Polymorphism detection and characterization of sperm cells chromatin remodeling associated genes in Murrah buffalo.

Seasonal variations significantly impact buffalo bull semen production and quality, particularly during the summer months. Understanding the genetic basis of these changes is important for managing bull fertility and improving sperm quality. The present study focused on characterizing and identifying polymorphisms in chromatin remodeling genes, protamines (PRMs) and Transition Nuclear Proteins (TNPs) in Murrah buffalo bulls with varying semen quality due to seasonal effects. Our findings revealed none of the coding region variation in PRM1, PRM2, TNP1, and TNP2, these genes are highly conserved in buffalo. Two intronic variants were identified, including G16C in PRM1 intron 1 and intronic SNP in PRM2 intron 1 (G96A). The complete CDS of consensus sequence of bubaline PRM1 was 86.3% identical and 94.1% similar to the bovine PRM1. Whereas the complete CDS of consensus sequence of bubaline TNP2 was 78.2% identical and 91.0% similar to bovine TNP2. Further, no statistically significant differences in the fold change of TNP1, TNP2, PRM1, and PRM2 levels between the hot summer SNA and SA groups and the winter SNA and SA groups This study represents the first comprehensive report on the characterization of bubaline PRM1 (complete CDS), PRM2 (partial CDS), TNP1 (partial CDS), and TNP2 (complete CDS) genes in buffalo sperm cells. Results of the study, clearly indicate that the genes associated with protamine (PRM1 and TNP2) are highly conserved in Bubalus bubalis. Understanding these genetic underpinnings can have implications for improving buffalo bull fertility and semen quality.

In Silico Analysis: Molecular Characterization and Evolutionary Study of CLCN Gene Family in Buffalo.

Chloride channels (ClCs) have received global interest due to their significant role in the regulation of ion homeostasis, fluid transport, and electrical excitability of tissues and organs in different mammals and contributing to various functions, such as neuronal signaling, muscle contraction, and regulating the electrolytes' balance in kidneys and other organs. In order to define the chloride voltage-gated channel (CLCN) gene family in buffalo, this study used in silico analyses to examine physicochemical properties, evolutionary patterns, and genome-wide identification. We identified eight CLCN genes in buffalo. The ProtParam tool analysis identified a number of important physicochemical properties of these proteins, including hydrophilicity, thermostability, in vitro instability, and basic nature. Based on their evolutionary relationships, a phylogenetic analysis divided the eight discovered genes into three subfamilies. Furthermore, a gene structure analysis, motif patterns, and conserved domains using TBtool demonstrated the significant conservation of this gene family among selected species over the course of evolution. A comparative amino acid analysis using ClustalW revealed similarities and differences between buffalo and cattle CLCN proteins. Three duplicated gene pairs were identified, all of which were segmental duplications except for CLCN4-CLCN5, which was a tandem duplication in buffalo. For each gene pair, the Ka/Ks test ratio findings showed that none of the ratios was more than one, indicating that these proteins were likely subject to positive selection. A synteny analysis confirmed a conserved pattern of genomic blocks between buffalo and cattle. Transcriptional control in cells relies on the binding of transcription factors to specific sites in the genome. The number of transcription factor binding sites (TFBSs) was higher in cattle compared to buffalo. Five main recombination breakpoints were identified at various places in the recombination analysis. The outcomes of our study provide new knowledge about the CLCN gene family in buffalo and open the door for further research on candidate genes in vertebrates through genome-wide studies.

Disentangling river and swamp buffalo genetic diversity: initial insights from the 1000 Buffalo Genomes Project.

More people in the world depend on water buffalo for their livelihoods than on any other domesticated animals, but its genetics is still not extensively explored. The 1000 Buffalo Genomes Project (1000BGP) provides genetic resources for global buffalo population study and tools to breed more sustainable and productive buffaloes. Here we report the most contiguous swamp buffalo genome assembly (PCC_UOA_SB_1v2) with substantial resolution of telomeric and centromeric repeats, ∼4-fold more contiguous than the existing reference river buffalo assembly and exceeding a recently published male swamp buffalo genome. This assembly was used along with the current reference to align 140 water buffalo short-read sequences and produce a public genetic resource with an average of ∼41 million single nucleotide polymorphisms per swamp and river buffalo genome. Comparison of the swamp and river buffalo sequences showed ∼1.5% genetic differences, and estimated divergence time occurred 3.1 million years ago (95% CI, 2.6-4.9). The open science model employed in the 1000BGP provides a key genomic resource and tools for a species with global economic relevance.

Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Genetic and Phenotypic Trends for Production and Reproduction Traits in Murrah Buffaloes.

The objective of the study was to estimate genetic and phenotypic trends for first lactation production and reproduction traits in Murrah buffaloes. The information of pedigree and targeted traits of 640 Murrah buffaloes was collected for the period from 1997 to 2020. The first lactation production traits included first lactation milk yield (FLMY), 305 days first lactation milk yield (305FLMY), first lactation length (FLL), first lactation peak yield (FPY) whereas reproduction traits included first service period (FSP), first calving interval (FCI) and first dry period (FDP). Genetic and phenotypic trends were estimated. Phenotypic trends for FLMY, 305FLMY, FLL and FPY exhibited as 36.96 ± 8.58 kg/year, 31.93 ± 8.34 kg/year, 1.47 ± 0.55 days/year and 0.12 ± 0.02 kg/year, respectively and respective genetic trends exhibited as 3.73 ± 1.67 kg/year, 1.94 ± 0.76 kg/year, -0.15 ± 0.07 days/year and 0.01 ± 0.01 kg/year, respectively. It was revealed that there were significant (p < 0.05) and positive phenotypic trends for all production traits while genetic trends were significant (p < 0.05) for FLMY and 305FLMY traits. The phenotypic trends of studied reproductive traits indicated that only FDP trait significantly (p < 0.01) decreased (1.87 days/year) over time. For FSP and FCI traits, nonsignificant (p > 0.05) genetic and phenotypic trends indicated no change over time. This study highlighted potential challenges in maintaining reproductive efficiency alongside productivity improvements in Murrah buffaloes.

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.

Mitochondrial DNA D-loop SNPs unveil molecular signatures of milk production variation in Murrah buffalo.

BACKGROUND: The Murrah buffalo, pivotal in Asian agriculture, faces challenges in maximizing milk production despite significant breeding efforts. Recognizing its economic importance, this study investigates mtDNA D-loop variations in Murrah buffalo as potential indicators of milk production variability, addressing challenges in maximizing yield despite significant breeding efforts. METHODS AND RESULTS: Analyzing mtDNA D-loop sequences from 50 buffaloes, we categorized them into Low (Group 1), Medium (Group 2), and High ECM (Group 3) groups based on milk yields, fat and protein percentage of a 30-day period data. Somatic cell mtDNA D-loop analysis revealed distinct genetic variations, with significant differences among ECM groups. Group 2 showed higher SNP prevalence, group 3 had more insertions/deletions, and Group 1 exhibited the highest transition frequency. Notably, a consistent "C" deletion at the 714th position occurred in Groups 1 and 3, prevalent in 68% of Group 2. A G-A variation at the 93rd position was specific to the medium ECM group. Negative Tajima D values indicated unique variations in each group, with Group 1 having the highest number, and a specific SNP linked to Group 2 was identified. These SNPs in the D-loop region could impact mtDNA replication, influencing mitochondrial content among animals. Our results provide valuable insights into the role of mtDNA D-loop polymorphisms in milk production traits in Murrah buffalo. CONCLUSIONS: Our research highlights the potential for valuable markers of cellular energy efficiency in Murrah buffalo. Exploring diverse cytoplasmic backgrounds opens avenues for mtDNA-based selection strategies, enhancing milk production and optimizing genetic traits for the dairy industry.

Molecular identification and genetic variability of equine and bovine ocular setariasis in india: molecular profiling by mitochondrial genes.

BACKGROUND: Ocular setariasis is an ectopic infection caused by a parasite under the genus Setaria. Adult worms belong to the Setariidae family and typically reside in the peritoneal cavity of ungulates. However, immature forms of these species may aberrantly migrate to the eyes of cattle, buffalo, goats, horses and several other hosts, leading to corneal opacity and blindness. Here, we have distinguished the Setaria digitata collected from both equine and buffalo hosts based on the morphology, molecular profiling of mitochondrial cytochrome c oxidase subunit 1 (Cox1), cytochrome c oxidase subunit 3 (Cox3) and, Nicotinamide Adenine Dinucleotide dehydrogenase subunit 1 (NAD1) genes. METHODS AND RESULTS: A single filarial worm was collected from the eye of one equine and one bovine host. These worms were then processed for morphological examination and DNA isolation. Cox1, Cox3 and NAD1 genes were amplified using specific primers and subjected to custom sequencing. The sequences were then used for multiple sequence alignment, assessment of entropy, similarity and haplotype diversity analysis. Key morphological features confirmed the worms collected were male and female Setaria digitata from equine and buffalo hosts, respectively. Cox1, Cox3 and NAD1 gene sequence analysis showed a close association of S.digitata Indian isolates with its counterparts from Sri Lanka and China isolates. CONCLUSION: The phylogram of bovine S. digitata sequences shows a close relationship to other equine S. digitata sequences, indicating the need for further in-depth studies on the prevalence of infection across various host species and intermediate hosts. Although the sequence results suggest that S. digitata is likely the causative agent of ocular setariasis in India, additional samples are needed to confirm this conclusion. Comprehensive analysis of the transcriptome and proteome of S. digitata from both bovine and equine hosts is necessary to explore variations in host-parasite interactions. These findings will aid in future parasite identification, investigations into vector prevalence in India, and the development of control measures against ocular setariasis.

Estimation of the threshold for heat stress and genetic features for milk yield in Mehsana buffaloes in India.

Heat stress is one of the primary environmental factors that harm both the productivity and health of buffaloes. The current study was conducted to estimate the threshold of temperature humidity index (THI)1 and genetic features for milk yield of first-lactation Mehsana buffaloes using an univariate repeatability test-day model. The data included 130,475 first lactation test-day milk yield (FLTDMY) records of 13,887 Mehsana buffaloes and the daily temperature and humidity. The statistical model included herd test day as fixed effects, days-in-milk (DIM) classes, age of the animal, as well as random factors such as the additive genetic effect (AGE) of animal in general conditions (intercept), AGE of the buffaloes subjected to heat stress (slope), permanent environmental effect of animal in general conditions (intercept), permanent environmental effect of animal under heat stress conditions (slope) and random residual effect. It was expected that the general effects and the heat-tolerance effects would be correlated, represented by the present investigation's repeatability models. The variance components of FLTDMY in the present study were computed using the REML method. The threshold for THI was 78. At the THI below the threshold, the heritability estimated for the FLTDMY trait was 0.29, and the additive genetic variance (AGV) for heat stress conditions was 0. At THI of 83, AGV for heat stress conditions was highest for FLTDMY. The genetic correlation of general AGE to heat-tolerant AGE was -0.40. The results indicated that a consistent selection for milk production, avoiding the thermal tolerance, may diminish the thermal tolerance capacity of Mehsana buffaloes.

Evolutionary insights of interferon lambda genes in tetrapods.

Type III interferon (IFN), also known as IFN-λ, is an innate antiviral protein. We retrieved the sequences of IFN-λ and their receptors from 42 tetrapod species and conducted a computational evolutionary analysis to understand the diversity of these genes. The copy number variation (CNV) of IFN-λ was determined through qPCR in Indian cattle and buffalo. The tetrapod species feature intron-containing type III IFN genes. Some reptiles and placental mammals have 2 IFN-λ loci, while marsupials, monotremes, and birds have a single IFN-λ locus. Some placental mammals and amphibians exhibit multiple IFN-λ genes, including both intron-less and intron-containing forms. Placental mammals typically possess 3-4 functional IFN-λ genes, some of them lack signal peptides. IFN-λ of these tetrapod species formed 3 major clades. Mammalian IFN-λ4 appears as an ancestral form, with syntenic conservation in most mammalian species. The intron-less IFN-λ1 and both type III IFN receptors have conserved synteny in tetrapod. Purifying selection was noted in their evolutionary analysis that plays a crucial role in minimizing genetic diversity and maintaining the integrity of biological function. This indicates that these proteins have successfully retained their biological function and indispensability, even in the presence of the type I IFNs. The expansion of IFN-λ genes in amphibians and camels have led to the evolution of multiple IFN-λ. The CNV can arise from gene duplication and conversion events. The qPCR-based absolute quantification revealed that IFN-λ3 and IFN-λ4 have more than 1 copy in buffalo (Murrah) and 6 cattle breeds (Sahiwal, Tharparkar, Kankrej, Red Sindhi, Jersey, and Holstein Friesian). Overall, these findings highlight the evolutionary diversity and functional significance of IFN-λ in tetrapod species.

Genome-Wide Identification and Evolutionary and Mutational Analysis of the Bos taurus Pax Gene Family.

Bos taurus is known for its tolerance of coarse grains, adaptability, high temperature, humidity, and disease resistance. Primarily, cattle are raised for their meat and milk, and pinpointing genes associated with traits relevant to meat production can enhance their overall productivity. The aim of this study was to identify the genome, analyze the evolution, and explore the function of the Pax gene family in B. taurus to provide a new molecular target for breeding in meat-quality-trait cattle. In this study, 44 Pax genes were identified from the genome database of five species using bioinformatics technology, indicating that the genetic relationships of bovids were similar. The Pax3 and Pax7 protein sequences of the five animals were highly consistent. In general, the Pax gene of the buffalo corresponds to the domestic cattle. In summary, there are differences in affinity between the Pax family genes of buffalo and domestic cattle in the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies. We believe that Pax1/9 has an effect on the growth traits of buffalo and domestic cattle. The Pax3/7 gene is conserved in the evolution of buffalo and domestic animals and may be a key gene regulating the growth of B. taurus. The Pax2/5/8 subfamily affects coat color, reproductive performance, and milk production performance in cattle. The Pax4/6 subfamily had an effect on the milk fat percentage of B. taurus. The results provide a theoretical basis for understanding the evolutionary, structural, and functional characteristics of the Pax family members of B. taurus and for molecular genetics and the breeding of meat-production B. taurus species.

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.

Genome-wide comparative analyses highlight 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 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, including both water and food systems. 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 used. A total of 709, 309, 468, and 354 genes were detected to possess 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, myosin light chain kinase 3 (MYLK3), and 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 that 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.NEW & NOTEWORTHY Indian Chilika buffaloes are being maintained on extensive grazing system and have a unique ability to convert local salty vegetation into valuable human food. However, adaptability to saline habitat of Chilika buffalo has not been explored to date. Here, we identified genes and biological pathways involved, such as MAPK signaling, renin secretion, endocytosis, and oxytocin signaling pathway, underlying adaptability of Chilika buffalo to saline environment. This investigation shed light on the mechanisms underlying the buffalo's resilience in its native surroundings.

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.

Deciphering tissue-specific expression profiles of mitochondrial genome-encoded tRNAs and rRNAs through transcriptomic profiling in buffalo.

BACKGROUND: Mitochondria, essential for cellular energy production through oxidative phosphorylation (OXPHOS), integrate mt-DNA and nuclear-encoded genes. This cooperation extends to the mitochondrial translation machinery, involving crucial mtDNA-encoded RNAs: 22 tRNAs (mt-tRNAs) as adapters and two rRNAs (mt-rRNAs) for ribosomal assembly, enabling mitochondrial-encoded mRNA translation. Disruptions in mitochondrial gene expression can strongly impact energy generation and overall animal health. Our study investigates the tissue-specific expression patterns of mt-tRNAs and mt-rRNAs in buffalo. MATERIAL AND METHODS: To investigate the expression patterns of mt-tRNAs and mt-rRNAs in different tissues and gain a better understanding of tissue-specific variations, RNA-seq was performed on various tissues, such as the kidney, heart, brain, and ovary, from post-pubertal female buffaloes. Subsequently, we identified transcripts that were differentially expressed in various tissue comparisons. RESULTS: The findings reveal distinct expression patterns among specific mt-tRNA and mt-rRNA genes across various tissues, with some exhibiting significant upregulation and others demonstrating marked downregulation in specific tissue contexts. These identified variations reflect tissue-specific physiological roles, underscoring their significance in meeting the unique energy demands of each tissue. Notably, the brain exhibits the highest mtDNA copy numbers and an abundance of mitochondrial mRNAs of our earlier findings, potentially linked to the significant upregulation of mt-tRNAs in brain. This suggests a plausible association between mtDNA replication and the regulation of mtDNA gene expression. CONCLUSION: Overall, our study unveils the tissue-specific expression of mitochondrial-encoded non-coding RNAs in buffalo. As we proceed, our further investigations into tissue-specific mitochondrial proteomics and microRNA studies aim to elucidate the intricate mechanisms within mitochondria, contributing to tissue-specific mitochondrial attributes. This research holds promise to elucidate the critical role of mitochondria in animal health and disease.

Continent-wide genomic analysis of the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management.

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.

Unknown parent groups and truncated pedigree in single-step genomic evaluations of Murrah buffaloes.

Missing pedigrees may produce bias in genomic evaluations. Thus, strategies to deal with this problem have been proposed as using unknown parent groups (UPG) or truncated pedigrees. The aim of this study was to investigate the impact of modeling missing pedigrees under single-step genomic best linear unbiased prediction (ssGBLUP) evaluations for productive and reproductive traits in dairy buffalo using different approaches: (1) traditional BLUP without UPG (BLUP), (2) traditional BLUP including UPG (BLUP/UPG), (3) ssGBLUP without UPG (ssGBLUP), (4) ssGBLUP including UPG in the A and A22 matrices (ssGBLUP/A_UPG), (5) ssGBLUP including UPG in all elements of the H matrix (ssGBLUP/H_UPG), (6) BLUP with pedigree truncation for the last 3 generations (BLUP/truncated), and (7) ssGBLUP with pedigree truncation for the last 3 generations (ssGBLUP/truncated). Unknown parent groups were not used in the scenarios with truncated pedigree. A total of 3,717, 4,126, and 3,823 records of the first lactation for accumulated 305-d milk yield (MY), age at first calving (AFC), and lactation length (LL), respectively, were used. Accuracies ranged from 0.27 for LL (BLUP) to 0.46 for MY (BLUP), bias ranged from -0.62 for MY (ssGBLUP) to 0.0002 for AFC (BLUP/truncated), and dispersion ranged from 0.88 for MY (BLUP/A_UPG) to 1.13 for LL (BLUP). Genetic trend showed genetic gains for all traits across 20 years of selection, and the impact of including genomic information, UPG, or pedigree truncation under GEBV accuracies ranged among the evaluated traits. Overall, methods using UPG, truncation pedigree, and genomic information exhibited potential to improve GEBV accuracies, bias, and dispersion for all traits compared with other methods. Truncated scenarios promoted high genetic gains. In small populations with few genotyped animals, combining truncated pedigree or UPG with genomic information is a feasible approach to deal with missing pedigrees.

CRISPR-mediated editing of ß-lactoglobulin (BLG) gene in buffalo.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, ß-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (-/-) heterozygous, bi-allelic (-/-) homozygous, and mono-allelic (-/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (-/-) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

The evolution of contemporary livestock species: Insights from mitochondrial genome.

The domestication of animals marks a pivotal moment in human history, profoundly influencing our demographic and cultural progress. This process has led to significant genetic, behavioral, and physical changes in livestock species compared to their wild ancestors. Understanding the evolutionary history and genetic diversity of livestock species is crucial, and mitochondrial DNA (mtDNA) has emerged as a robust marker for investigating molecular diversity in animals. Its highly conserved gene content across animal species, minimal duplications, absence of introns, and short intergenic regions make mtDNA analysis ideal for such studies. Mitochondrial DNA analysis has uncovered distinct cattle domestication events dating back to 8000 years BC in Southwestern Asia. The sequencing of water buffalo mtDNA in 2004 provided important insights into their domestication history. Caprine mtDNA analysis identified three haplogroups, indicating varied maternal origins. Sheep, domesticated 12,000 years ago, exhibit diverse mtDNA lineages, suggesting multiple domestication events. Ovine mtDNA studies revealed clades A, B, C, and a fourth lineage, group D. The origins of domestic pigs were traced to separate European and Asian events followed by interbreeding. In camels, mtDNA elucidated the phylogeographic structure and genetic differentiation between wild and domesticated species. Horses, domesticated around 3500 BC, show significant mtDNA variability, highlighting their diverse origins. Yaks exhibit unique adaptations for high-altitude environments, with mtDNA analysis providing insights into their adaptation. Chicken mtDNA studies supported a monophyletic origin from Southeast Asia's red jungle fowl, with evidence of multiple origins. This review explores livestock evolution and diversity through mtDNA studies, focusing on cattle, water buffalo, goat, sheep, pig, camel, horse, yak and chicken. It highlights mtDNA's significance in unraveling maternal lineages, genetic diversity, and domestication histories, concluding with insights into its potential application in improving livestock production and reproduction dynamics.