova sheep. As expected, females displayed stronger resistance during the first and second experimental challenges. Differential systemic humoral immune responses were observed comparing sex groups, in which higher levels of specific antibodies targeting 24 kDa excretory-secretory (ES24) protein of H. contortus of IgG and IgM antibodies were respectively observed as predominant isotypes in males and females. The IgM levels were significantly correlated with phenotypes of resistance, evaluated by packed cell volume and fecal egg counts. To our knowledge this is the first study reporting divergent humoral responses profiles to H. contortus infection between male and female sheep. The impact of β-globin haplotypes was less pronounced in females compared to males. Notably, only males showed significant weight differences across haplotypes, with Hb-AA lambs being the heaviest. Additionally, Hb-AA males had significantly higher PCV (indicating better red blood cell health) and lower FEC (indicating lower parasite burden). These findings suggest a more pronounced effect of β-globin polymorphisms on H. contortus infection in males, potentially due to their generally weaker resistance compared to females. This study highlights the importance of sex and β-globin haplotypes in shaping immune responses to H. contortus infection. Specifically, IgM antibodies targeting the ES24 protein appear to play a crucial role in host-parasite interactions and may hold promise for therapeutic development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β-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 β-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 β-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 β-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 β-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., Competing Interests: Declaration of Competing Interest The authors report no declarations of competing interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Results with the use of hCG after synchronization protocol are still inconsistent, which may vary according to breed, season, day of application and dose of the drug used. The aim of the present study was to evaluate the functionality of luteal tissue and ovarian perfusion after hCG treatment during early luteal phase. Estrus-synchronized ewes were randomly assigned to receive i.m. injection of 300 IU of hCG (G-hCG; n = 40) or 1 mL of saline (G-Control; n = 32) on Day 7.5 after progesterone withdrawal. Ultrasonographic evaluations of the ovaries and ovarian and iliac arteries were performed on Days 7.5, 10.5, 13.5, and 21.5. The accessory corpus luteum (aCL) formation rate was 52.5% for G-hCG. There was interaction (p > 0.05) for treatment (G-hCG and G-Control), days (7.5, 10.5, 13.5 and 21.5) and PD (Pregnant and Non-pregnant) for the variables of biometric characteristics of the corpus luteum B-Mode and Color Doppler on days 7.5, 10.5, 13.5 and 21.5. There was no difference (p > 0.05) for pregnancy rates and mean fetuses per ewe between the treatment groups. It is concluded that the application of hCG 7.5 days after the hormonal protocol in Morada Nova ewes in a breeding season is efficient in inducing aCL formation and increasing luteal tissue biometry. However, there was no effect on pregnancy rate., Competing Interests: Declaration of Competing Interest I, Joedson Dantas Gonçalves, declare that I do not have any personal or financial interests that could influence or bias my actions, decisions, or judgments in any professional capacity. I have no affiliations, investments, or relationships that could create conflicts of interest. I acknowledge that conflicts of interest can compromise objectivity, fairness, and integrity, and I am committed to maintaining the highest level of ethical conduct in all my endeavors. I will disclose any potential conflicts of interest that may arise during the course of my work, and take appropriate steps to mitigate or address them transparently and in accordance with relevant laws, regulations, and organizational policies. Furthermore, I understand that any actions or decisions I take in my professional role should be based on merit, relevant expertise, and the best interests of the individuals, organizations, or entities that I serve. I will act in a fair, impartial, and transparent manner, and avoid any actions that could compromise my ability to fulfill my professional duties objectively and with integrity. hereby affirm that the above declaration accurately reflects my current understanding and commitment to upholding high standards of integrity, transparency, and ethical conduct in all my professional endeavors., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Association between ovine β-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity β A ➔ β C 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 β-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 β-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 β A 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 β A allele of β-globin haplotype., (© 2023 John Wiley & Sons Ltd.)

Haemonchus contortus is the most prevalent and important gastrointestinal nematode (GIN) in small ruminants. Since it reduces the packed cell volume (PCV), causing anemia, early diagnosis can be used for targeted selective treatment (TST) of sheep, reducing antiparasitic drug use and anthelmintic resistance. This study aimed to predict PCV values through near-infrared spectroscopy (NIRS) and to develop a classification and diagnosis model of H. contortus infection using PCV values, eggs per gram of feces (EPG) counts and mean daily weight gain (DWG). A total of 1728 spectra were collected from blood samples of 216 lambs with a portable NIR spectroscope. In parallel, other parameters indicative of infection were measured: PCV by hematocrit, FAffa MAlan CHArt (FAMACHA) scores, EPG and DWG. To evaluate the relationship between NIRS spectra and the evaluated parameters, principal component analysis (PCA) was used for an exploratory analysis, regression by the partial least squares method (PLS) for the prediction of PCV values via NIRS, and PCA linear discriminant analysis (PCA-LDA) as a classification model for diagnosis. The absorption peaks in the NIRS region associated with the excitation of overtones of nitrogen-hydrogen (N-H) functional groups of proteins had a strong impact on the principal components (PCs), indicating that blood proteins, especially hemoglobin, can be estimated by the NIRS technique. The model for predicting PCV by PLS presented a standard error of prediction of 2.53, root-mean-square error of 2.48, and coefficient of determination of 0.84, indicating good correlation between the PCV values predicted by the model and the PCV obtained by hematocrit. The PCA-LDA model presented 93.33% sensitivity and 82.18% accuracy, both higher than those of the FAMACHA method, as was expected for resilient Morada Nova lambs. The multivariate models associated with the NIRS technique reported here can be used in the future as a quick and versatile tool for H. contortus infection diagnosis and TST application in lambs., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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.

The objective of the study was to evaluate energy or energy/protein supplementation in the ewe diet, in the last third of gestation, on maternal placental and endocrine characteristics, as well as its effects on the behavior of neonatal lambs and productive performance until weaning. A total of 128 ewes were used, and the experimental diet was fed from 100 days gestation until lambing, with the birth of 172 lambs. The ewes were distributed into three treatments: CTL (control, n = 43) with metabolizable energy (ME) and crude protein (CP) intake according to NRC (1985); ME (energy supplementation, n = 44) plus 21% ME; and MECP (energy/protein supplementation, n = 41) plus 26% ME and CP. Body weight, body condition score, serum hormone concentrations, placental characteristics, lamb performance and behavior, and production efficiency of the ewe from lambing to weaning were measured. ME and MECP ewes were heavier before (p = 0.006) and just after lambing (p = 0.002) and had higher serum triiodothyronine (p = 0.001) and cortisol (p = 0.004) concentrations on the day of lambing. ME ewes had higher placental efficiency (p = 0.036) and lower total cotyledon weight (p = 0.011). ME and MECP diets increased both lamb birth weight (p = 0.015) and weaning weight (p = 0.009). Production efficiency at birth and at weaning was not influenced (p > 0.05) by treatments. Lamb behavior was influenced by the ME and MECP diets, reducing the time to kneel (p ≤ 0.05), to try to stand (p ≤ 0.05), and the latency to stand (p ≤ 0.005). It is concluded that overnutrition in the last third of gestation was positive for the ovine production system, with improved production rates, hormonal profile, placental characteristics, and neonatal behavior.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study evaluated the economic impact of gastrointestinal nematode (GIN) infection in Morada Nova lambs under different parasite chemical control conditions. For this, 246 lambs, in the rainy and dry season, were randomized into groups according to their anthelmintic treatment with levamisole: control (CT: no treatment); routine treatment (RT: treated every 42 days); and targeted selective treatment (TST: treated according to the average daily weight gain, DWG). From 63 days of age (D63) to D210, the lambs were weighed and monitored for GIN infection parameters. Spending on anthelmintics in the production system was 1.3% of the total economic result. The economic result per animal (R$ 5.00 = US$ 1.00) was higher in the RT group, amounting to US$ 6.60 in the rainy and US$ 5.69 in the dry season, due to higher DWG. Thus, RT presented economic results 14.4% and 10.9% higher than CT, and 7.2% and 1.9% higher than TST, in the rainy and dry season, respectively. However, fast development of resistance made RT unfeasible. Here, the economic impact of GIN infection on a national scale is discussed, demonstrating its importance and the impossibility of profitable and sustainable sheep production without adequate control.

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&#95;rs427671974, OAR2&#95;rs419988472, OAR5&#95;rs424070217, and OAR17&#95;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.

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 LC 50 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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9 SOV , n = 21) or 12-day (G-12 SOV , n = 21) progesterone (P 4 )-based estruses synchronization protocol coupled with superovulatory treatment with decreasing doses of porcine follicle-stimulating hormone (133 mg of pFSH given over 3 days). Non-surgical embryo recovery (NSER) was performed 6-7 days after the onset of oestrus. Total antral follicle count doubled from the first to the sixth pFSH dose in both groups (p < .05). Oestrus responses did not vary between the two groups of animals (95.2%). Corpora lutea (CL) were detected in 85.0% and 60.0% of ewes that previously manifested oestrus behaviour in G-9 SOV and G-12 SOV respectively. NSER was successfully completed in 86.2% of ewes that had CL (p > .05). The mean number of CL per ewe/successfully flushed donor ewe was greater (p < .05) in G-12 SOV (12.3 ± 1.7/12.1 ± 1.9) than in G-9 SOV (7.9 ± 1.4/8.2 ± 1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p > .05) in G-12 SOV (5.8 ± 1.9 and 3.7 ± 1.7) than G-9 SOV (3.5 ± 1.1 and 0.8 ± 0.3 respectively). The total follicle count (all follicles ≥2 mm in diameter) at the sixth pFSH dose (at P 4 -device removal) was positively correlated (p < .05) with the number of CL (r = .95) and viable embryos (r = .91) in G-12 SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12 SOV group had better superovulatory responses compared with G-9 SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P 4 for 12 days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes., (© 2021 Wiley-VCH GmbH.)

Deletion of pre-adult β C -globin in sheep harboring BB haplotype of β-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 β-globin haplotype identification, and association between resistance against H. contortus and presence of β A allele was observed in Morada Nova sheep. Thus, this study aimed to better investigate the differences between β-globin haplotypes and to develop a conventional multiplex PCR, as an alternative to qPCR assay for β-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 β-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 β-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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The effect of genetic crossing and nutritional management on weight gain and the concentration of minerals and trace elements in the carcass, blood, leather, and viscera of sheep were evaluated. Several statistical strategies were used to evaluate the different elemental composition characteristics of pure breed animals, i.e., White Dorper (ODO), Ile de France (OIF), Texel (OTX), and Santa Inês (OSI), as well as their crossbreeds 1/2 White Dorper and 1/2 Santa Inês (ODS), 1/2 Ile de France, and 1/2 Santa Inês (OIS), 1/2 Texel × 1/2 Santa Inês (OTS). Three different diets were evaluated AL (ad libitum), R75, and R63 (75 and 63 g of dry matter/kg of the animal metabolic weight, respectively). Levels of Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn were determined by inductively coupled plasma optical emission spectrometry (ICP OES). The concentration of inorganic elements in the different body components was not affected by the diet (P > 0.05), and OTX and OTS were the breeds with the highest weight gain. Random forest importance models demonstrated that Zn in the carcass, K, Ca, and Zn in blood, and K in leather are most associated with separating the different evaluated sheep's breeds. Crossbreeding the native Santa Inês breed with sheep of exotic breeds produces animals well adapted to confinement., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

This study assessed the feasibility of in vivo embryo production and nonsurgical embryo recovery (NSER) in Morada Nova ewes (an endangered native Brazilian breed of sheep) subjected to different estrus synchronization and/or superovulation protocols. Ewes received intravaginal sponges soaked with 60 mg medroxyprogesterone acetate (MAP), which were kept in place for six (G6; n  = 12), nine (G9; n  = 12), or 12 (G12; n  = 12) days. Half of the ewes in each group remained estrus synchronized only ( SYNCH ) and the other half was superovulated ( SOV ) with 133 mg porcine follicle-stimulating hormone (pFSH). There were no differences ( p  > 0.05) in antral follicle counts determined with ultrasonography 60 hours before MAP sponge removal (or at the time of the first pFSH dose) among G6 (6.4 ± 0.9), G9 (6.2 ± 0.7), and G12 (5.5 ± 0.6). Estrus responses and NSER success rates did not vary ( p  > 0.05) among the three progestin-treatment groups of ewes for either estrus-induced or superovulated animals. The onset of estrus occurred 10-12 hours later ( p  < 0.01) in G9 SYNCH ewes compared with G6 SYNCH and G12 SYNCH, and the duration of estrus was ∼19 hours greater ( p  < 0.01) in G9 SOV than in G6 SOV . The average duration of the NSER procedure was 32.6 ± 1.3 minutes. At least one structure was recovered in 85.7% of synchronized and in 87.5% of superovulated ewes. Viable embryo recovery rates were also similar ( p  > 0.05) for G6 (1.0 ± 0.3 and 2.5 ± 1.5), G9 (1.3 ± 0.5 and 4.8 ± 2.0), and G12 groups (1.0 ± 0.3 and 4.8 ± 2.3; estrus-synchronized and superovulated ewes, respectively). In conclusion, progestogen pretreatment of different durations and NSER can be employed in Morada Nova ewes, resulting in reasonable viable embryo recovery rates in both estrus-synchronized and superovulated animals. Therefore, both techniques are suitable for use in commercial settings as well as small ruminant conservation programs.

Two β-globin allelic haplotypes (A and B) were identified in domestic sheep, wherein animals which are homozygous for β B allele (BB haplotype) have a deletion of pre-adult β C -globin and consequently are less tolerant to anemia and hypoxia. Since Haemonchus contortus infection, is associated with severe anemia, studies performed from 1960s to 1990s investigated the association between β-globin haplotype and resistance against this parasite. However, the findings were controversial, pointing out from increased resistance in animals harboring the β A allele to inexistence of association. Thus, our study aimed to develop a qPCR for β-globin haplotype identification, and to evaluate the association between β-globin haplotype and resistance against H. contortus in a group of sheep submitted to artificial infection with this parasite. A total of 286 lambs of Morada Nova breed were experimentally challenged with 4000 H. contortus L3 and monitored for 112 days from weaning. Significantly improved (p < 0.05) phenotypic profiles (lower fecal egg counts, higher packed cell volume and birthweight) were observed for AA haplotype animals, especially when compared to BB animals, while AB animals were similar to BB. This is the first report of a qPCR assay for ovine β-globin haplotype identification. In view of significant differences of phenotypic profiles between haplotype groups, the developed qPCR may constitute an important tool for sheep producers to improve genetic selection of parasite resistant animals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotropin (eCG) im and 37.5 μg d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P 4 ) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gastrointestinal nematodes are a major constraint in sheep production. Breeding for resistance has proven to be an effective and feasible approach to address this problem. The use and investigation of genetic markers for resistance traits could accelerate genetic progress and lead to a better understanding of underlying molecular mechanisms. Thus, the aim of this study was to evaluate if five single nucleotide polymorphisms SNPs OAR2&#95;14765360, OAR6&#95;81718546, OAR11&#95;62887032, OAR12&#95;69606944 and OAR15&#95;59871543 are associated with resistance and resilience traits in a flock of the Morada Nova sheep breed. Lambs were submitted to two consecutive parasite challenges by oral infection with 4000 infective larvae L 3 ) of Haemonchus contortus. Fecal egg counts (FEC), packed cell volume (PVC) and body weight were measured every one or two weeks for 42 days in each trial. DNA samples from 287 lambs, 131 ewes and 4 rams were amplified by ARMS-PCR or PCR-RFLP and genotypes were determined. Analysis of variance (ANOVA) was used for association analyses between genotypes and phenotypes. In case of significant association, the allele substitution effect was calculated based on a linear model. OAR2&#95;14765360 and OAR12&#95;69606944 were associated with FEC, and OAR12&#95;69606944 also had significant effects on PCV and weight gain, showing favourable associations of the CC genotype with all evaluated traits. Both OAR6&#95;81718546 and OAR11&#95;62887032 were associated with weight gain, and OAR6&#95;81718546 had an additional effect on PCV. OAR15&#95;59871543 was not polymorphic in the population. OAR6&#95;81718546 and OAR12&#95;69606944 presented significant allele substitution effects of -1.06 ± 0.52 kg for the T allele on final body weight and 0.74 ± 0.32 for the C allele in PCV of the same sampling date, respectively. This is the first report of SNPs associated with gastrointestinal nematode resistance in this sheep breed. Our findings support the existence of quantitatice trait loci (QTL) for resistance and resilience in linkage disequilibrium with the polymorphic SNPs and suggest their future use for explorations of these traits in Morada Nova sheep., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H . contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L 3 . 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L 3 , and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1β expression levels ( P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed ( P < 0.05). Higher levels ( P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels ( P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H . contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response., Competing Interests: The authors have no conflict of interest., (Copyright © 2019 João Henrique Barbosa Toscano et al.)

Morada Nova breed sheep are without wool, tropicalized, small-sized animals, known for their high-quality meat and skin. Their body development naturally depends on the genetic potential and adequate nutritional support, which suggests that the offer of high-energy density diets positively influences their productive indicators. Thus, the present study investigated the effect of a high-energy diet for the Morada Nova lambs on body development and testicular function, considering their histomorphometric characteristics and seminal quality. Forty-two males (19.2 weeks, 20.7 ± 3.5 kg) were equally divided into two groups and fed with 2.05 Mcal (G7, n = 21) or 2.37 Mcal (G24, n = 21) of metabolizable energy/day, equivalent to 7% and 24% above the minimum for growing lambs. The animals were confined for 23 weeks (W0 to W23). Weight and body score differed significantly from the W1 (P < 0.05). From the W5, thoracic perimeter, body length, wither height, and rump attributes were higher in G24 (P < 0.05). The scrotal circumference and testicular volume were higher in G24 from the W3 (P < 0.05). Although testosterone levels were not affected (P = 0.05), the highest energy intake increased the diameter of the seminiferous tubules and the development of the epididymal epithelium (P < 0.05). This positively influenced the seminal quality and reduced the minor defects (21.87% vs. 17.13%) and the total spermatic defects (26.34% vs. 21.78%, P < 0.05). Thus, it is possible to employ higher levels of dietary energy for Morada Nova young males to express higher productive efficiency and earlier reproductive attributes of interest.

Morada Nova is a Brazilian hair sheep breed that is well adapted to the country's mainly tropical climate and has good potential for meat and leather production. This breed is reported to be resistant to Haemonchus contortus infection, a highly desired characteristic due to the large impact of this parasite on sheep farming. Therefore, the present study aimed to characterize 287 recently weaned Morada Nova lambs and 123 ewes in relation to their resistance against H. contortus . The animals were dewormed and 15 days later artificially infected with 4000 H. contortus L 3 (D0). They were individually monitored by periodic assessment of fecal egg count (FEC), packed cell volume (PCV), and live weight (LW). On D42, the sheep were again dewormed and submitted to a new parasitic challenge, following the same scheme. The animals of each category (lambs and ewes) were ranked according to individual mean FEC values, and classified as resistant (R, 20%), intermediate (I, 60%), or susceptible (S, 20%) to H. contortus infection. At weaning, high FEC were observed in all three phenotypes (P > 0.05). After the artificial infections, there was a significant difference (P < 0.05) among the three lamb phenotypes for the mean FEC (R < I < S), PCV (R > I > S), and LW (R = I > S). The infection levels (FEC) were negatively correlated with PCV (r = -0.66; P < 0.001), and LW (r = -0.30; P < 0.001). Despite this, the lambs were resilient, since more than 88% of these animals maintained the PCV above 24%, even when heavily infected. The importance of selective parasite control before weaning to reduce the negative impact on slaughter weight was evidenced, taking into account the high positive correlation between LW at weaning and final LW (r = 0.73; P < 0.001). The ewes, in turn, were strongly resistant to the parasite. Despite highly significant differences (P < 0.001) for mean FEC between phenotypes (R < I < S), 98% of the ewes maintained FEC below 4000 EPG. Their health was not affected, since PCV and LW did not differ between phenotypes, and these parameters were not significantly correlated with FEC (P > 0.05). With the phenotypic characterization performed here, it is possible to introduce procedures for parasite control in Morada Nova flocks, facilitating the target-selective treatment approach. The results of this study can also support improvement of meat production by the Morada Nova breed., (© 2019 The Authors.)

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous-induced Santa Inês ewes (6-day treatment with progestin-releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6-7 days after oestrous onset and mating. All ewes received 37.5 μg of d-cloprostenol via latero-vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non-surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17-40 min) and ~96% of flushing fluid could be recovered (range: 85%-100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep., (© 2019 Blackwell Verlag GmbH.)

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.

Gastrointestinal nematodes significantly affect the ovine industry, and Haemonchus contortus is considered the most pathogenic parasite in tropical regions. This situation is aggravated when the main strategy to control worms fails because of the genetic resistance that parasites acquire against anthelmintics. Aiming to anticipate the events involved in anthelmintic resistance, we induced monepantel resistance in H. contortus by in vivo subdosing of sheep hosts. Four successive passages of a monepantel-susceptible H. contortus isolate in Santa Ines or Ile de France sheep hosts resulted in three monepantel-resistant (efficacy varying from 0 to 58.5%) H. contortus isolates. Sheep hosts were treated from 0.075 mg/kg to the therapeutic dose of 2.5 mg/kg of monepantel in 19-26 rounds of selection for 112-133 weeks. Success in inducing H. contortus resistance to monepantel may have been affected by worm burden and by host-parasite interactions, including a possible effect of the breed of sheep hosts. We conclude that subdosing of sheep, although time-consuming, is an efficient in vivo strategy for the induction of monepantel resistance in H. contortus. The resistant parasites can be used in further studies to elucidate the genetic and biochemical events involved in the acquisition of anthelmintic resistance.

For different ovine breeds to maximize their reproductive capacity in countries with tropical climate, it is important to evaluate their potential for thermal resilience and consequences on their reproductive traits. Therefore, the objective of this study was to evaluate the effect of thermal environment temperatures of climate seasons in a tropical climate region on the surface temperatures of the scrotum, testicular biometric characteristics, seminal quality and serum testosterone concentration of rams of different genotypes. Breeders of four different genotypes (Dorper, n = 8, Texel, n = 8, Santa Inês, n = 9 and Morada Nova, n = 8) were used throughout the four climate seasons. Higher thermal challenge was recorded in the spring and summer. In the summer increase in scrotal surface temperature was detected by infrared thermography (P < 0.05), mainly in the regions of the distal testicular pole and tail of the epididymis. The animals of the Texel genotype had higher rectal temperature in the summer. In spring, this genotype also had the highest testicular pole (32.2 ± 0.5 °C; P < 0.05) and distal (29.9 ± 0.4 °C; P < 0.05) temperatures and a higher mean testicular temperature (31.7 ± 0.4 °C; P < 0.05). The Morada Nova genotype showed a higher surface temperature gradient between testicular poles (2.96 ± 0.1 °C; P < 0.05), especially in spring. Genotype-dependent thermal sensitivity was detected for the thermal gradient between the testicular poles, reflecting the seminal quality. There was a positive correlation of the thermal gradient between testicular poles with sperm membrane integrity and negative correlation with total sperm defects. The Texel genotype showed less progressive motility and higher percentage of sperm defects. There was no difference in testosterone concentration between genotypes and in the different seasons (P > 0.05). Thus, the indigenous genotypes showed a greater capability to maintain the scrotum-testicular thermoregulation. Dorper animals resembled the indigenous sheep genotypes, in terms of seminal characteristics, unlike Texel animals, which showed lower adaptability and lower seminal quality., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Morada Nova is a Brazilian hair sheep breed that is well adapted to the country's mainly tropical climate and has good potential for meat and leather production. This breed is reported to be resistant to Haemonchus contortus infection, a highly desired characteristic due to the large impact of this parasite on sheep farming. Therefore, the present study aimed to characterize 287 recently weaned Morada Nova lambs and 123 ewes in relation to their resistance against H. contortus. The animals were dewormed and 15 days later artificially infected with 4000 H. contortus L 3 (D0). They were individually monitored by periodic assessment of fecal egg count (FEC), packed cell volume (PCV), and live weight (LW). On D42, the sheep were again dewormed and submitted to a new parasitic challenge, following the same scheme. The animals of each category (lambs and ewes) were ranked according to individual mean FEC values, and classified as resistant (R, 20%), intermediate (I, 60%), or susceptible (S, 20%) to H. contortus infection. At weaning, high FEC were observed in all three phenotypes (P>0.05). After the artificial infections, there was a significant difference (P<0.05) among the three lamb phenotypes for the mean FEC (RI > S), and LW (R=I > S). The infection levels (FEC) were negatively correlated with PCV (r = -0.66; P<0.001), and LW (r = -0.30; P<0.001). Despite this, the lambs were resilient, since more than 88% of these animals maintained the PCV above 24%, even when heavily infected. The importance of selective parasite control before weaning to reduce the negative impact on slaughter weight was evidenced, taking into account the high positive correlation between LW at weaning and final LW (r=0.73; P<0.001). The ewes, in turn, were strongly resistant to the parasite. Despite highly significant differences (P<0.001) for mean FEC between phenotypes (R0.05). With the phenotypic characterization performed here, it is possible to introduce procedures for parasite control in Morada Nova flocks, facilitating the target-selective treatment approach. The results of this study can also support improvement of meat production by the Morada Nova breed., (Copyright © 2019. Published by Elsevier B.V.)