Quantitative proteomics reveals significant variation in host responses of cattle with differing buffalo fly susceptibility.

Background: Control of buffalo flies (Haematobia irritans exigua, BFs) relies mainly on chemical methods; however, resistance to insecticides is widespread in BF populations. Breeding for resistance to BFs represents a possible alternative, but direct phenotyping of animals is laborious and often inaccurate. The availability of reliable diagnostic biomarker(s) to identify low BF carrier cattle would facilitate rapid and accurate selection for genetic improvement. However, limited information is available regarding differences amongst cattle in host responses to BF infestation. Methods: This study investigated the variation in Brangus cattle serum proteomic profiles before (naïve) and after peak BF exposure, in low (LF) and high BF burden (HF) cattle. Cattle were phenotyped for susceptibility based on BF counts on multiple dates using visual and photographic techniques. The relative abundance of serum proteins in cattle before and after exposure to BFs was analysed using sequential window acquisition of all theoretical fragment ion mass spectrometry (SWATH-MS). Results: Exposure to BFs elicited similar responses in HF and LF cattle, with 79 and 70 proteins, respectively, showing significantly different abundances post exposure as compared to their relevant naïve groups. The comparison of serum samples from naïve HF and LF cattle identified 44 significantly differentially abundant (DA) proteins, while 37 significantly DA proteins were identified from the comparison between HF and LF cattle post-exposure to BFs. Proteins with higher abundance in naïve LF cattle were enriched in blood coagulation mechanisms that were sustained after exposure to BFs. Strong immune response mechanisms were also identified in naïve LF cattle, whereas these responses developed in HF cattle only after exposure to BF. High BF cattle also showed active anticoagulation mechanisms in response to BF exposure, including downregulation of coagulation factor IX and upregulation of antithrombin-III, which might facilitate BF feeding. Conclusion: Underlying differences in the abundance of proteins related to blood coagulation and immune response pathways could potentially provide indirect indicators of susceptibility to BF infestation and biomarkers for selecting more BF-resistant cattle.

Comparison of colostrum and milk extracellular vesicles small RNA cargo in water buffalo.

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to "cell differentiation" and "microvillus assembly", while for mEV "cardiac and blood vessel development" and "mitochondria" emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing.

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

Probiotic potential of autochthonous Lactobacillus species from buffalo calves in controlling multidrug resistant Escherichia coli.

The aim of this study was to investigate the probiotic potential of autochthonous Lactobacillus species isolated from buffalo calves against multidrug-resistant Escherichia coli. A total of 252 rectal swabs were collected from healthy neonatal buffalo calves under 30 days old from six districts of Andhra Pradesh, India in a completely randomized design from August 2019 to August 2021, of which 190 Lactobacillus strains were isolated based on cultural, morphological, biochemical and molecular tests. Out of 190 isolates, 57 showed high levels of auto-aggregation (> 80.00%) and hydrophobicity (> 60.00%) and 51 of the 57 isolates had a zone of inhibition greater than 15.00 mm in diameter against multidrug-resistant E. coli in an Agar well diffusion assay. Among the 51 isolates, 36 were found to be acid and bile tolerant and showed varying levels of sensitivity to antibiotics such as erythromycin, clindamycin, tetracycline, chloramphenicol, and ampicillin. Among the 36 isolates, Limosilactobacillus reuteri 178, L. reuteri 209, L. fermentum 182, L. fermentum 211, and Lactiplanti-bacillus plantarum 34 were non-hemolytic, and none of the isolates were able to hydrolyse gelatine. Therefore, these five autochthonous Lactobacillus species may be used in probiotic or synbiotic formulations against multidrug resistant E. coli in buffalo calves.

LPS-Induced Mitochondrial Dysfunction Reduces Oocyte Maturation and Developmental Competence of Buffalo Embryos via ROS Mediated TLR4 Signalling.

PROBLEM: Lipopolysaccharide (LPS) from gram-negative bacteria has reportedly been associated with infectious diseases like metritis, which has a substantial adverse effect on animal reproductive performance and causes serious financial losses for the dairy sector. The current work aimed to establish the impact of LPS on in vitro oocyte maturation and subsequent in vitro developmental competence of oocytes, as well as to investigate the explanatory molecular mechanism underlying this effect. METHOD OF STUDY: Buffalo cumulus-oocyte complexes (COCs) were challenged with 0, 5, 10 and 20 µg/mL LPS during IVM followed by IVF and IVC. Cytoplasmic and nuclear maturation, cleavage and blastocyst rate, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP, ΔΨm) and transcript abundance of genes related to inflammation, antioxidation and apoptosis were evaluated. RESULTS: The maturation and subsequent embryonic development competency were found to be significantly (p ≤ 0.05) reduced with the addition of 10 and 20 µg/mL LPS to IVM media. ROS production accompanied by a decreased ΔΨm was recorded in LPS-treated oocytes in comparison to the control group (p ≤ 0.05). Our results were further supported by the transcriptional expression of proinflammatory (TLR4, CD14 and RPS27A) and apoptotic gene (Caspase 3) which were found to be significantly increased while antioxidant genes (SOD2 and GPX1) were decreased significantly in matured oocytes and blastocyst after LPS exposure. CONCLUSIONS: The deleterious effects of LPS are mediated through ROS generation, which triggers inflammatory processes via the TLR4 pathway and impairs oocyte maturation and subsequent embryonic development.

The Physicochemical, Textural, Microbiological and Sensory Properties of Skimmed Buffalo Milk Yoghurt with Tragacanth Gum During Storage.

Research background: In the food industry, research interest in the functional effects of natural polysaccharides from plants has increased in recent years. Tragacanth gum is used in dairy products because of its stabilising, thickening, fat-replacing and prebiotic properties. However, skimmed milk is considered a significant commercial loss in the production of buffalo clotted cream. Therefore, the aim of the present study is to investigate the potential of tragacanth gum in the production of yoghurt from buffalo milk residues with different concentrations of tragacanth gum (0.5, 1 and 1.5 g/L). Experimental approach: Skimmed buffalo milk with different concentrations of tragacanth gum was pasteurised and, after cooling at 45 °C, a starter culture was added to each sample. All samples were fermented to a pH=4.80±0.2. The gross composition, acidity, water activity, water-holding capacity, whey separation, mass fractions of organic acids and volatile aroma compounds, counts of total aerobic mesophilic bacteria, yeasts and moulds, Lactococcus spp. and Lactobacillus spp. as well as sensory and textural properties were analysed during 15 days storage. Results and conclusions: The results showed that the use of tragacanth gum increased the dry matter mass fraction, water-holding capacity and mass fraction of proteins in the samples, while whey separation decreased as the concentration of gum increased. The addition of gum improved textural properties and hardness of the yoghurt. In terms of consistency, the sample with 1 g/L tragacanth gum was the most reliable. In the control group, the total aerobic mesophilic bacteria count was highest on the first and last day of storage. According to the results of the sensory evaluation, the sample with 0.5 g/L tragacanth gum was the most favourable. Novelty and scientific contribution: Research has shown that the use of stabilisers in varying ratios improves the quality of yoghurt made from fat-free buffalo milk, which is a by-product of industrial production. So instead of ending up as industrial waste, it is recycled and its value is increased.

The proteomic landscape of sperm surface deciphers its maturational and functional aspects in buffalo.

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

Within-Host Viral Growth and Immune Response Rates Predict Foot-and-Mouth Disease Virus Transmission Dynamics for African Buffalo.

AbstractInfectious disease dynamics operate across biological scales: pathogens replicate within hosts but transmit among populations. Functional changes in the pathogen-host interaction thus generate cascading effects across organizational scales. We investigated within-host dynamics and among-host transmission of three strains (SAT-1, -2, -3) of foot-and-mouth disease viruses (FMDVs) in their wildlife host, African buffalo. We combined data on viral dynamics and host immune responses with mathematical models to ask the following questions: How do viral and immune dynamics vary among strains? Which viral and immune parameters determine viral fitness within hosts? And how do within-host dynamics relate to virus transmission? Our data reveal contrasting within-host dynamics among viral strains, with SAT-2 eliciting more rapid and effective immune responses than SAT-1 and SAT-3. Within-host viral fitness was overwhelmingly determined by variation among hosts in immune response activation rates but not by variation among individual hosts in viral growth rate. Our analyses investigating across-scale linkages indicate that viral replication rate in the host correlates with transmission rates among buffalo and that adaptive immune activation rate determines the infectious period. These parameters define the virus's relative basic reproductive number (ℛ0), suggesting that viral invasion potential may be predictable from within-host dynamics.

p67 gene alleles sequence analysis reveals Theileria parva parasites associated with East Coast fever and Corridor disease in buffalo from Zambia.

Theileriosis caused by Theileria parva infections is responsible for high cattle mortalities in Zambia. Although infected buffalo are a risk to cattle, the characterization of T. parva parasites occurring in this host in Zambia has not been reported. Furthermore, considering the advances in the development of a p67 subunit vaccine, the knowledge of p67 genetic and antigenic diversity in both cattle and buffalo associated T. parva is crucial. Therefore, blood samples from buffalo (n=43) from Central, Eastern and Southern provinces, and cattle (n=834) from Central, Copperbelt, Eastern, Lusaka, and Southern provinces, were tested for T. parva infection and the parasites characterized by sequencing the gene encoding the p67 antigen. About 76.7 % of buffalo and 19.3 % of cattle samples were PCR positive for T. parva. Three of the four known p67 allele types (1, 2 and 3) were identified in parasites from buffalo, of which two (allele types 2 and 3) are associated with T. parva parasites responsible for Corridor disease. Only allele type 1, associated with East Coast fever, was identified from cattle samples, consistent with previous reports from Zambia. Phylogenetic analysis revealed segregation between allele type 1 sequences from cattle and buffalo samples as they grouped separately within the same sub-clade. The high occurrence of T. parva infection in buffalo samples investigated demonstrates the risk of Corridor disease infection, or even outbreaks, should naïve cattle co-graze with infected buffalo in the presence of the tick vector. In view of a subunit vaccine, the antigenic diversity in buffalo associated T. parva should be considered to ensure broad protection. The current disease control measures in Zambia may require re-evaluation to ensure that cattle are protected against buffalo-derived T. parva infections. Parasite stocks used in 'infection and treatment' immunization in Zambia, have not been evaluated for protection against buffalo-derived T. parva parasites currently circulating in the buffalo population.

The virome of bubaline (Bubalus bubalis) tonsils reveals an unreported bubaline polyomavirus.

Water buffalo (Bubalus bubalis) farming is increasing in many regions of the world due to the species' ability to thrive in environments where bovine cattle would struggle. Despite water buffaloes being known for their resistance to diseases, there is a lack of data about the diversity of the microbiome of the species. In this study, we examined the virome diversity in palatine tonsils collected from animals from the island of Marajó, northern Pará state, Brazil, which harbors the largest bubaline flock in the country. Tonsil fragments from 60 clinically healthy bubalines were randomly selected from a sample of 293 animals. The samples were purified, extracted, and randomly amplified with phi29 DNA polymerase. After amplification, the products were purified and sequenced. Circular DNA viruses were predominant in the tonsils' virome. Sequences of genome segments representative of members of the genera Alphapolyomavirus (including a previously unreported bubaline polyomavirus genome) and Gemycircularvirus were identified, along with other not yet classified circular virus genomes. As the animals were clinically healthy at the time of sampling, such viruses likely constitute part of the normal tonsillar virome of water buffaloes inhabiting the Ilha do Marajó biome.

Influence of additive and maternal effects on production and reproduction traits in Murrah buffaloes: Insights from Bayesian analysis.

The aim of this research was to assess genetic parameters for first lactation production and reproduction traits in Murrah buffaloes by employing additive and maternal effects. Data on pedigree and specific traits of 640 Murrah buffaloes were gathered from 1997 to 2020. These traits encompassed first lactation milk yield (FLMY), 305-day first lactation milk yield (305FLMY), first lactation length (FLL), first lactation peak yield (FPY), first service period (FSP), first calving interval (FCI) and first dry period (FDP). Genetic evaluations employed six univariate animal models, accounting for both direct and maternal effects, facilitated by THRGIBBS1F90 and POSTGIBBSF90 programs. Fixed factors included in the analysis were period of calving, season of calving and age at first calving. The Bayesian estimates for direct heritability, derived from the most suitable model, were as follows: FLMY: 0.28 ± 0.01, 305FLMY: 0.30 ± 0.01, FLL: 0.19 ± 0.01, FPY: 0.18 ± 0.01, FSP: 0.12 ± 0.01, FCI: 0.14 ± 0.01 and FDP: 0.12 ± 0.01. Maternal effects were found significant, ranging from 5% to 10%, in first lactation traits under Model 2 and Model 5. Additionally, positive and significant genetic and phenotypic correlations were observed among the studied traits. In conclusion, selection based on 305-day first lactation milk yield suggests potential for genetic enhancement in Murrah buffaloes, advocating its inclusion in breeding programmes to bolster early performance. Also, consideration of maternal influences is necessary for genetic progress of animals.

The Potential of Infrared Thermography for Early Pregnancy Diagnosis in Nili-Ravi Buffaloes.

This study was designed to explore the potential of infrared thermography (IRT) as an alternate approach for early pregnancy diagnosis in buffaloes. The surface temperature (ST) of different regions (eyes, muzzle, flanks, and vulva) was determined in 27 buffaloes using IRT from the day of artificial insemination (AI; Day 0), and measurement was repeated every fourth day until Day 24 post-AI. From all regions, the ST in each thermograph was recorded at three temperature values (maximum, average, minimum). Pregnancy status was confirmed through ultrasonography on Day 30, and animals were retrospectively grouped as pregnant or non-pregnant for analysis of thermographic data. In pregnant buffaloes, all three values of ST were significantly greater (p ≤ 0.05) for the left flank, while, in the left eye and vulva, only the maximum and average values were significantly greater. By contrast, the maximum ST of the muzzle was significantly lower (p ≤ 0.05) in pregnant buffaloes compared to non-pregnant buffaloes. However, the ST of the right eye and right flank did not show significant temperature variation at any value. These findings suggest that IRT has the potential to identify thermal changes associated with pregnancy in buffaloes at an early stage.

Low Effectiveness of Mid-Infrared Spectroscopy Prediction Models of Mediterranean Italian Buffalo Bulk Milk Coagulation Traits.

This study evaluated the potential use of mid-infrared spectroscopy to predict milk coagulation traits in bulk milk from Mediterranean Italian buffaloes. A total of 1736 bulk milk samples from 55 farms in central Italy were collected during the official milk quality testing system. The prediction models were developed based on modified partial least-squares regression with 75% of the samples and validated with the remaining samples. All bulk milk samples coagulated between 7.37 and 29.45 min. Average values for milk coagulation traits in the calibration set were 17.71 min, 3.29 min, and 38.83 mm for rennet coagulation time, curd firming time, and curd firmness, respectively. The validation set included samples with similar mean and standard deviation for each trait. The prediction models showed the greatest coefficient of determination of external validation (0.57) and the ratio of prediction to deviation (1.52) for curd firmness. Similar fitting statistics of the prediction models were obtained for rennet coagulation time and curd firming time. In conclusion, the prediction models for all three coagulation traits were below the threshold to consider the prediction models adequate even for rough screening of the samples.

Genome-wide comparative analyses highlights selection signatures underlying saline adaptation in Chilika buffalo.

Chilika, a native buffalo breed of the Eastern coast of India, is mainly distributed around the Chilika brackish water lake connected with the Bay of Bengal Sea. This breed possesses a unique ability to delve deep into the salty water of the lake and stay there to feed on local vegetation of saline nature. Adaptation to salinity is a genetic phenomenon, however, the genetic basis underlying the salinity tolerance is still limited in animals specifically in livestock. The present study explores the genetic evolution that unveils the Chilika buffalo's adaptation to the harsh saline habitat (water and food system). For this study, whole genome resequencing data on 18 Chilika buffalo and for comparison 10 Murrah buffalo of normal habitat were generated. For identification of selection sweeps, intrapopulation and interpopulation statistics were employed. A total of 709, 309, 468, and 354 genes were detected having selection sweeps in Chilika buffalo using the nucleotide diversity (θπ), Tajima's D, nucleotide diversity ratio (θπ-ratio), and FST methods, respectively. Further analysis revealed a total of 23 genes including EXOC6B, VPS8, LYPD1, VPS35, CAMKMT, NCKAP5, COMMD1, MYLK3, B3GNT2 were found to be common by all the methods. Furthermore, functional annotation study of identified genes provided pathways such as MAPK signaling, renin secretion, endocytosis, oxytocin signaling pathway, etc. Gene network analysis enlists hub genes, provide insights into their interactions with each other. In conclusion, this study has highlighted the genetic basis underlying the local adaptive function of Chilika buffalo under saline environment.

Thiols-rich peptide from water buffalo horn keratin alleviates oxidative stress and inflammation through co-regulating Nrf2/Hmox-1 and NF-κB signaling pathway.

Water buffalo horn (WBH), a traditional Chinese medicine, is known for its antipyretic, anti-inflammatory and antioxidant properties. This study aims to investigate the therapeutic potential of WBH keratin (WBHK) and its derived thiol-rich peptide fractions (SHPF) for oxidative stress and inflammation. WBHK and SHPF were prepared and tested using various models including LPS-induced fever in rabbits, H2O2-induced oxidative damage in bEnd.3 cells, TNF-α-induced inflammation in bEnd.3 cells and LPS-induced inflammation in RAW 264.7 cells. Expression of key markers, such as Nrf2, Hmox-1 and NF-κB, were analyzed using qRT-PCR, ELISA and Western blotting. Label-free quantitative proteomic analysis was used to identify key differential proteins associated with the efficacy of SHPF. Our results demonstrated that treatment with WBHK significantly reduced body temperature after 0.5 h of administration in the fever rabbit model. SHPF could alleviate cellular inflammatory injury and oxidative damage by activating the key transcription factor Nrf2 and increasing the expression level of Hmox-1. SHPF could inhibit the NF-κB pathway by reducing IκB phosphorylation. It was also found that SHPF could reduce pro-inflammatory cytokine (IL-6, COX-2 and PGE2) and inhibit the expression of VCAM-1, ICAM-1, IL-6 and MCP-1. Proteomics analysis showed that SHPF could inhibit HMGB1 expression and release. The results indicated that SHPF could significantly reduce inflammation and oxidative stress by regulating the Nrf2/Hmox-1 and NF-κB pathways. These findings suggest the potential therapeutic applications of WBH components in the treatment of oxidative stress and inflammation-related diseases.

Genomic analysis reveals the association of KIT and MITF variants with the white spotting in swamp buffaloes.

BACKGROUND: Swamp-type buffaloes with varying degrees of white spotting are found exclusively in Tana Toraja, South Sulawesi, Indonesia, where spotted buffalo bulls are highly valued in accordance with the Torajan customs. The white spotting depigmentation is caused by the absence of melanocytes. However, the genetic variants that cause this phenotype have not been fully characterized. The objective of this study was to identify the genomic regions and variants responsible for this unique coat-color pattern. RESULTS: Genome-wide association study (GWAS) and selection signature analysis identified MITF as a key gene based on the whole-genome sequencing data of 28 solid and 39 spotted buffaloes, while KIT was also found to be involved in the development of this phenotype by a candidate gene approach. Alternative candidate mutations included, in addition to the previously reported nonsense mutation c.649 C > T (p.Arg217*) and splice donor mutation c.1179 + 2T > A in MITF, a nonsense mutation c.2028T > A (p.Tyr676*) in KIT. All these three mutations were located in the genomic regions that were highly conserved exclusively in Indonesian swamp buffaloes and they accounted largely (95%) for the manifestation of white spotting. Last but not the least, ADAMTS20 and TWIST2 may also contribute to the diversification of this coat-color pattern. CONCLUSIONS: The alternative mutations identified in this study affect, at least partially and independently, the development of melanocytes. The presence and persistence of such mutations may be explained by significant financial and social value of spotted buffaloes used in historical Rambu Solo ceremony in Tana Toraja, Indonesia. Several de novo spontaneous mutations have therefore been favored by traditional breeding for the spotted buffaloes.

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications.

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10-9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10-9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10-9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10-9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines.

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.

Use of a commercial feed supplement based on diatom earth and yeast products on oxidative status and in vitro immune response in buffaloes during peripartum.

The transition period is a critical metabolic phase for dairy ruminants, especially those with high production levels. In spite of this, little is still known about dairy water buffalo. The aim of this study was to evaluate the effect of a commercial feed additive based on diatomaceous earth and hydrolyzed yeasts on health status, milk quality and immune response of buffalo cows during the transition period. Eighty healthy Water buffaloes (Bubalus bubalis) of Italian Mediterranean breed were included in the trial. They were subdivided in two groups: one group received the additive (n = 40) while the control group (n=40) received a placebo. The trial lasted 120 days, from 60 days before calving to 60 days in milk. Blood samples were collected from each buffalo at -60d (60 days from the expected calving), -30 d, 0 d (calving), +15 d, +30 d, and +60 d (respectively, i.e., 15, 30 and 60 days in milking). The biochemical as well as the oxidative profile, and the antioxidant power and enzymatic activity were evaluated in the samples obtained. Moreover, acute phase proteins, reactive proteins and Interleukine plasma levels were determined. Peripheral blood mononuclear cells (PBMC) and monocytes were isolated and viability, reactive oxygen species (ROS) and reactive nitrogen species (RNS) were measured on PMBC and monocytes. The introduction of additives enhanced the total antioxidant capacity and enzyme activity, while no differences were observed in oxidation products throughout the trial. Additionally, it significantly reduced the synthesis of ROS in polymorphonuclear cells, supporting a potential positive response in animals experiencing inflammation. The impact of oxidation on the products was not evident. Despite higher enzyme levels in plasma, this did not necessarily correspond to significantly increased enzymatic activity, but rather indicated a higher potential. From these results, it was evident that the transition period in buffaloes differs notably from what reported in literature for cows, probably due to the absence of common postpartum production diseases in dairy cows and lower metabolic challenges linked to lower milk production in buffaloes. Few parameters exhibited notable changes during the transition period in buffaloes, notably certain antioxidant enzymes, PBMC viability, PBMC ROS production, and Hp levels.

Effect of S. cerevisiae strain KA500 supplementation on feed performance, feed efficiency, and digestion ability in feedlot buffaloes.

Live yeasts have favorable characteristics for use in animal feed, and may become a beneficial tool to improve digestive efficiency in buffaloes (Bubalus bubalis). The productive performance, feed efficiency, and digestion ability of buffaloes fed diets supplemented with yeast (Saccharomyces cerevisiae strain KA500) were evaluated. Eighteen male Murrah buffaloes, with initial weight 250 ± 31 kg (mean ± standard deviation), and aged approximately 12 months, were randomly assigned to one of two treatments. The treatments included experimental feed containing 10 g of the live yeast capable of forming 2 × 1010 colony forming units (CFU) and control (feed with no added yeast). The daily weight gain tended to be lower (p = 0.07) in buffaloes supplemented with yeast. There was a reduction in daily dry matter intake (DMI) and in % yield of live weight in buffaloes supplemented with yeast. There was no effect of live yeast supplementation on weight gain/kg dry matter intake, height at withers or rump, body condition score, total weight gain, carcass yield, plasma urea nitrogen concentrations, purine derivatives, and plasma glucose concentrations. The digestibility of dry matter (DM) and organic matter (OM) were lower (p < 0.05) with the supplementation of live yeast, although live yeast supplementation did not affect the digestibility of neutral detergent fiber (NDF) and non-NDF OM. The strain and dosage of live yeast used did not have a positive effect on buffalo performance and digestibility of dietary nutrients.