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.

Analysis of miRNAs in milk of four livestock species.

BACKGROUND: Milk is essential for mammalian nutrition because it provides vital nutrients for growth and development. Milk composition, which is influenced by genetic and environmental factors, supports lactation, a complex process crucial for milk production and quality. Recent research has focused on noncoding RNAs, particularly microRNAs (miRNAs), which are present in body fluids and regulate gene expression post-transcriptionally. This study comprehensively characterizes miRNAs in milk of four livestock species, namely Bubalus bubalis, Capra hircus, Equus asinus, and Ovis aries and identifies potential target genes. RESULTS: High-throughput sequencing of milk RNA resulted in distinct read counts across species: B. bubalis (8,790,441 reads), C. hircus (12,976,275 reads), E. asinus (9,385,067 reads), and O. aries (7,295,297 reads). E. asinus had the highest RNA mapping rate (94.6%) and O. aries the lowest (84.8%). A substantially greater proportion of miRNAs over other small RNAs was observed for the donkey milk sample (7.74%) compared to buffalo (0.87%), goat (1.57%), and sheep (1.12%). Shared miRNAs, which included miR-200a, miR-200b, miR-200c, and miR-23a among others, showed varying expression levels across species, confirmed by qPCR analysis. Functional annotation of predicted miRNA target genes highlighted diverse roles, with an enrichment in functions linked to metabolism and immunity. Pathway analysis identified immune response pathways as significant, with several miRNAs targeting specific genes across species, suggesting their regulatory function in milk. CONCLUSIONS: Both conserved and species-specific miRNAs were detected in milk of the investigated species. The identified target genes of these miRNAs have important roles in neonatal development, adaptation, growth, and immune response. Furthermore, they influence milk and meat production traits in livestock.

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.

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.

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.

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes.

BACKGROUND: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte.

INTRODUCTION: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. METHODS: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. RESULTS: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. CONCLUSION: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

BACKGROUND: Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. METHODS AND RESULTS: Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures [38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. CONCLUSION: It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

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.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

An abattoir study of the prevalence of foot lesions and claw measurements in water buffalo in Egypt.

BACKGROUND: Lameness has been associated with compromised animal welfare and reduced productivity in dairy cattle herds worldwide. However, little is known about the prevalence of claw lesions in the dairy buffalo population in Egypt. Furthermore, the optimum measurements for claw trimming in buffalo are unknown. A cross-sectional cadaver study was conducted where 135 pair buffalo hind feet were collected from 4 slaughterhouses and examined for the presence of claw lesions. The proportion and associated 95% confidence interval (CI) of each type of lesion were calculated. A separate set of healthy claws (n = 26) underwent ultrasonography (US) and computed tomography (CT). The agreement between US and CT measurements was assessed using Passing-Bablok regression and intraclass correlation coefficient. The CT measurements were used to calculate trimming recommendations. RESULTS: At least one lesion was identified in 242 claws (89.6%, 95% CI = 85.4-93.0). In healthy claws, poor to moderate agreement was identified between US and CT measurements which could be due a sample size of the study. The average ± standard deviation (SD) minimum recommended external wall length of the lateral and medial claws in heifers was 7.1 ± 0.36 cm and 7.5 ± 0.35 cm, respectively. The average ± SD minimum recommended external wall length in buffaloes over five years of age was 8.2 ± 0.27 cm and 8.4 ± 0.39 cm for the lateral and medial claws, respectively. CONCLUSIONS: The study found a high prevalence of claw lesions in buffalo in Egypt, the clinical significance of which requires further elucidation. Recommended measurements will help guide claw trimming in buffalo to minimise lesions.

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.

Effects of apigenin and trans-ferulic acid on microscopic and oxidative stress parameters in the semen of water buffalo bulls during cryopreservation.

Cryopreservation involves exposing sperm to stressful conditions that affect cell viability. The high quality of the Azerbaijani water buffalo's by-products, such as buffalo milk, makes it a species of significant importance. Our focus is on protecting its genetic resources by preserving its sperm, as their numbers will decrease in the coming years and they are at risk of extinction. This study's goal was to ascertain how apigenin (A) and trans-ferulic acid (t-FA) affected the semen quality of Azari water buffalo bulls under cryopreservation. Pooled buffalo sperm (n = 35 ejaculations) were diluted in a Tris-based diluent also containing varying amounts of apigenin (0.2, 0.4, 0.6, and 0.8 mM) and trans-ferulic acid (2.5, 5, 10 and 20 mM). Following a freeze-thaw procedure, samples were assayed for total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione activity (GSH), glutathione peroxidase (GPx), progressive motility and total motility, motility properties, plasma membrane functionality, and viability. Sixty days after insemination, the rectal examination was performed on 38 buffaloes that had undergone sexual breeding to confirm pregnancy. The results of the study show that the addition of A-0.2, A-0.4, and t-FA-10 to buffalo semen increases the percentage of intact plasma membrane, motility, and sperm viability, as well as the levels of GSH, GPx, CAT. and TAC. In addition, there is a decrease in MDA and DNA damage after cryopreservation. Furthermore, the results show that 0.4 mM apigenin significantly increases conception rates compared to the control group. The base extender of Tris supplemented with A (0.4 and 0.2 mM) and t-FA (10 mM) improves the antioxidant indices of both frozen and thawed buffalo sperm, which in turn improves post-thawing sperm quality and in vivo fertility improves buffalo sperm.

Preovulatory follicular dynamics and ovulatory events following the use of GnRH 84 h after medroxyprogesterone acetate sponge removal in postpartum buffaloes.

Herein, we evaluated the effects of Gonadotropin hormone-releasing hormone (GnRH) administration 84 h after medroxyprogesterone acetate (MAP) sponge removal on follicular growth, ovulation timing, and pregnancy per artificial insemination (AI) in cosynchronized postpartum Nili Ravi buffaloes. In this study, 58 Nili Ravi postpartum buffaloes (DIM = 103 ± 1.64) were randomly divided into two treatment groups (n = 29/treatment): GnRH-TAI-84 and TAI-84. All buffaloes were administered a MAP sponge for seven days. Upon MAP sponge removal, all the subjects received prostaglandin F2α (PGF2α), and Timed AI (TAI) was performed 84 hours after sponge removal. In the GnRH-TAI-84 group, the buffaloes received GnRH alongside insemination, whereas in the TAI-84 group, the buffaloes were inseminated without GnRH administration. Follicle diameter and blood estradiol levels were measured every 6 h from 72-108 h after MAP sponge removal. The animals were checked for pregnancy using ultrasonography 40 days after AI. Animals subjected to the GnRH-TAI-84 protocol had a higher follicular growth rate and preovulatory follicle size than those in the TAI-84 group. The follicular diameter was also larger in animals that received GnRH-TAI-84 than in those that received TAI-84 90 and 96 h after MAP sponge removal. Buffaloes in the GnRH-TAI-84 group had lower estradiol concentrations at 90, 96, 102, and 108 h than those in the TAI-84 group. Ovulation in GnRH-TAI-84 buffaloes occurred 11 h earlier than that in buffaloes from the TAI-84 group. A shorter interval between AI and ovulation in GnRH-TAI-84 buffaloes (14 h vs. 25 h) led to greater pregnancies per AI (62% vs. 17%) compared to buffaloes from the TAI-84 group.

Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

Weighted single step GWAS reveals genomic regions associated with economic traits in Murrah buffaloes.

The objective of the present study was to identify genomic regions influencing economic traits in Murrah buffaloes using weighted single step Genome Wide Association Analysis (WssGWAS). Data on 2000 animals, out of which 120 were genotyped using a double digest Restriction site Associated DNA (ddRAD) sequencing approach. The phenotypic data were collected from NDRI, India, on growth traits, viz., body weight at 6M (month), 12M, 18M and 24M, production traits like 305D (day) milk yield, lactation length (LL) and dry period (DP) and reproduction traits like age at first calving (AFC), calving interval (CI) and first service period (FSP). The biallelic genotypic data consisted of 49353 markers post-quality check. The heritability estimates were moderate to high, low to moderate, low for growth, production, reproduction traits, respectively. Important genomic regions explaining more than 0.5% of the total additive genetic variance explained by 30 adjacent SNPs were selected for further analysis of candidate genes. In this study, 105 genomic regions were associated with growth, 35 genomic regions with production and 42 window regions with reproduction traits. Different candidate genes were identified in these genomic regions, of which important are OSBPL8, NAP1L1 for growth, CNTNAP2 for production and ILDR2, TADA1 and POGK for reproduction traits.

Diversity assessment of a lesser known buffalo population from Central India and its comparative evaluation reveals presence of sufficient genetic variation and absence of selection.

Planned breeding and conservation strategies for a lesser-known population require an assessment of complete genetic diversity and population structure analysis in addition to its morphometric characteristics. In the present study, a comparative analysis of the genetic structure of a rare buffalo population, namely Chhattisgarhi, was extensively studied using a panel of FAO-recommended microsatellite markers along with well-established breeds namely Murrah, Nili-Ravi, Gojri, Kalahandi, and Nagpuri. Mode shift analysis indicated the absence of genetic bottleneck in the recent past. Assessment of genetic diversity indices across all loci indicated the presence of sufficient genetic variation within and between populations. Analysis of molecular variance between the six different buffalo populations attributed 19.05% of the variations to between-population differentiation. Cluster analyses using DAPC and Bayesian approach along with the phylogenetic tree based on UPGMA grouped six populations into three groups. The Chhattisgarhi population was revealed to be genetically closer to Nagpuri and Kalahandi populations. The study reveals the presence of sufficient genetic diversity within the Chhattisgarhi population and indicates the absence of a systematic selection program. We suggest improvement and conservation programs should be planned for this breed in the near future through short-term selection.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Exome-wide comparative analyses revealed differentiating genomic regions for performance traits in Indian native buffaloes.

In India, 20 breeds of buffalo have been identified and registered, yet limited studies have been conducted to explore the performance potential of these breeds, especially in the Indian native breeds. This study is a maiden attempt to delineate the important variants and unique genes through exome sequencing for milk yield, milk composition, fertility, and adaptation traits in Indian local breeds of buffalo. In the present study, whole exome sequencing was performed on Chhattisgarhi (n = 3), Chilika (n = 4), Gojri (n = 3), and Murrah (n = 4) buffalo breeds and after stringent quality control, 4333, 6829, 4130, and 4854 InDels were revealed, respectively. Exome-wide FST along 100-kb sliding windows detected 27, 98, 38, and 35 outlier windows in Chhattisgarhi, Chilika, Gojri, and Murrah, respectively. The comparative exome analysis of InDels and subsequent gene ontology revealed unique breed specific genes for milk yield (CAMSAP3), milk composition (CLCN1, NUDT3), fertility (PTGER3) and adaptation (KCNA3, TH) traits. Study provides insight into mechanism of how these breeds have evolved under natural selection, the impact of these events on their respective genomes, and their importance in maintaining purity of these breeds for the traits under study. Additionally, this result will underwrite to the genetic acquaintance of these breeds for breeding application, and in understanding of evolution of these Indian local breeds.

Analysis of OCT4 and PGP9.5 gene expression in prenatal and postnatal buffalo (Bubalus bubalis) testes.

This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells' regulation and potential applications.