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.

Comparative analyses of copy number variations between swamp buffaloes and river buffaloes.

As an important source of genomic variation, copy number variation (CNV) contributes to environmental adaptation in worldwide buffaloes. Despite this importance, CNV divergence between swamp buffaloes and river buffaloes has not been studied previously. Here, we report 21 152 CNV regions (CNVRs) in 141 buffaloes of 20 breeds detected through multiple CNV calling strategies. Only 248 CNVRs were shared between river buffalo and swamp buffalo, reflecting great variation of CNVRs between the two subspecies. Population structure analysis based on CNVs successfully separated the two buffalo subspecies. We further assessed CNV divergence by calculating FST for genome-wide CNVs. Totally, we identified 110 significantly divergent CNV segments and 44 putatively selected genes between river buffaloes and swamp buffaloes. In particular, LALBA, a key gene controlling milk production in cattle, presented a highly differentiated CNV in the promoter region, which makes it a strong functional candidate gene for differences between swamp buffaloes and river buffaloes in traits related to milk production. Our study provides useful information of CNVs in buffaloes, which may help explain the genetic differences between the two subspecies.

Genetic admixture and population structure analysis of Indian water buffaloes (Bubalus bubalis) using STR markers.

BACKGROUND: India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different buffalo populations of India, using 20 highly polymorphic microsatellite markers has been generated for the genetic diversity analysis. The buffalo populations of Eastern Odisha state, were the primary focus. METHODS AND RESULTS: The minimal spanning network based on Bruvo's distance, PCA (Principal Component Analysis) based on the Fst (Fixation Index) values, and genetic admixture analysis using both the STRUCTURE and 'snapclust' were performed. The analysis could identify the Manda population as distinct from other Odisha buffalo breeds as well as adjoining Chhattisgarhi buffalo breeds. The total observed number of alleles ranged between 143 (Manda) and 301 (Paralakhemundi) with an average of 204 alleles per breed. The Sambhalpuri buffalo population also clustered into two separate subpopulations, half of the unique sub-population located geographically south-wards, displayed no admixture with any of the adjacent buffalo populations. The Manda buffalo population has shown sufficient allelic richness and heterozygosity under random mating being practiced in the field conditions. CONCLUSIONS: The study has led to the identification of the Manda as a distinct buffalo population, and the germplasm has been registered as a new Indian buffalo breed. Whereas, the Sambhalpuri population requires elaborate analysis to confirm the existence of two distinct sub-populations.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo.

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo.

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

The genome of the lowland anoa (Bubalus depressicornis) illuminates the origin of river and swamp buffalo.

Two types of domestic water buffalo are currently recognized: the river buffalo from the Indian subcontinent and Mediterranean countries and the swamp buffalo from China and Southeast Asia. To test the hypothesis of two separate species of water buffalo, we sequenced the genome of the lowland anoa, Bubalus depressicornis, which is a dwarf wild buffalo endemic to Sulawesi, and two genomes of swamp buffalo, and made comparisons with 12 additional genomes. Three genomic data sets were constructed to infer phylogenetic relationships: the mitochondrial genome (15,468 bp; maternal transmission), two concatenated Y-chromosomal genes, AMELY and DDX3Y (20,036 bp; paternal transmission), and a selection of 30 nuclear genes representing all cattle chromosomes (364,887 bp; biparental transmission). The comparisons between our 30 nuclear gene sequences obtained by read mapping and those directly extracted from Bos taurus and Bubalus bubalis genome assemblies show that the mapping approach revealed higher levels of heterozygosity at both nucleotide sites and indels (insertions and deletions) (0.09-0.15%), as well as several sequence errors (0.07%). Our phylogenetic and molecular dating analyses provide strong evidence that the lowland anoa, river buffalo, and swamp buffalo are three distinct taxa which separated rapidly from each other during the Pleistocene epoch. We therefore conclude that two species of domestic water buffalo should be distinguished: Bubalus bubalis for the river buffalo and Bubalus kerabau for the swamp buffalo. The new classification can have deep implications for understanding the evolution and selection of domesticated forms and for the conservation and management of wild buffalo populations in South and Southeast Asia.

Melatonin supplementation improved cryopreserved Thai swamp buffalo semen.

The production of reactive oxygen species (ROS) during cryopreservation process impairs the sperm characteristics and fertilizing ability. However, melatonin, an antioxidant, could protect spermatozoa against this cell damage during cryopreservation. Therefore, we attempted to evaluate whether the melatonin supplementing in the semen extender could improve the sperm quality of swamp buffalo during cryopreservation. The semen collected from six swamp buffalo bulls were diluted with tris-citrate egg yolk extender supplementing with 0, 0.1, 0.5, 1.0, 2.0 and 3.0 mM of melatonin. The parameters of sperm viability and motility were evaluated using computer-assisted semen analyser (CASA) after cryopreservation on days 1, 7, 15 and 30. The group supplemented with 1.0 mM melatonin exhibited the higher viability after cryopreservation on days 1, 7, 15 and 30 with 58.346 ± 2.1a , 57.586 ± 2.0a , 55.082 ± 1.8a and 55.714 ± 1.8a , respectively, and showed the best results of motility parameters. However, higher concentration of melatonin at 3.0 mM impaired all the parameters. In conclusion, the addition of melatonin at 1 mM to semen extender could exert the best protection against sperm damage in swamp buffalo bull during cryopreservation.

Expression and localization of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in corpora lutea during the estrous cycle and pregnancy in Thai swamp buffalo (Bubalus bubalis).

In female mammals, luteal cells rapidly proliferate and form corpora lutea (CLs) after ovulation. The corpus luteum (CL) plays crucial roles in establishing and maintaining pregnancy. To gain further insights into the role of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptosis factor that is structurally similar to procaspase-8 but lacks proteolytic enzyme activity, we examined the expression in CLs of Thai swamp buffalos (Bubalus bubalis) during the early, mid, and late stage of the estrous cycle and pregnancy. cFLIP short form and long form (cFLIPS and cFLIPL, respectively) mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction and western blotting, respectively. cFLIPS mRNA levels were low in the mid and late stages of the estrous cycle and increased during pregnancy (P < 0.05). cFLIPL mRNA was highly expressed in CLs during pregnancy and was lower in the mid and late stages of the estrous cycle. The level of cFLIPS protein was high in CLs during pregnancy and low levels were noted in the mid stage of the estrous cycle (P < 0.05). Higher levels of cFLIPL protein were demonstrated in CLs during pregnancy and lower levels were found in CLs during the early stage of the estrous cycle. Strong positive immunohistochemical staining for cFLIPS/L proteins was observed in luteal cells during pregnancy. The present findings revealed that cFLIP was at the highest level in CLs during pregnancy, and this may act as a dominant survival anti-apoptotic factor by inhibiting intracellular apoptosis signal transduction in luteal cells of CLs during pregnancy.

Asian water buffalo: domestication, history and genetics.

The domestic Asian water buffalo (Bubalus bubalis) is found on all five continents, with a global population of some 202 million. The livelihoods of more people depend on this species than on any other domestic animal. The two distinct types (river and swamp) descended from different wild Asian water buffalo (Bubalus arnee) populations that diverged some 900 kyr BP and then evolved in separate geographical regions. After domestication in the western region of the Indian subcontinent (ca. 6300 years BP), the river buffalo spread west as far as Egypt, the Balkans and Italy. Conversely, after domestication in the China/Indochina border region ca. 3000-7000 years BP, swamp buffaloes dispersed through south-east Asia and China as far as the Yangtze River valley. Molecular and morphological evidence indicates that swamp buffalo populations have strong geographic genetic differentiation and a lack of gene flow, but strong phenotypic uniformity. In contrast, river buffalo populations show a weaker phylogeographic structure, but higher phenotypic diversity (i.e. many breeds). The recent availability of a high-quality reference genome and of a medium-density marker panel for genotyping has triggered a number of genome-wide investigations on diversity, evolutionary history, production traits and functional elements. The growing molecular knowledge combined with breeding programmes should pave the way to improvements in production, environmental adaptation and disease resistance in water buffalo populations worldwide.

Multiple domestication of swamp buffalo in China and South East Asia.

The domestication of swamp buffalo (Bubalus bubalis carabanesis) has been discussed for years and still remained unclear. To obtain a better understanding of where, when and how the swamp buffaloes were domesticated, the complete mitochondrial DNA D-loop sequences of 1,788 individuals from China, Vietnam, Laos, Thailand, Burma, Bangladesh and India were investigated. Our results revealed swamp buffalo with abundant genetic diversity. The lineage SA of swamp buffalo may be first domesticated in Lower Yangtze and then migrated following two different routes: one migrated along the Upper Yangtze to Southwest China (L1); the other migrated to Southeast China to Southeast Asia through Guangxi province (L2). During the migration process several later domestication events may be inspired in the Upper Yangtze (SB3), Southwest China and North of Southeast Asia (SB1 and SB2). In this study, we proposed a non-independent multiple domestication pattern in swamp buffalo.

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos.

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 µM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 µM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 µM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 µM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4-8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.

Y-chromosome haplotype analysis revealing multiple paternal origins in swamp buffaloes of China and Southeast Asia.

To further probe into the paternal origins and domestication area of swamp buffaloes, we examined Y-chromosome diversity of 482 bulls representing 22 populations from China and Southeast Asia. A total of 40 bovine Y-chromosome-specific microsatellite (Y-STR) markers were screened in this study. The results showed that seven Y-STR markers (UMN2405, UMN0504, UMN0103, UMN1307, BC1.2, UMN0304 and INRA008) were specific and polymorphic in male swamp buffaloes, which can define 9 Y-haplotypes corresponding to four Y-haplogroups (Y1, Y2, Y3 and Y4). Haplogroup Y1 was predominant (83.4%) in all swamp buffalo populations, indicating haplogroup Y1 was the major domestication event of swamp buffalo. In addition, the abundant genetic background and backbone of haplogroup Y1 suggested Yangtze Valley as the major domestication area of swamp buffalo. Interestingly, haplogroup Y4 was only confined in Hainan Island which was more ancient than other haplogroups. We hypothesized that haplogroup Y4 was the descendants of the wild Asian buffalo trapped on Hainan Island in prehistoric glacier period and preserved by later introgression into domesticated cows after the domestication. In conclusion, our findings revealed four divergent paternal origins in swamp buffaloes based on Y-STR markers.

Tropical legume supplementation influences microbial protein synthesis and rumen ecology.

Four rumen-fistulated male swamp buffaloes, 5-year-old with initiated live weight at 360 ± 12 kg, were randomly assigned according to a 4 × 4 Latin square design to investigate the effect of feeding high level of dried Leucaena leaf (DLL) on feed intake, fermentation efficiency and microbial protein synthesis. The dietary treatments were the feeding levels of DLL at 0, 2, 4 and 6 kg/head/day. All buffaloes were supplemented with concentrate mixtures at 0.1% of body weight, and rice straw was fed ad libitum with the availability of water and mineral block at all time. The results revealed that the total feed intake and nutrient digestibility were significantly improved with the increasing levels of DLL feeding, and the highest was in the buffaloes consuming DLL at 6 kg/head/day. Feeding high levels of DLL did not affect on ruminal pH and temperature, while ammonia nitrogen, blood urea nitrogen and volatile fatty acid concentration were significantly enhanced. Moreover, methane production was dramatically reduced by increasing levels of DLL feeding. Total direct counts of the micro-organism population were increased with the increasing levels of DLL feeding. According to the application of quantitative PCR to quantity cellulolytic bacteria (16S rRNA) targets, it was found that the population of total bacteria and Fibrobactor succinogenes was affected by treatments, while Ruminococcus flavefaciens and methanogen population were significantly decreased as buffaloes were fed with DLL. The nitrogen balance and microbial nitrogen supply were remarkably improved with the increasing levels of DLL feeding. Based on this study, it could be concluded that high levels of DLL feeding at 6 kg/head/day could enhance feed intake, nutrient digestibility, rumen fermentation efficiency and microbial protein synthesis in swamp buffaloes fed on rice straw without any adverse effect.

Separation and quantification of milk casein from different buffalo breeds.

Understanding the milk protein expression profile in different buffalo breeds plays an important role in improving hybrid selection and determining the effects on milk protein synthesis. The aim of this research is to compare the differences in milk protein content, composition and distribution between River buffalo and their crossbreeds for hybrid screening. Four groups of milk samples that included Nili-Ravi (N), Murrah (M), a Nili-Ravi-Murrah crossbreed (M-N), and a crossbreed of river buffalo with local swamp buffalo (C) were collected. The protein composition of the buffalo milk was determined by RP-HPLC. A gel-based proteomic approach consisting of two-dimensional gel electrophoresis coupled with mass spectrometry was utilised for the detailed protein characterisation of milk from different breeds. The results of this analysis showed that the river/swamp buffalo crossbreed (C) displayed the highest content of total protein (4·46%) and κ-casein (11·14%) but the lowest content of α-lactalbumin (6·79%). By selecting 23 different protein spots among the four types of milk that contained the most spots corresponding to κ-casein, ß-casein and αs1-casein, correlations between the crossbreeds, protein polymorphism and phosphorylation could be made. The results of this study indicate that crossbreeding a swamp buffalo with a river buffalo has a notable effect on the protein content and composition that may be exploited for producing high-quality raw milk in food technology applications and dairy food production.

Comparative Proteomic Analysis of Mature and Immature Oocytes of the Swamp Buffalo (Bubalus bubalis).

Maternal protein components change markedly during mammalian oogenesis. Many of these proteins have yet to be characterized and verified. In this study, a proteomics approach was used to evaluate changes in proteins during oogenesis in the Swamp Buffalo (Bubalus bubalis). Proteins from 500 immature oocytes and 500 in vitro matured oocytes were subjected to two-dimensional electrophoresis, and more than 400 spots were detected. Image analysis indicated that 17 proteins were differentially expressed between the two groups. Eight proteins were identified by mass spectrometry. In mature oocytes, three proteins were down-regulated: major vault protein (MVP), N-acetyllactosaminide ß-1,6-N-acetylglucosaminyl-transferase (GCNT-2), and gem-associated protein (GEMIN)8, whereas five other proteins, heat shock protein (HSP)60, Ras-responsive element-binding protein 1 (RREB-1), heat shock cognate 71 kDa protein (HSC71), hemoglobin subunit α (HBA), and BMP-2-inducible protein kinase (BMP-2K), were up-regulated. The expression profiles of HSP60 and GEMIN8 were further verified by Western blotting. The changes in HSP60 protein expression demonstrate the increasing need for mitochondrial protein importation to facilitate macromolecular assembly during oocyte maturation. The down-regulation of GEMIN8 production implies that RNA splicing is impaired in mature oocytes.

Effects of Supplementation of Eucalyptus (E. Camaldulensis) Leaf Meal on Feed Intake and Rumen Fermentation Efficiency in Swamp Buffaloes.

Four rumen fistulated swamp buffaloes were randomly assigned according to a 4×4 Latin square design to investigate the effects of Eucalyptus (E. Camaldulensis) leaf meal (ELM) supplementation as a rumen enhancer on feed intake and rumen fermentation characteristics. The dietary treatments were as follows: T1 = 0 g ELM/hd/d; T2 = 40 g ELM/hd/d; T3 = 80 g ELM/hd/d; T4 = 120 g ELM/hd/d, respectively. Experimental animals were kept in individual pens and concentrate was offered at 0.3% BW while rice straw was fed ad libitum. The results revealed that voluntary feed intake and digestion coefficients of nutrients were similar among treatments. Ruminal pH, temperature and blood urea nitrogen concentrations were not affected by ELM supplementation; however, ELM supplementation resulted in lower concentration of ruminal ammonia nitrogen. Total volatile fatty acids, propionate concentration increased with the increasing level of EML (p<0.05) while the proportion of acetate was decreased (p<0.05). Methane production was linearly decreased (p<0.05) with the increasing level of ELM supplementation. Protozoa count and proteolytic bacteria population were reduced (p<0.05) while fungal zoospores and total viable bacteria, amylolytic, cellulolytic bacteria were unchanged. In addition, nitrogen utilization and microbial protein synthesis tended to increase by the dietary treatments. Based on the present findings, it is suggested that ELM could modify the rumen fermentation and is potentially used as a rumen enhancer in methane mitigation and rumen fermentation efficiency.

Effects of eucalyptus crude oils supplementation on rumen fermentation, microorganism and nutrient digestibility in swamp buffaloes.

This study was conducted to investigate the effects of eucalyptus (E. Camaldulensis) crude oils (EuO) supplementation on voluntary feed intake and rumen fermentation characteristics in swamp buffaloes. Four rumen fistulated swamp buffaloes, body weight (BW) of 420±15.0 kg, were randomly assigned according to a 2×2 factorial arrangement in a 4×4 Latin square design. The dietary treatments were untreated rice straw (RS) without EuO (T1) and with EuO (T2) supplementation, and 3% urea-treated rice straw (UTRS) without EuO (T3) and with EuO (T4) supplementation. The EuO was supplemented at 2 mL/h/d in respective treatment. Experimental animals were kept in individual pens and concentrate mixture was offered at 3 g/kg BW while roughage was fed ad libitum. Total dry matter and roughage intake, and apparent digestibilites of organic matter and neutral detergent fiber were improved (p<0.01) by UTRS. There was no effect of EuO supplementation on feed intake and nutrient digestibility. Ruminal pH and temperature were not (p>0.05) affected by either roughage sources or EuO supplementation. However, buffaloes fed UTRS had higher ruminal ammonia nitrogen and blood urea nitrogen as compared with RS. Total volatile fatty acid and butyrate proportion were similar among treatments, whereas acetate was decreased and propionate molar proportion was increased by EuO supplementation. Feeding UTRS resulted in lower acetate and higher propionate concentration compared to RS. Moreover, supplementation of EuO reduced methane production especially in UTRS treatment. Protozoa populations were reduced by EuO supplementation while fungi zoospores remained the same. Total, amylolytic and cellulolytic bacterial populations were increased (p<0.01) by UTRS; However, EuO supplementation did not affect viable bacteria. Nitrogen intake and in feces were found higher in buffaloes fed UTRS. A positive nitrogen balance (absorption and retention) was in buffaloes fed UTRS. Supplementation of EuO did not affect nitrogen utilization. Both allantoin excretion and absorption and microbial nitrogen supply were increased by UTRS whereas efficiency of microbial protein synthesis was similar in all treatments. Findings of present study suggested that EuO could be used as a feed additive to modify the rumen fermentation in reducing methane production both in RS and UTRS. Feeding UTRS could improve feed intake and efficiency of rumen fermentation in swamp buffaloes. However, more research is warranted to determine the effect of EuO supplementation in production animals.

Effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes.

The objective of this study was to investigate the effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes. Four, 4-yr old rumen fistulated swamp buffaloes were randomly assigned to receive four dietary treatments according to a 2×2 factorial arrangement in a 4×4 Latin square design. Factor A was carbohydrate source; cassava chip (CC) and CC+rice bran at a ratio 3:1 (CR3:1), and factor B was level of cottonseed meal (CM); 109 g CP/kg (LCM) and 328 g CP/kg (HCM) in isonitrogenous diets (490 g CP/kg). Buffaloes received urea-treated rice straw ad libitum and supplemented with 5 g concentrate/kg BW. It was found that carbohydrate source did not affect feed intake, nutrient intake, digested nutrients, nutrient digestibility, ammonia nitrogen concentration, fungi and bacterial populations, or microbial protein synthesis (p>0.05). Ruminal pH at 6 h after feeding and the population of protozoa at 4 h after feeding were higher when buffalo were fed with CC than in the CR3:1 treatment (p<0.05). Buffalo fed with HCM had a lower roughage intake, nutrient intake, population of total viable and cellulolytic bacteria and microbial nitrogen supply than the LCM fed group (p<0.05). However, nutrient digestibility, ruminal pH, ammonia concentration, population of protozoa and fungi, and efficiency of microbial protein synthesis were not affected by cottonseed meal levels (p>0.05). Based on this experiment, concentrate with a low level of cottonseed meal could be fed with cassava chips as an energy source in swamp buffalo receiving rice straw.

The diversity and quality of forages and their potency as herbal anthelmintic for swamp buffalo in Brebes District, Central Java.

Background and Aim: Swamp buffaloes play an important role in the rural economy of Indonesia. They consume various forages during their grazing time, including those with anti-parasitic potential. However, the information about the type and quality of forages and their potential as a natural anthelmintic for swamp buffalo is very limited. This study aimed to identify the diversity, quality, and anthelmintic potency of forages consumed by swamp buffaloes reared in Bantarkawung Subdistrict, Brebes District, Central Java Province, Indonesia. Materials and Methods: Samples of forages were obtained during three observation periods of the study, with a 12-week interval between each period. Forage diversity was evaluated by identifying its consumption by swamp buffaloes during their grazing activity in the field and feeding time in their shed. The quality of forages was analyzed using proximate analysis to measure their dry matter (DM), crude fiber (CF), crude protein (CP), crude fat (CFat), and ash contents. This is followed by the calculation of their total digestible nutrient based on the proximate analysis results. Botanical composition analysis was then conducted to measure the predominance of forages consumed by the livestock during their grazing activity. Literature reviews were carried out to explore forage's anthelmintic activity. Results: The results showed that swamp buffaloes consume nine species of forage in the shed and 47 in the grazing area, including nine legumes, 18 grass, and 20 others. Swamp buffaloes consumed forages of lower quality, which contained high CF contents and varying levels of other nutrients below their daily nutritional needs. The grazing activity allowed swamp buffaloes to consume a higher variety of forages with better nutritional quality, thereby enabling them to meet their nutritional needs. Legumes and other forages served as the major protein sources, providing CP of 20.03% DM and 11.53% DM, and CF levels of 17.01% DM and 20.35% DM, respectively. The results also showed that the consumption of these forages increased during the rainy season. The predominant species of legumes consumed were Leucaena leucocephala and Acacia spp., while Alternanthera sessilis and Merremia umbellata were the predominant species of other forages. A total of 13 of the 47 species could potentially be used as natural anthelmintic due to their secondary metabolites, namely, tannin, flavonoid, saponin, terpenoid, diterpenoid, and mimosine. These compounds exert anthelmintic effects by inhibiting egg-hatching and larval development, as well as damaging the surface structure of both larvae and adult worms, ultimately leading to the death of the parasites. Conclusion: Overall, swamp buffaloes consumed more variety of forages during grazing compared to when they were kept in sheds. While the low-variety and low-quality forages provisioned for swamp buffaloes in their shed resulted in a low nutrient intake below their daily requirement. Furthermore, daily grazing activities allowed swamp buffaloes to fulfill and supplement their need by consuming a variety of grasses, legumes, and other forages in their respective grazing areas. Some of these forages also have the potential to become natural anthelmintic because they contain secondary metabolites, such as tannins, flavonoids, saponins, terpenoids, diterpenoids, and mimosine.

An Analysis of DNA Sequence Polymorphism in the Swamp Buffalo Toll-like Receptor (TLR2) Gene.

Toll-like receptors (TLRs) are transmembrane proteins important for directing immune responses. Their primary role is to recognize pathogens based on single-nucleotide polymorphism (SNP) characteristics. TLR2 is categorized as a pattern recognition receptor (PRR) that is important for the recognition of pathogens. Nucleotide variation in the coding region determines the conformation of the TLR protein, affecting its protein domain efficiency. This study aimed to identify SNPs in the coding region of TLR2 to enhance available genetic tools for improving health and production in swamp buffalo. A total of 50 buffaloes were randomly sampled from the northeastern part of Thailand for genomic DNA extraction and sequencing. Nucleotide sequences were aligned and compared with cattle and river buffalo based on the database. The results showed, there were 29 SNP locations in swamp buffalo and 14 different locations in both cattle and buffaloes. Haplotype analysis revealed that 27 haplotypes occurred. Swamp buffalo were identified from 13 SNPs based on biallelic analysis, which found eight synonymous and five nonsynonymous SNPs. Nucleotide diversity (π) was 0.16, indicating genetic diversity. Genetic diversity (haplotype diversity; HD) was high at 0.99 ± 0.04. This indicates a high probability that the two sample haplotypes are different. The π and HD values are important indicators of the genetic diversity of the swamp buffalo population. In summary, the Thai swamp buffalo population detected a polymorphism of the coding region of the TRL2 gene. Therefore, further, in-depth study of the relationship between these genes in the immune system and disease resistance should be recommended.