Long divergent haplotypes introgressed from wild sheep are associated with distinct morphological and adaptive characteristics in domestic sheep.

The worldwide sheep population comprises more than 1000 breeds. Together, these exhibit a considerable morphological diversity, which has not been extensively investigated at the molecular level. Here, we analyze whole-genome sequencing individuals of 1,098 domestic sheep from 154 breeds, and 69 wild sheep from seven Ovis species. On average, we detected 6.8%, 1.0% and 0.2% introgressed sequence in domestic sheep originating from Iranian mouflon, urial and argali, respectively, with rare introgressions from other wild species. Interestingly, several introgressed haplotypes contributed to the morphological differentiations across sheep breeds, such as a RXFP2 haplotype from Iranian mouflon conferring the spiral horn trait, a MSRB3 haplotype from argali strongly associated with ear morphology, and a VPS13B haplotype probably originating from urial and mouflon possibly associated with facial traits. Our results reveal that introgression events from wild Ovis species contributed to the high rate of morphological differentiation in sheep breeds, but also to individual variation within breeds. We propose that long divergent haplotypes are a ubiquitous source of phenotypic variation that allows adaptation to a variable environment, and that these remain intact in the receiving population probably due to reduced recombination.

Screening of hair follicle telogen-associated circRNAs in sheep and construction of their ceRNA network.

Sheep breeds with hair-shedding traits have many advantages over non-shedding sheep breeds, not only because of reduced shearing labor and feeding management costs but also because it reduces in vitro parasites and improves adaptability to summer heat stress. The wool of Dorper sheep naturally sheds in spring due to the periodic growth of hair follicles. CircRNAs primarily regulate the morphogenesis of hair follicles through the ceRNA mechanism. In this study, five 2-year-old Dorper ewes with extreme hair-shedding phenotype (S) and three Dorper ewes with non-shedding (N) phenotype were selected for subsequent analyses. For RNA extraction, skin tissues were collected on 27th September 2019 (S1, N1), 3rd January 2020 (S2, N2), and 17th March 2020 (S3, N3), which were then subjected to RNA-seq. RNA-seq technology revealed 20,185 novel circRNAs in the hair follicles of Dorper sheep. Among them, 1450 circRNAs were differentially expressed (DE). Clustering heatmap and expression pattern analyses were performed on DE circRNAs, which indicated 78 circRNAs with T pattern (Telogen, highly expressed in telogen), and the source genes for candidate circRNAs were further screened by functional enrichment analysis, which identified 13 crucial genes enriched in pathways associated with hair follicle development. Additionally, a ceRNA regulatory network comprising 4 circRNAs, 11 miRNAs, and 13 target genes was constructed. Overall, this study screened circRNAs that may be associated with the telogen phase of hair follicles in sheep, providing a relevant theoretical basis for wool shedding in sheep and for breeding Dorper sheep with automatic wool shedding.

Genome-wide association study identifies genetic variants underlying footrot in Portuguese Merino sheep.

BACKGROUND: Ovine footrot caused by Dichelobacter nodosus (D. nodosus) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses. RESULTS: Phenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction (p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes. CONCLUSION: This is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

Construction of a high-density genetic map and QTL localization of body weight and wool production related traits in Alpine Merino sheep based on WGR.

BACKGROUND: The Alpine Merino is a new breed of fine-wool sheep adapted to the cold and arid climate of the plateau in the world. It has been popularized in Northwest China due to its superior adaptability as well as excellent production performance. Those traits related to body weight, wool yield, and wool fiber characteristics, which are economically essential traits in Alpine Merino sheep, are controlled by QTL (Quantitative Trait Loci). Therefore, the identification of QTL and genetic markers for these key economic traits is a critical step in establishing a MAS (Marker-Assisted Selection) breeding program. RESULTS: In this study, we constructed the high-density genetic linkage map of Alpine Merino sheep by sequencing 110 F1 generation individuals using WGR (Whole Genome Resequencing) technology. 14,942 SNPs (Single Nucleotide Polymorphism) were identified and genotyped. The map spanned 2,697.86 cM, with an average genetic marker interval of 1.44 cM. A total of 1,871 high-quality SNP markers were distributed across 27 linkage groups, with an average of 69 markers per LG (Linkage Group). Among them, the smallest genetic distance is 19.62 cM for LG2, while the largest is 237.19 cM for LG19. The average genetic distance between markers in LGs ranged from 0.24 cM (LG2) to 3.57 cM (LG17). The marker density in the LGs ranged from LG14 (39 markers) to LG1 (150 markers). CONCLUSIONS: The first genetic map of Alpine Merino sheep we constructed included 14,942 SNPs, while 46 QTLs associated with body weight, wool yield and wool fiber traits were identified, laying the foundation for genetic studies and molecular marker-assisted breeding. Notably, there were QTL intervals for overlapping traits on LG4 and LG8, providing potential opportunities for multi-trait co-breeding and further theoretical support for selection and breeding of ultra-fine and meaty Alpine Merino sheep.

No correlative evidence of costs of infection or immunity on leucocyte telomere length in a wild population of Soay sheep.

Telomere length (TL) is a biomarker hypothesized to capture evolutionarily and ecologically important physiological costs of reproduction, infection and immunity. Few studies have estimated the relationships among infection status, immunity, TL and fitness in natural systems. The hypothesis that short telomeres predict reduced survival because they reflect costly consequences of infection and immune investment remains largely untested. Using longitudinal data from a free-living Soay sheep population, we tested whether leucocyte TL was predicted by infection with nematode parasites and antibody levels against those parasites. Helminth parasite burdens were positively associated with leucocyte TL in both lambs and adults, which is not consistent with TL reflecting infection costs. We found no association between TL and helminth-specific IgG levels in either young or old individuals which suggests TL does not reflect costs of an activated immune response or immunosenescence. Furthermore, we found no support for TL acting as a mediator of trade-offs between infection, immunity and subsequent survival in the wild. Our results suggest that while variation in TL could reflect short-term variation in resource investment or environmental conditions, it does not capture costs of infection and immunity, nor does it behave like a marker of an individual's helminth-specific antibody immune response.

Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy.

The role of the glycosylation status of PrPC in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrPC. Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrPC-expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrPC is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Investigating pedigree- and SNP-associated components of heritability in a wild population of Soay sheep.

Estimates of narrow sense heritability derived from genomic data that contain related individuals may be biased due to the within-family effects such as dominance, epistasis and common environmental factors. However, for many wild populations, removal of related individuals from the data would result in small sample sizes. In 2013, Zaitlen et al. proposed a method to estimate heritability in populations that include close relatives by simultaneously fitting an identity-by-state (IBS) genomic relatedness matrix (GRM) and an identity-by-descent (IBD) GRM. The IBD GRM is identical to the IBS GRM, except relatedness estimates below a specified threshold are set to 0. We applied this method to a sample of 8557 wild Soay sheep from St. Kilda, with genotypic information for 419,281 single nucleotide polymorphisms. We aimed to see how this method would partition heritability into population-level (IBS) and family-associated (IBD) variance for a range of genetic architectures, and so we focused on a mixture of polygenic and monogenic traits. We also implemented a variant of the model in which the IBD GRM was replaced by a GRM constructed from SNPs with low minor allele frequency to examine whether any additive genetic variance is captured by rare alleles. Whilst the inclusion of the IBD GRM did not significantly improve the fit of the model for the monogenic traits, it improved the fit for some of the polygenic traits, suggesting that dominance, epistasis and/or common environment not already captured by the non-genetic random effects fitted in our models may influence these traits.

Neonatal resuscitation with continuous chest compressions and high frequency percussive ventilation in preterm lambs.

BACKGROUND: Cerebral oxygen delivery (cDO2) is low during chest compressions (CC). We hypothesized that gas exchange and cDO2 are better with continuous CC with high frequency percussive ventilation (CCC + HFPV) compared to conventional 3:1 compressions-to-ventilation (C:V) resuscitation during neonatal resuscitation in preterm lambs with cardiac arrest induced by umbilical cord compression. METHODS: Fourteen lambs in cardiac arrest were randomized to 3:1 C:V resuscitation (90CC + 30 breaths/min) per the Neonatal Resuscitation Program guidelines or CCC + HFPV (120CC + HFPV continuously). Intravenous epinephrine was given every 3 min until return of spontaneous circulation (ROSC). RESULTS: There was no difference in the incidence and time to ROSC between both groups. Median (IQR) PaCO2 was significantly lower with CCC + HFPV during CC, at ROSC and 15 min post-ROSC-[104 (99-112), 83 (77-99), and 43 (40-64)], respectively compared to 3:1 C:V-[149 (139-167), 153 (143-168), and 153 (138-178) mmHg. PaO2 and cDO2 were higher with CCC + HFPV during CC and at ROSC. PaO2 was similar 15 min post-ROSC with a lower FiO2 in the CCC + HFPV group 0.4 (0.4-0.5) vs. 1 (0.6-1). CONCLUSION: In preterm lambs with perinatal cardiac-arrest, continuous chest compressions with HFPV does not improve ROSC but enhances gas exchange and increases cerebral oxygen delivery compared to 3:1 C:V during neonatal resuscitation. IMPACT STATEMENT: Ventilation is the most important intervention in newborn resuscitation. Currently recommended 3:1 compression-to-ventilation ratio is associated with hypercarbia and poor oxygen delivery to the brain. Providing uninterrupted continuous chest compressions during high frequency percussive ventilation is feasible in a lamb model of perinatal cardiac arrest, and demonstrates improved gas exchange and oxygen delivery to the brain. This is the first study in premature lambs evaluating high frequency percussive ventilation with asynchronous chest compressions and lays the groundwork for future clinical studies to optimize gas exchange and hemodynamics during chest compressions in newborns.

Total liquid ventilation in an ovine model of extreme prematurity: a randomized study.

BACKGROUND: This study aimed at comparing cardiorespiratory stability during total liquid ventilation (TLV)-prior to lung aeration-with conventional mechanical ventilation (CMV) in extremely preterm lambs during the first 6 h of life. METHODS: 23 lambs (11 females) were born by c-section at 118-120 days of gestational age (term = 147 days) to receive 6 h of TLV or CMV from birth. Lung samples were collected for RNA and histology analyses. RESULTS: The lambs under TLV had higher and more stable arterial oxygen saturation (p = 0.001) and cerebral tissue oxygenation (p = 0.02) than the lambs in the CMV group in the first 10 min of transition to extrauterine life. Although histological assessment of the lungs was similar between the groups, a significant upregulation of IL-1a, IL-6 and IL-8 RNA in the lungs was observed after TLV. CONCLUSIONS: Total liquid ventilation allowed for remarkably stable transition to extrauterine life in an extremely preterm lamb model. Refinement of our TLV prototype and ventilation algorithms is underway to address specific challenges in this population, such as minimizing tracheal deformation during the active expiration. IMPACT: Total liquid ventilation allows for remarkably stable transition to extrauterine life in an extremely preterm lamb model. Total liquid ventilation is systematically achievable over the first 6 h of life in the extremely premature lamb model. This study provides additional incentive to pursue further investigation of total liquid ventilation as a transition tool for the most extreme preterm neonates.

Lung ultrasound detects regional aeration inhomogeneity in ventilated preterm lambs.

BACKGROUND: Inhomogeneous lung aeration is a significant contributor to preterm lung injury. EIT detects inhomogeneous aeration in the research setting. Whether LUS detects inhomogeneous aeration is unknown. The aim was to determine whether LUS detects regional inhomogeneity identified by EIT in preterm lambs. METHODS: LUS and EIT were simultaneously performed on mechanically ventilated preterm lambs. LUS images from non-dependent and dependent regions were acquired and reported using a validated scoring system and computer-assisted quantitative LUS greyscale analysis (Q-LUSMGV). Regional inhomogeneity was calculated by observed over predicted aeration ratio from the EIT reconstructive model. LUS scores and Q-LUSMGV were compared with EIT aeration ratios using one-way ANOVA. RESULTS: LUS was performed in 32 lambs (~125d gestation, 128 images). LUS scores were greater in upper anterior (non-dependent) compared to lower lateral (dependent) regions of the left (3.4 vs 2.9, p = 0.1) and right (3.4 vs 2.7, p < 0.0087). The left and right upper regions also had greater LUS scores compared to right lower (3.4 vs 2.7, p < 0.0087) and left lower (3.7 vs 2.9, p = 0.1). Q-LUSMGV yielded similar results. All LUS findings corresponded with EIT regional differences. CONCLUSION: LUS may have potential in measuring regional aeration, which should be further explored in human studies. IMPACT: Inhomogeneous lung aeration is an important contributor to preterm lung injury, however, tools detecting inhomogeneous aeration at the bedside are limited. Currently, the only tool clinically available to detect this is electrical impedance tomography (EIT), however, its use is largely limited to research. Lung ultrasound (LUS) may play a role in monitoring lung aeration in preterm infants, however, whether it detects inhomogeneous lung aeration is unknown. Visual LUS scores and mean greyscale image analysis using computer assisted quantitative LUS (Q-LUSMGV) detects regional lung aeration differences when compared to EIT. This suggests LUS reliably detects aeration inhomogeneity warranting further investigation in human trials.

Postnatal steroids as lung protective and anti-inflammatory in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization precede bronchopulmonary dysplasia (BPD). Glucocorticoids are anti-inflammatory and reduce ventilator requirements in preterm infants. However, high-dose glucocorticoids inhibit alveolarization. The effect of glucocorticoids on lung function and structure in preterm newborns exposed to antenatal inflammation is unknown. We hypothesise that postnatal low-dose dexamethasone reduces ventilator requirements, prevents inflammation and BPD-like lung pathology, following antenatal inflammation. METHODS: Pregnant ewes received intra-amniotic LPS (E.coli, 4 mg/mL) or saline at 126 days gestation; preterm lambs were delivered 48 h later. Lambs were randomised to receive either tapered intravenous dexamethasone (LPS/Dex, n = 9) or saline (LPS/Sal, n = 10; Sal/Sal, n = 9) commencing <3 h after birth. Respiratory support was gradually de-escalated, using a standardised protocol aimed at weaning from ventilation towards unassisted respiration. Tissues were collected at day 7. RESULTS: Lung morphology and mRNA levels for inflammatory mediators were measured. Respiratory support requirements were not different between groups. Histological analyses revealed higher tissue content and unchanged alveolarization in LPS/Sal compared to other groups. LPS/Dex lambs exhibited decreased markers of pulmonary inflammation compared to LPS/Sal. CONCLUSION: Tapered low-dose dexamethasone reduces the impact of antenatal LPS on ventilation requirements throughout the first week of life and reduces inflammation and pathological thickening of the preterm lung IMPACT: We are the first to investigate the combination of antenatal inflammation and postnatal dexamethasone therapy in a pragmatic study design, akin to contemporary neonatal care. We show that antenatal inflammation with postnatal dexamethasone therapy does not reduce ventilator requirements, but has beneficial maturational impacts on the lungs of preterm lambs at 7 days of life. Appropriate tapered postnatal dexamethasone dosing should be explored for extuabtion of oxygen-dependant neonates.

Phenotype transition from wild mouflon to domestic sheep.

The domestication of animals started around 12,000 years ago in the Near East region. This "endless process" is characterized by the gradual accumulation of changes that progressively marked the genetic, phenotypic and physiological differences between wild and domesticated species. The main distinctive phenotypic characteristics are not all directly attributable to the human-mediated selection of more productive traits. In the last decades, two main hypotheses have been proposed to clarify the emergence of such a set of phenotypic traits across a variety of domestic species. The first hypothesis relates the phenotype of the domesticated species to an altered thyroid hormone-based signaling, whereas the second one relates it to changes in the neural crest cells induced by selection of animals for tameness. These two hypotheses are not necessarily mutually exclusive since they may have contributed differently to the process over time and space. The adaptation model induced by domestication can be adopted to clarify some aspects (that are still controversial and debated) of the long-term evolutionary process leading from the wild Neolithic mouflon to the current domestic sheep. Indeed, sheep are among the earliest animals to have been domesticated by humans, around 12,000 years ago, and since then, they have represented a crucial resource in human history. The aim of this review is to shed light on the molecular mechanisms and the specific genomic variants that underlie the phenotypic variability between sheep and mouflon. In this regard, we carried out a critical review of the most recent studies on the molecular mechanisms that are most accredited to be responsible for coat color and phenotype, tail size and presence of horns. We also highlight that, in such a complicate context, sheep/mouflon hybrids represent a powerful and innovative model for studying the mechanism by which the phenotypic traits related to the phenotypic responses to domestication are inherited. Knowledge of these mechanisms could have a significant impact on the selection of more productive breeds. In fact, as in a journey back in time of animal domestication, the genetic traits of today's domestic species are being progressively and deliberately shaped according to human needs, in a direction opposite to that followed during domestication.

Genistein effect in cultured ovine ovarian granulosa cells.

Genistein, an isoflavone has the potential to mimic, augment, or dysregulate the steroid hormone production pathways. We hypothesized that genistein affects the granulosa cell (GCs) functions through a series of biochemical, molecular, and genomic cascades. The present study was conducted to evaluate the impact of genistein exposure on GCs viability, apoptosis, and steroidogenesis. The present study involved 3/5 days of exposure to genistein on GCs collected from abattoir-derived ovine ovaries at doses of 0, 1, 10, 25, 50, and 100 µM. The harvested GCs were used for growth, cytotoxicity, and gene expression studies related to apoptosis, growth, and steroidogenesis. We observed that genistein had both stimulatory at 10 and 25 µM levels as well as inhibitory effects at 50 and 100 µM levels on the growth and proliferation of GCs. Genistein significantly decreased the levels of 17ß-estradiol at higher exposure (50 and 100 µM), whereas the progesterone level increased significantly as the genistein exposure increased. Additionally, genistein could also alter the mRNA expression of the steroidogenic receptor, enzymes, proteins, and growth-related genes suggesting that genistein could potentially alter the steroidogenic pathways. We conclude that genistein can interfere with cell survival and steroidogenesis by exhibiting a dose-dependent biphasic response on the viability, growth-related parameters, and the synthesis of 17ß-estradiol in the cultured GCs.

The impact of different levels of wheat diets on hepatic oxidative stress, immune response, and lipid metabolism in Tibetan sheep (Ovis aries).

BACKGROUND: Compared with corn, wheat contains higher crude protein, amino acids concentration. However, wheat contains a mass of anti-nutritional factors, resulting in increased of the digesta viscosity and impaired the intestinal function in ruminant. OBJECTIVE: This study aimed to investigate the effects of substitution of different amounts of wheat for corn on hepatic metabolism in the Tibetan lamb. METHODS: Ninety Tibetan lambs (Body weight = 12.37 ± 0.92 kg) were randomly assigned to three groups: 0% wheat diet (Control), 10% wheat diet (Low group), and 15% wheat diet (High group). The feeding trial lasted for 130 d, including a 10 d adaption period. Hepatic gene expression profiling was performed via RNA sequencing after the conclusion of the feeding trials. RESULTS: Results showed that greater level of glutathione peroxidase levels in L group compared with those of the C and H groups (P < 0.05). The immune indexes, including interleukin-1ß (IL-1ß), immunoglobulin A (IgA), and IgM were also elevated in L group compared with the other groups (P < 0.05). Compared with H group, the hepatocytes were arranged radially, and hepatic plates anastomosed with each other to form a labyrinth-like structure in L group. Transcriptomic analysis showed 872 differentially expressed genes (DEG) between H and L group, of which 755 were down-regulated and 117 were up-regulated. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 32 pathways were significantly enriched (Q-value < 0.05), such as the cAMP signaling pathway, Th1 and Th2 cell differentiation, leukocyte transendothelial migration, platelet activation and adipocytokine signaling pathway. Additionally, the expression of comment DEGs were verified via quantitative reverse-transcription polymerase chain reaction. CONCLUSION: In summary, our findings suggest that wheat can be supplemented up to 10% in Tibetan sheep, contributing to improve the hepatic oxidative stress, immune response and lipid metabolism through regulating the expression of related genes.

Chestnut tannin extract modulates growth performance and fatty acid composition in finishing Tan lambs by regulating blood antioxidant capacity, rumen fermentation, and biohydrogenation.

Tannins as plant extracts have emerged as promising and potential alternatives for antibiotics in modern livestock cultivation systems. This study investigates the effect of dietary chestnut tannin extract (CTE) in finishing Tan lambs. Twenty-seven male Tan lambs were randomly divided into three groups: (1) control group (CON; basal diet); (2) low-dose CTE group (LCTE; basal diet + 2 g/kg CTE, dry matter [DM] basis); (3) high-dose CTE group (HCTE; basal diet + 4 g/kg CTE, DM basis). The HCTE group exhibited markedly higher average daily gain (ADG) and DM intake than CON (P < 0.01). The ruminal total volatile fatty acid concentration increased linearly with increasing CTE supplementation (P < 0.01), while the opposite trend was observed for butyrate molar proportion (P < 0.01). Upon increasing CTE dosage, plasma glucose, high-density lipoprotein cholesterol, glutathione peroxidase, and superoxide dismutase content increased linearly (P < 0.05), whereas low-density lipoprotein cholesterol and urea nitrogen decreased linearly or quadratically (P < 0.05), respectively. A linear increase was also observed in ruminal t6 C18:1 and t9, c12 C18:2 proportions (P < 0.01), and plasma C18:2n-6 and n-6 polyunsaturated fatty acids proportions with increased CTE supplementation (P < 0.01). In the longissimus dorsi muscle, the atherogenic index decreased linearly (P < 0.05), while c11 C18:1 and C20:5n-3 increased linearly (P < 0.05). Moreover, c9, t11 conjugated linoleic acids proportion increased in subcutaneous fat with CTE supplementation (P < 0.01). In conclusion, Dietary CTE enhances the ADG of finishing Tan lambs in a dose-dependent manner, modulates plasma metabolites and antioxidant capacity, and improves rumen fermentation and body fatty acid composition. These results provide a reference for the rational application of CTE in ruminant production.

Analysis of fecal microbiome and metabolome changes in goats with pregnant toxemia.

BACKGROUND: Pregnancy toxemia is a common disease, which occurs in older does that are pregnant with multiple lambs in the third trimester. Most of the sick goats die within a few days, which can seriously impact the economic benefits of goat breeding enterprises. The disease is believed to be caused by malnutrition, stress, and other factors, that lead to the disorder of lipid metabolism, resulting in increased ketone content, ketosis, ketonuria, and neurological symptoms. However, the changes in gut microbes and their metabolism in this disease are still unclear. The objective of this experiment was to evaluate the effect of toxemia of pregnancy on the fecal microbiome and metabolomics of does. RESULTS: Eight pregnant does suspected of having toxemia of pregnancy (PT group) and eight healthy does during the same pregnancy (NC group) were selected. Clinical symptoms and pathological changes at necropsy were observed, and liver tissue samples were collected for pathological sections. Jugular venous blood was collected before morning feeding to detect biochemical indexes. Autopsy revealed that the liver of the pregnancy toxemia goat was enlarged and earthy yellow, and the biochemical results showed that the serum levels of aspartate aminotransferase (AST) and ß-hydroxybutyric acid (B-HB) in the PT group were significantly increased, while calcium (Ca) levels were significantly reduced. Sections showed extensive vacuoles in liver tissue sections. The microbiome analysis found that the richness and diversity of the PT microbiota were significantly reduced. Metabolomic analysis showed that 125 differential metabolites were screened in positive ion mode and enriched in 12 metabolic pathways. In negative ion mode, 100 differential metabolites were screened and enriched in 7 metabolic pathways. CONCLUSIONS: Evidence has shown that the occurrence of pregnancy toxemia is related to gut microbiota, and further studies are needed to investigate its pathogenesis and provide research basis for future preventive measures of this disease.

Diversity of Rickettsia species in collected ticks from Southeast Iran.

Rickettsia occurs worldwide and rickettsiosis is recognized as an emerging infection in several parts of the world. Ticks are reservoir hosts for pathogenic Rickettsia species in humans and domestic animals. Most pathogenic Rickettsia species belong to the spotted Fever Group (SFG). This study aimed to identify and diagnose tick fauna and investigate the prevalence of Rickettsia spp. in ticks collected from domestic animals and dogs in the rural regions of Kerman Province, Southeast Iran. In this study, tick species (fauna) were identified and 2100 ticks (350 pooled samples) from two genera and species including Rhipicephalus linnaei (1128) and Hyalomma deteritum (972) were tested to detect Rickettsia genus using Real-time PCR. The presence of the Rickettsia genus was observed in 24.9% (95%CI 20.28-29.52) of the pooled samples. Sequencing and phylogenetic analyses revealed the presence of Rickettsia aeschlimannii (48.98%), Rickettsia conorii israelensis (28.57%), Rickettsia sibirica (20.41%), and Rickettsia helvetica (2.04%) in the positive samples. The results showed a significant association between county variables and the following variables: tick spp. (p < 0.001), Rickettsia genus infection in ticks (p < 0.001) and Rickettsia spp. (p < 0.001). In addition, there was a significant association between tick species and host animals (dogs and domestic animals) (p < 0.001), Rickettsia spp infection in ticks (p < 0.001), and Rickettsia spp. (p < 0.001). This study indicates a high prevalence of Rickettsia spp. (SFG) in ticks of domestic animals and dogs in rural areas of Kerman Province. The health system should be informed of the possibility of rickettsiosis and the circulating species of Rickettsia in these areas.

Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Cysteine supplementation pre-freeze and post-thaw improves integrity and reduces oxidative stress in cryopreserved ram spermatozoa.

Cryopreserved ram sperm is highly sensitive to oxidative stress by reactive oxygen species (ROS) which impair sperm function and integrity. Antioxidants such as cysteine can mitigate the effect of ROS, although the optimal concentration or timing of supplementation is unknown. This study aimed to determine the effect of concentration and timing of cysteine supplementation on the integrity and function of cryopreserved ram spermatozoa. Nine ejaculates were collected from three Texel rams then cryopreserved and supplemented with cysteine (0, 0.5, or 1.0 mg/mL) added pre-freeze (PF), post-thaw (PT) or pre-freeze and post-thaw (PF + PT) generating seven treatments: 1) control 0 mg/mL, 2) PF 0.5 mg/mL, 3) PF 1 mg/mL, 4) PT 0.5 mg/mL, 5), PT 1.0 mg/mL, 6) PF + PT 0.5 mg/mL and 7) PF + PT 1.0 mg/mL. Sperm motility, viability, acrosome integrity, ROS production and penetrability through artificial cervical mucus were assessed post-thaw. Cysteine supplementation reduced ROS production which thereby improved spermatozoa motility, viability, acrosome integrity and penetrability (p < 0.001) Sperm integrity for all parameters was greatest in spermatozoa treated PF + PT with 1.0 mg/mL cysteine, although treatment pre-freeze or post-thaw also improved integrity beyond the control. This study has identified that 1.0 mg/mL cysteine is most beneficial and has highlighted the importance of preventing oxidative stress in spermatozoa post-thaw. These finding can help to mitigate the detrimental effect of cryopreservation on spermatozoa and aid the development of cryopreservation protocols in sheep.

Inflating Pressure and Not Expiratory Pressure Initiates Lung Injury at Birth in Preterm Lambs.

Rationale: Inflation is essential for aeration at birth, but current inflating pressure settings are without an evidence base. Objectives: To determine the role of inflating pressure (ΔP), and its relationship with positive end-expiratory pressure (PEEP), in initiating early lung injury pathways in the preterm lamb lung. Methods: Preterm (124 to 127 d) steroid-exposed lambs (n = 45) were randomly allocated (8-10 per group) to 15 minutes of respiratory support with placental circulation and 20 or 30 cm H2O ΔP, with an initial high PEEP (maximum, 20 cm H2O) recruitment maneuver known to facilitate aeration (dynamic PEEP), and compared with dynamic PEEP with no ΔP or 30 cm H2O ΔP and low (4 cm H2O) PEEP. Lung mechanics and aeration were measured throughout. After an additional 30 minutes of apneic placental support, lung tissue and bronchoalveolar fluid were analyzed for regional lung injury, including proteomics. Measurements and Main Results: The 30 cm H2O ΔP and dynamic PEEP strategies resulted in quicker aeration and better compliance but higher tidal volumes (often >8 ml/kg, all P < 0.0001; mixed effects) and injury. ΔP 20 cm H2O with dynamic PEEP resulted in the same lung mechanics and aeration, but less energy transmission (tidal mechanical power), as ΔP 30 cm H2O with low PEEP. Dynamic PEEP without any tidal inflations resulted in the least lung injury. Use of any tidal inflating pressures altered metabolic, coagulation and complement protein pathways within the lung. Conclusions: Inflating pressure is essential for the preterm lung at birth, but it is also the primary mediator of lung injury. Greater focus is needed on strategies that identify the safest application of pressure in the delivery room.