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.

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.

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.

Complete genome sequence of Indonesian probiotic strain Lactiplantibacillus plantarum subsp. plantarum Dad-13.

Previous studies have investigated the probiotic properties of L. plantarum subsp. plantarum Dad-13. Nevertheless, genomic sequence data from previous studies were not yet available to support each probiotic characteristic. This study focused on the complete genome sequence of the strain to validate its role in specific probiotic properties.

Investigating the infection dynamics and molecular detection of Cryptosporidium in Buffaloes in Sylhet, Bangladesh.

Cryptosporidiosis, a zoonotic infection impacting both livestock and humans, is inadequately understood in terms of its prevalence and transmission dynamics involving buffaloes in Bangladesh. This research, conducted in the Sylhet division, aimed to explore the prevalence and potential risk factors influencing Cryptosporidium spp. in the faecal samples of 392 buffaloes. Detection of the parasite utilized modified Ziehl-Neelsen staining, with molecular identification achieved through nested PCR (nPCR). The comprehensive analysis revealed 9.18% (36/392) prevalence at the individual animal level and 40.48% (17/42) at the herd level. Age-based analysis revealed fluctuating infection rates of Cryptosporidium spp. in buffaloes across distinct age brackets, with rates of 22.61% in those aged 0-6 months, 5.00% in those aged 6-12 months, and 1.03% in those aged 12-18 months. Diarrheic buffaloes showed a significantly (p < 0.001) higher infection rate (26.67%; 28/105) compared to non-diarrheic buffaloes (2.79%; 8/287). In risk factor analysis, binary logistic regression revealed that buffaloes aged 0-6 months were experiencing a likelihood that is 14.84 times higher to be affected by Cryptosporidium in contrast to their older counterparts (OR = 14.85; p = 0.02). Additionally, diarrhoeic buffaloes were found to be more susceptible to Cryptosporidium compared to healthy buffaloes (OR = 17.50; p < 0.001). A higher stocking density was associated with an increased likelihood of infection in buffaloes (OR = 11.20; p = 0.01). The results of this study emphasize the necessity for targeted interventions, considering factors like diarrheic condition and stocking density, to effectively manage and control cryptosporidiosis in Bangladesh.

Revealing microbial diversity in buffalo milk with high somatic cell counts: implications for mastitis diagnosis and treatment.

Mastitis is one of the most serious diseases that threatens the health of dairy animals. The somatic cell count (SCC) in milk is widely used to monitor mastitis. This study aimed to reveal the diversity of microorganisms in buffalo milk with high somatic cell count (SCC ≥ 3 × 105 cells/mL, n = 30) and low somatic cell count (SCC ≤ 5 × 104 cells/mL, n = 10), and identify the dominant bacteria that cause mastitis in a local buffalo farm. We also investigated the potential method to treat bacterial mastitis. The V3-V4 region of 16 S rDNA was sequenced. Results showed that, compared to the milk with low SCC, the high SCC samples showed lower microbial diversity, but a high abundance of bacteria and operational taxonomic units (OTUs). By in vitro isolation and culture, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were found to be the leading pathogens, which is consistent with the 16 S rDNA sequencing data. We further isolated 3 of the main pathogens and established a pathogen detection method based on ELISA. In addition, the antibacterial effects of 10 antimicrobials and 15 Chinese herbal extracts were also investigated. Results showed that the microbial has developed tolerance to several of the antimicrobials. While the water extracts of Chinese herbal medicine such as Galla Chinensis, Coptis chinensis Franch, Terminalia chebula Retz, and Sanguisorba officinalis L can effectively inhibit the growth of main pathogens. This study provides novel insight into the microbial diversity in buffalo milk and a reference for the prevention, diagnosis, and treatment of mastitis.

Nuclear Genome-Encoded Mitochondrial OXPHOS Complex I Genes in Female Buffalo Show Tissue-Specific Differences.

Buffalo physiology intricately balances energy, profoundly influencing health, productivity, and reproduction. This study explores nuclear-mitochondrial crosstalk, revealing OXPHOS Complex I gene expression variations in buffalo tissues through high-throughput RNA sequencing. Unveiling tissue-specific disparities, the research elucidates the genomic landscape of crucial energy production genes, with broader implications for veterinary and agricultural progress. Post-slaughter, tissues from post-pubertal female buffaloes underwent meticulous processing and RNA extraction using the TRIzol method. RNA-Seq library preparation and IlluminaHiSeq 2500 sequencing were performed on QC-passed samples. Data underwent stringent filtration, mapping to the Bubalus bubalis genome using HISAT2. DESeq2 facilitated differential expression gene (DEG) analysis focusing on 57 Mitocarta 3-derived genes associated with OXPHOS complex I. Nuclear-encoded mitochondrial protein transcripts of OXPHOS complex 1 exhibited tissue-specific variations, with 51 genes expressing significantly across tissues. DEG analysis emphasized tissue-specific expression patterns, highlighting a balanced OXPHOS complex I subunit expression in the kidney vs. brain. Gene Ontology (GO) enrichment showcased mitochondria-centric terms, revealing distinct proton motive force-driven mitochondrial ATP synthesis regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses emphasized Thermogenesis and OXPHOS pathways, enriching our understanding of tissue-specific energy metabolism. Noteworthy up-regulation of NDUFB10 in the heart and kidney aligned with heightened metabolic activity. Brain-specific up-regulation of NDUFAF6 indicated a focus on mitochondrial function, while variations in NDUFA11 and ACAD9 underscored pivotal roles in the heart and kidney. GO and KEGG analyses highlighted tissue-specific mitochondrial ATP synthesis and NADH dehydrogenase processes, providing molecular insights into organ-specific metabolic demands and regulatory mechanisms. Our study unveils conserved and tissue-specific nuances in nuclear-encoded mitochondrial OXPHOS complex I genes, laying a foundation for understanding diverse energy demands and potential health implications.

Biomolecule-Producing Probiotic Bacterium Lactococcus lactis in Free or Nanoencapsulated Form for Endometritis Treatment and Fertility Improvement in Buffaloes.

A Lactococcus (L.) lactis strain producing antimicrobial and anti-inflammatory biomolecules (mainly 1,4-Diaza-2,5-dioxobicyclo[4.3.0]nonanes and pyrazine-derivatives) was tested for its capacity to cure clinical endometritis in buffaloes compared to conventional antibiotic-based treatment. Clinical endometritis-diagnosed buffaloes (n = 16/group) were infused intrauterine with four doses of 109 CFU-free (FLC group) or nanoencapsulated L. lactis (NLC group) and compared to those that received three doses of saline + a single dose of 500 mg cephapirin benzathin (AB group) or four doses of saline (control, C group) every other day. Endometrium samples were analyzed for cytological (polymorphonuclear cells, PMN), bacteriological, and proinflammatory mRNA expression. Uterine wash and blood samples were collected to determine proinflammatory cytokine concentrations and metabolites in the blood samples. The reproductive performance of buffaloes was assessed. Compared to the C group, the AB and NLC groups had the lowest percentage of PMN, followed by those in the FLC group (p < 0.05). All treated buffaloes had significantly lower numbers of pathogens than the control buffaloes. Compared to control, all treatments significantly down-regulated endometrial proinflammatory encoding mRNA expression. The concentrations of IL1B, TNFAIP7, and leukocyte esterase activity in the uterine washings were significantly decreased in the AB and NLC groups compared to the C and FLC groups. All treatments significantly decreased concentrations of serum proinflammatory cytokines compared to control. Both the AB and NLC groups had significantly lower concentrations of serum NEFA than the C and FLC groups. The percentage of control buffaloes having an echogenic uterus and PVD score > 2 was significantly higher than those in the treated buffaloes with higher numbers of corpora lutea, higher conception rates, and shorter days open than control buffaloes (p < 0.05). In conclusion, L. lactis-producing antimicrobial and anti-inflammatory metabolites reduce uterine inflammatory responses and improve fertility in buffaloes.

A Characterization and Functional Analysis of Peroxisome Proliferator-Activated Receptor Gamma Splicing Variants in the Buffalo Mammary Gland.

Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21-encoded protein had PPAR ligand-binding domains (NR_LBD_PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes (ACACA, CD36, ACSL1, GPAT, AGPAT6, DGAT1) was significantly modified (p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21. All kinds of FAs detected in this study were significantly decreased (p < 0.05) after RNAi of PPARG-X17 or PPARG-X21. Overexpression of PPARG-X17 or PPARG-X21 significantly decreased (p < 0.05) the SFA content, while significantly increased (p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.

Maternal genetic diversity and phylogenetic analysis of Indian riverine and swamp buffaloes: insights from complete mitochondrial genomes.

This study explored the genetic diversity and evolutionary history of riverine and swamp buffaloes in India, utilizing complete mitochondrial genome sequences. Through comprehensive sampling across varied agro-climatic zones, including 91 riverine buffaloes from 12 breeds and 6 non-descript populations, along with 16 swamp buffaloes of the Luit breed, this study employed next-generation sequencing techniques to map the mitogenomic landscape of these subspecies. Sequence alignments were performed with the buffalo mitochondrial reference genome to identify mitochondrial DNA (mtDNA) variations and distinct maternal haplogroups among Indian buffaloes. The results uncovered the existence of 212 variable sites in riverine buffaloes, yielding 67 haplotypes with high haplotype diversity (0.991), and in swamp buffaloes, 194 variable sites resulting in 12 haplotypes, displaying haplotype diversity of 0.950. Phylogenetic analyses elucidated the genetic relationships between Indian buffaloes and the recognized global haplogroups, categorizing Indian swamp buffaloes predominantly into the SA haplogroup. Intriguingly, the haplogroup SB2b was observed for the first time in swamp buffaloes. Conversely, riverine buffaloes conformed to established sub-haplogroups RB1, RB2, and RB3, underscoring the notion of Northwestern India as a pivotal domestication site for riverine buffaloes. The study supports the hypothesis of independent domestication events for riverine and swamp buffaloes, highlighting the critical role of genetic analysis in unraveling the complex evolutionary pathways of domestic animals. This investigation contributes to the global understanding of buffalo mitogenome diversity, offering insights into this important livestock species' domestication and dispersal patterns.

Age-related compositional and functional changes in the adult and breastfed buffalo rumen microbiome.

Introduction: The buffalo is an important domestic animal globally, providing milk, meat, and labor to more than 2 billion people in 67 countries. The rumen microorganisms of buffaloes play an indispensable role in enabling the healthy functionality and digestive function of buffalo organisms. Currently, there is a lack of clarity regarding the differences in the composition and function of rumen microorganisms among buffaloes at different growth stages. Methods: In this study, metagenomics sequencing technology was applied to examine the compositional and functional differences of rumen microorganisms in adult and breastfed buffaloes. Results: The results revealed that the rumen of adult buffaloes had significantly higher levels of the following dominant genera: Prevotella, UBA1711, RF16, Saccharofermentans, F23-D06, UBA1777, RUG472, and Methanobrevibacter_A. Interestingly, the dominant genera specific to the rumen of adult buffaloes showed a significant positive correlation (correlation>0.5, p-value<0.05) with both lignocellulose degradation-related carbohydrate-active enzymes (CAZymes) and immune signaling pathways activated by antigenic stimulation. The rumen of breastfed buffaloes had significantly higher levels of the following dominant genera: UBA629, CAG- 791, Selenomonas_C, Treponema_D, Succinivibrio, and RC9. Simultaneously, the rumen-dominant genera specific to breastfed buffaloes were significantly positively correlated (correlation>0.5, p-value<0.05) with CAZymes associated with lactose degradation, amino acid synthesis pathways, and antibiotic-producing pathways. Discussion: This indicates that rumen microorganisms in adult buffaloes are more engaged in lignocellulose degradation, whereas rumen microorganisms in breastfed buffaloes are more involved in lactose and amino acid degradation, as well as antibiotic production. In conclusion, these findings suggest a close relationship between differences in rumen microbes and the survival needs of buffaloes at different growth stages.

Acceptance of Muffins (Sweet and Savory) with the Addition of T. molitor, A. diaperinus, A. domesticus, R. differens, Considering Psychological Factors (Food Neophobia Scale, Consumer Attitude).

The aim of the study was to analyze the acceptance of muffins containing a 15% addition of powder from four edible insect species (Alphitobius diaperinus, Tenebrio molitor, Acheta domesticus, Ruspolia differens) in both savory and sweet versions, focusing on the psychological factors influencing their consumption. The study involved 106 adult consumers. Initially, the level of food neophobia (FNS) among participants was determined. Over 80% displayed low to medium levels of neophobia. Similar results were obtained when assessing attitudes towards insects, with most participants showing positive and ambivalent attitudes. Based on these findings, the acceptance of insect-based muffins was evaluated. The level of acceptance of insects varied and depended mainly on taste, smell, and texture. Participants with lower levels of neophobia and positive attitudes towards consuming insects generally rated the insect muffins higher compared to those with higher levels of neophobia and negative attitudes. The sweet versions of insect powder muffins were rated higher, which also indicates preferences and dietary habits. Products with grasshopper powder (GS, GCL) were rated the lowest for both taste versions. Conversely, products based on buffalo worms (BS, BCL) were seen as having the greatest potential for acceptance. Understanding consumer attitudes, neophobia, and levels of acceptance provides valuable insights for designing new insect-based foods.

Interferon-Tau in Maternal Peripheral Blood and Its Relationship with Progesterone and Pregnancy-Associated Glycoproteins in the Early Phases of Gestation in Water Buffalo.

The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal-placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis.

Multi-omics analysis of five species of milk and specific composition links within each species.

The gold standard of milk is human milk, not cow milk. The present study expects to explored the comprehensive nutritional value of different kinds of milk and the differences between them through multi-omics analysis and found functional components that are more similar to human milk. This study employed untargeted LC-MS/MS metabolomics, untargeted LC-MS/MS lipidomics, and 4D label-free proteomics analysis techniques. The findings revealed substantial disparities in metabolites, lipids, and proteins among the five types of milk. Notably, pig milk exhibited a remarkable abundance of N-acetylneuraminic acid (Neu5Ac) and specific polar lipids. Yak milk stood out with significantly elevated levels of creatine and lipoprotein lipase (LPL) compared to other species. Buffalo milk boasted the highest concentrations of L-isoleucine, echinocystic acid, and alkaline phosphatase, tissue-nonspecific isozyme (ALPL). The concentrations of iminostilbene and osteopontin (OPN) were higher in cow milk.

Effect of supplementing epinephrine in maturation media on in-vitro developmental competence of cattle and buffalo oocytes.

During in-vitro maturation, the oocyte experiences stressful conditions that likely compromise its development. Epinephrine is a catecholamine that plays a vital role during cellular stress by scavenging free radicals. The hypothesis is that epinephrine addition in maturation media improves the developmental competence of oocytes in cattle and buffalo. The objectives of the experiments were to investigate the effect of epinephrine addition in maturation media on nuclear maturation, developmental competence, and oocyte mRNA abundance of genes related to antioxidants and growth pathways in cattle and buffalo. In experiment 1, cattle oocytes were matured for 24 h in maturation media supplemented with increasing concentrations of epinephrine 0, 0.01, 1.0, and 100 µM. Oocytes were cultured to assess cleavage at 48 h and blastocyst on day 7 of the culture. The cumulus-oocyte complexes (COCs) expansion, nuclear maturation, and oocyte mRNA abundance of genes (SOD1, GPX4, GDF9, CASP9) were evaluated. In experiment 2, buffalo oocytes were matured and assessed for development and mRNA abundance as described for cattle. In addition, the blastomere number was counted in the hatched blastocyst. The data were analyzed using GLIMMIX and MIXED procedures of SAS. Results revealed that the supplementation of epinephrine increased (P ≤ 0.03) the COCs expansion, nuclear maturation, and developmental competence of oocytes in cattle. Interestingly, all the responses were maximized (quadratic effect; P ≤ 0.08) at 1 µM concentrations. The mRNA abundance of genes in cattle oocytes was not affected by the treatment. The experiment in buffalo revealed that epinephrine increased blastocyst formation without affecting COCs expansion, and nuclear maturation. The higher blastocyst was achieved at 0.01 µM concentrations of epinephrine. Interestingly, the addition of epinephrine increased the mRNA abundance of genes related to antioxidant pathways (SOD1, GPX4). Moreover, supplementation of epinephrine increased the blastomere count of the hatched blastocyst in buffalo. In conclusion, epinephrine addition in maturation media benefited oocyte development in cattle and blastocyst yield in buffalo at 1 and 0.01 µM concentrations, respectively. It appears that the addition of epinephrine affected different cellular pathways, COCs expansion, and nuclear maturation in cattle and increased antioxidant genes for buffalo.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull's ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen. RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively). CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

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.

Detection of Toxoplasma gondii infection in buffaloes (Bubalus bubalis) and cattle (Bos taurus) at the Tabriz abattoir, Iran.

BACKGROUND: Toxoplasma gondii is a widely prevalent zoonotic protozoan parasite in humans and warm-blooded animals worldwide. Infection of humans by this parasite can result in severe clinical symptoms, particularly in individuals with congenital toxoplasmosis or immunocompromised patients. Contamination mainly occurs through foodborne routes, especially the consumption of raw or undercooked meat from animals. OBJECTIVES: The aim of this study was to use PCR to detect T. gondii in tissues and organs of buffaloes and cattle slaughtered at Tabriz slaughterhouse, in Iran. METHODS: Fifty grams of heart, thigh, diaphragm and tongue from 50 buffaloes and 100 cattle slaughtered at the Tabriz industrial slaughterhouse were selected for sampling using a combination of convenience sampling. The samples were tested using a previously published PCR method. RESULTS: Out of the 150 animal samples, T. gondii was detected in 10 (6.7%, 95%CI: 3.2-11.9), including one buffalo (2%, 95%CI: 0.1-10.6) and nine cattle (9%, 95%CI: 4.2-16.4). There was no statistically significant difference in the rate of T. gondii infection among cattle based on age and sex (p > 0.05). CONCLUSIONS: The results indicated a potential risk of T. gondii transmission to humans through the consumption of infected meat. Therefore, appropriate and effective preventive measures should be taken to limit the transmission of this parasite to humans, and the consumption of raw and undercooked meat should be discouraged.

Hygiene Assessment of Buffalo Milking Parlours in Campania Region, Italy: A Preliminary Study by Using ATP Luminometry and Bacteriological Investigation.

Careful cleaning of a milking parlour and its equipment is fundamental to guarantee good raw milk quality and prevent the dissemination of bacteria and improve animal welfare. This study aimed to investigate, using an ATP-bioluminescence assay and bacteriological analysis, the bacterial contamination of milking parlours on milking parlour surfaces of buffalo farms in the Campania Region, evaluating the seasonal dynamics during the year 2022. Eight farms were selected by the Italian ClassyFarm system, which assesses the level of animal welfare and biosecurity according to risk analysis. Before sampling, all dairy farm owners filled out a questionnaire on milking management, animal hygiene, and health. The questionnaires evidenced similar cleaning procedures but an absence of a standardised cleaning protocol among the different farms. ATP bioluminescence results evidenced similar levels of contamination in all the selected buffalo farms, and the season comparison showed no significant differences. A variation in the percentages of bacterial isolates during the different seasons was observed, with a higher prevalence of Enterobacteriaceae (38%) in summer. A small number of samples exhibited an absence of bacterial growth. Identifying bacteria is crucial for understanding the microorganisms present in the milking parlour, yet employing ATP luminometry could offer broad and accurate applications in buffalo milking parlours. In conclusion, the use of ATP bioluminescence for evaluating the hygiene of a buffalo milking parlour could represent a further important advancement in dairy farming technology.