Comparative analysis of rumen metagenome, metatranscriptome, fermentation and methane yield in cattle and buffaloes fed on the same diet.

A study to compare the rumen microbial community composition, functional potential of the microbiota, methane (CH4) yield, and rumen fermentation was conducted in adult male cattle and buffaloes fed on the same diet. A total of 41 phyla, 169 orders, 374 families, and 1,376 microbial genera were identified in the study. Bacteroidetes and Firmicutes were the two most dominant bacterial phyla in both cattle and buffaloes. However, there was no difference in the abundance of Bacteroidetes and Firmicutes in the rumen metagenome of cattle and buffaloes. Based on the abundance, the Proteobacteria was the 3rd largest phylum in the metagenome, constituting 18-20% in both host species. Euryarchaeota was the most abundant phylum of the methanogens, whereas Methanobacteriales and Methanobrevibacter were the most abundant orders and genera in both species. The methanogen abundances were not different between the two host species. Like the metagenome, the difference between the compositional and functional abundances (metagenome vs. metatranscriptome) of the Bacteroidetes and Firmicutes was not significant, whereas the proteobacteria were functionally less active than their metagenomic composition. Contrary to the metagenome, the Euryarchaeota was the 3rd most functional phylum in the rumen and constituted ~15% of the metatranscriptome. Methanobacteriales were the most functional methanogens, accounting for more than 2/3rd of the total archaeal functionality. These results indicated that the methanogens from Euryarchaeota were functionally more active as compared to their compositional abundance. The CH4 yield (g/kg DMI), CH4 emission (g/kg DDM), dry matter (DM) intake, and rumen fermentation did not vary between the two host species. Overall, the study established a substantial difference between the compositional abundances and metabolic functionality of the rumen microbiota; however, feeding cattle and buffaloes on the same diet resulted in similar microbiota composition, metabolic functionality, and CH4 yield. Further studies are warranted to investigate the effect of different diets and environments on the composition and metabolic functionality of the rumen microbiota.

Interleukin-6 stimulates in vitro development of late-stage ovine embryos.

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72-192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0-192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8-16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ±â€¯1.52%) compared to the control (21.1 ±â€¯1.11%), and 5 ng/ml (25.9 ±â€¯2.98%) or 25 ng/ml (16.5 ±â€¯0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ±â€¯1.57%) and blastocyst formation (14.5 ±â€¯1.28%) compared to the control group (65.8 ±â€¯1.35% and 21.5 ±â€¯0.97%, respectively). In 8-16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

Comparative Rumen Metagenome and CAZyme Profiles in Cattle and Buffaloes: Implications for Methane Yield and Rumen Fermentation on a Common Diet.

A study was undertaken to compare the rumen microbial community composition, methane yield, rumen fermentation, and CAZyme profiles between cattle and buffaloes. The primary aim of this study was to ascertain the impact of the host species on the above when diet and environmental factors are fixed. A total of 43 phyla, 200 orders, 458 families, and 1722 microbial genera were identified in the study. Bacteroidetes was the most prominent bacterial phylum and constituted >1/3rd of the ruminal microbiota; however, their abundances were comparable between cattle and buffaloes. Firmicutes were the second most abundant bacteria, found to be negatively correlated with the Bacteroidetes. The abundances of Firmicutes as well as the F/B ratio were not different between the two host species. In this study, archaea affiliated with the nine phyla were identified, with Euryarchaeota being the most prominent. Like bacterial phyla, the abundances of Euryarchaeota methanogens were also similar between the cattle and buffaloes. At the order level, Methanobacteriales dominated the archaea. Methanogens from the Methanosarcinales, Methanococcales, Methanomicrobiales, and Methanomassiliicoccales groups were also identified, but at a lower frequency. Methanobrevibacter was the most prevalent genus of methanogens, accounting for approximately three percent of the rumen metagenome. However, their distribution was not different between the two host species. CAZymes affiliated with five classes, namely CBM, CE, GH, GT, and PL, were identified in the metagenome, where the GH class was the most abundant and constituted ~70% of the total CAZymes. The protozoal numbers, including Entodiniomorphs and Holotrichs, were also comparable between the cattle and buffaloes. Results from the study did not reveal any significant difference in feed intake, nutrient digestibility, and rumen fermentation between cattle and buffaloes fed on the same diet. As methane yield due to the similar diet composition, feed ingredients, rumen fermentation, and microbiota composition did not vary, these results indicate that the microbiota community structure and methane emissions are under the direct influence of the diet and environment, and the host species may play only a minor role until the productivity does not vary. More studies are warranted to investigate the effect of different diets and environments on microbiota composition and methane yield. Further, the impact of variable productivity on both the cattle and buffaloes when the diet and environmental factors are fixed needs to be ascertained.

Effect of an anti-methanogenic supplement on enteric methane emission, fermentation, and whole rumen metagenome in sheep.

A study was conducted to investigate the impact of an anti-methanogenic product supplementation on enteric methane emissions, whole rumen metagenome and ruminal fermentation in sheep. Twelve adult male sheep were randomly divided into two groups of six animals each. Animals were fed ad libitum on a total mixed ration either without (CON) or with an anti-methanogenic supplement (Harit Dhara-HD). The anti-methanogenic supplement contained 22.1% tannic acid in a 3: 1 ratio of condensed and hydrolysable tannins. The supplementation of product revealed a significant reduction in daily enteric methane emission (21.9 vs. 17.2 g/d) and methane yield (23.2 vs. 18.2) without affecting the nutrient intake and digestibility. However, the propionate concentration in the HD treatment group was significantly higher than in the CON group. On the contrary, the ammonia nitrogen concentration was lower. The anti-methanogenic supplement significantly decreased the ruminal protozoa in the HD treatment group. Whole rumen metagenome analysis revealed that the core bacterial (Bacteroidetes and Firmicutes) and archaeal communities (Methanobrevibacter and Methanosarcina) were comparable between the CON and HD treatment groups. However, the supplementation of anti-methanogenic product led to a considerable reduction in the abundance of Proteobacteria, whereas the abundance of Lentisphaerae was greater. The supplementation significantly decreased the abundance of Methanocaldococcus, Methanococcoides, Methanocella, and Methanoregula methanogens. A total of 36 KO related to methanogenesis were identified in this study. The activities of formate dehydrogenase (EC 1.8.98.6) and tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86) were significantly lowered by the anti-methanogenic product supplementation in sheep. In conclusion, the anti-methanogenic supplement has the potential to decrease enteric methane emission (~22%) at the recommended level (5% of DM) of supplementation. The contribution of minor methanogens vulnerable to supplementation to rumen methanogenesis is not known; hence, the culturing of these archaea should be taken on priority for determining the impact on overall rumen methanogenesis.

Effect of Long-Term Supplementation With Silkworm Pupae Oil on the Methane Yield, Ruminal Protozoa, and Archaea Community in Sheep.

Supplementation with lipids and oils is one of the most efficient strategies for reducing enteric methane emission. However, high costs and adverse impacts on fiber degradation restrict the use of conventional oils. Silkworm pupae, a non-conventional oil source rarely used for human consumption in India, could be one of the cheaper alternatives for methane mitigation. The objective of this study was to investigate the effect on sheep of long-term supplementation (180 days) of silkworm pupae oil (SWPO) with two distinct supplementation regimes (daily and biweekly) on daily enteric methane emission, methane yield, nutrient digestibility, rumen fermentation, ruminal archaea community composition, and protozoal population. The effect of the discontinuation of oil supplementation on enteric methane emission was also investigated. Eighteen adult male sheep, randomly divided into three groups (n = 6), were provisioned with a mixed diet consisting of 10.1% crude protein (CP) and 11.7 MJ/kg metabolizable energy formulated using finger millet straw and concentrate in a 55:45 ratio. SWPO was supplemented at 2% of dry matter intake (DMI) in test groups either daily (CON) or biweekly (INT), while no oil was supplemented in the control group (CTR). DMI (p = 0.15) and CP (p = 0.16) in the CON and INT groups were similar to that of the CTR group; however, the energy intake (MJ/kg) in the supplemented groups (CON and INT) was higher (p < 0.001) than in CTR. In the CON group, body weight gain (kg, p = 0.02) and average daily gain (g, p = 0.02) were both higher than in the CTR. The daily methane emission in the CON (17.5 g/day) and INT (18.0 g/day) groups was lower (p = 0.01) than the CTR group (23.6 g/day), indicating a reduction of 23-25% due to SWPO supplementation. Similarly, compared with the CTR group, methane yields (g/kg DMI) in test groups were also significantly lower (p < 0.01). The transient nature of the anti-methanogenic effect of SWPO was demonstrated in the oil discontinuation study, where daily methane emission reverted to pre-supplementation levels after a short period. The recorded methanogens were affiliated to the families Methanobacteriaceae, Methanomassilliicoccaceae, and Methanosarcinaceae. The long-term supplementation of oil did not induce any significant change in the rumen archaeal community, whereas minor species such as Group3b exhibited differing abundance among the groups. Methanobrevibacter, irrespective of treatment, was the largest genus, while Methanobrevibacter gottschalkii was the dominant species. Oil supplementation in CON and INT compared with CTR decreased (p < 0.01) the numbers of total protozoa (× 107 cells/ml), Entodiniomorphs (× 107 cells/ml), and Holotrichs (× 106 cells/ml). SWPO continuous supplementation (CON group) resulted in the largest reduction in enteric methane emission and relatively higher body weight gain (p = 0.02) in sheep.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility.

Deciphering sperm transcriptome is the key to understanding the molecular mechanisms governing peri-fertilization, embryonic development, and pregnancy establishment. This study aimed to profile sperm transcriptome to identify signature transcripts regulating male fertility. Semen samples were collected from 47 bulls with varied fertility rates. The sperm total RNA was isolated (n = 8) and subjected to transcriptome sequencing. Based on the expression pattern obtained from RNA profiling, the bulls were grouped (p = 0.03) into high-fertile and sub-fertile, and signature transcripts controlling sperm functions and fertility were identified. The results were validated using the OMIM database, qPCR, and sperm function tests. The sperm contains 1100 to 1700 intact transcripts, of which BCL2L11 and CAPZA3 were abundant and associated (p < 0.05) with spermatogenesis and post-embryonic organ morphogenesis. The upregulated genes in the acrosome integrity and functional membrane integrity groups had a close association with the fertility rate. The biological functions of these upregulated genes (p < 0.05) in the high-fertile bulls were associated with spermatogenesis (AFF4 and BRIP1), sperm motility (AK6 and ATP6V1G3), capacitation and zona binding (AGFG1), embryo development (TCF7 and AKIRIN2), and placental development (KRT19). The transcripts involved in pathways regulating embryonic development such as translation (EEF1B2 and MTIF3, p = 8.87E-05) and nonsense-mediated decay (RPL23 and RPL7A, p = 5.01E-27) were upregulated in high-fertile bulls. The identified transcripts may significantly impact oocyte function, embryogenesis, trophectoderm development, and pregnancy establishment. In addition, the study also reveals that the genes governing sperm functional membrane integrity and acrosome integrity have a prospective effect on male fertility.

IGF-1 treatment during in vitro maturation improves developmental potential of ovine oocytes through the regulation of PI3K/Akt and apoptosis signaling.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4-8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3ß, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos.

Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined treatment with IL-7 (5 ng/ml) and Tempol (10 and 25 mM) were comparable with that of the control. The proportion of NBT stained area did not differ significantly between the 8-cell embryos and morula, but was found significantly greater in the degenerating embryos. In conclusion, the developed NBT staining method was found effective for detecting and interpreting the level of intracellular ROS in oocytes, cumulus cells and embryos. This method can be used as an alternative to the DCFH-DA staining method.

In vitro evaluation of graded level of Silkworm pupae (Bombyx mori) oil on methane production, fermentation characteristics, and protozoal populations.

AIM: The present study was undertaken to evaluate the effect of variable levels of silkworm pupae oil and roughage: concentrate ratio on in vitro methane production, fermentation characteristics, and rumen protozoa population. MATERIALS AND METHODS: In vitro gas production study (24 h) was performed with graded levels of silkworm pupae oil, namely, 0.5, 1, 2, 4, and 5% of the basal diet and four variable dietary regimes consisting roughage and concentrate in different proportions (70:30, 60:40, 50:50, and 40:60). At the end of incubation, gas samples were analyzed for methane, while fermented rumen liquor was used for protozoa enumeration. A separate set of incubations was carried out for the determination of in vitro dry matter digestibility. RESULTS: Results from the in vitro studies revealed no adverse impact of the silkworm pupae oil supplementation up to 2% level on total gas production. However, supplementation beyond 2% has shown a reduction in total gas production. Incubation with variable levels (0.5-5%) of silkworm pupae oil with different dietary regimes indicated negligible (3-5%) to a substantial reduction (25-30%) on methane production. A graded decrement in methane production was recorded with increasing levels of silkworm pupae oil. Similarly, the protozoal populations were decreased from 10 to 51.5% with graded levels of silkworm pupae oil in different dietary regimes as studies did not reveal any significant (p>0.05) variation between 2 and 4% of oil supplementation. CONCLUSION: The silkworm pupae oil supplementation at 2% level decreases methane production by 12-15% without any adverse impact on feed fermentation. Oil supplementation may have a more pronounced effect on methane reduction if added to high roughage diet at in vitro conditions. However, in vivo, studies in ruminants are warranted to confirm the methane reduction with silkworm pupae oil supplementation.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

Production of Short Chain Fructo-oligosaccharides from Inulin of Chicory Root Using Fungal Endoinulinase.

Short chain fructo-oligosaccharides (SC-FOS) are the potential prebiotics possessing diverse applications in both food and feed industries. The present study was aimed to extract inulin from chicory roots followed by its conversion into SC-FOS applying endoinulinase from Aspergillus fumigatus. The inulin was extracted from chicory roots through boiling in hot water, followed by precipitation with ethanol at room temperature or freezing condition. Maximum yield (42%) of inulin was obtained with three volumes of chilled absolute ethanol at room temperature. HPLC analysis of enzymatic hydrolysate detected kestose (GF2), nystose (GF3), and other FOS having higher degree of polymerization (DP). Maximum GF2 (5.79 mg/ml) was detected at temperature 50 °C, pH 5.5 with 2 U of enzyme dose after 6 h of hydrolysis; while maximum GF3 (4.33 mg/ml) was recorded at 60 °C, 5.5 pH with 0.5 U enzyme dose after 2 h of hydrolysis. Nevertheless, complete hydrolysis of inulin was noticed with 99% total oligosaccharide yield at 55 °C, 5.5 pH with 0.5 U enzyme dose after 4 h of hydrolysis with negligible amount of mono- and di-saccharides. The present finding demonstrated the process for higher yield of inulin from chicory roots followed by its conversion into SC-FOS applying fungal endoinulinase.

Interleukin-7 improves in vitro maturation of ovine cumulus-oocyte complexes in a dose dependent manner.

Interleukin-7 (IL-7) mediated signals are linked to development, proliferation, survival and differentiation of cells. Recent evidences indicate its role in oocyte maturation process as well. Nevertheless, the underlying mechanisms of IL-7 involvement in oocyte maturation are not well characterized. In addition, currently no information is available on the effect of exogenous IL-7 on oocyte maturation in ovine or any other species. In this study, the effect of IL-7 supplementation during in vitro maturation (IVM) on the maturation rate, production of reactive oxygen species (ROS) and gene expression of ovine cumulus-oocyte complexes (COC) was assessed. IL-7 (0.5, 1, 2, 5 and 10 ng/ml) was supplemented in IVM medium at the beginning (0 h) and maturation rate of COC was assessed at the completion of IVM (24 h). The maturation rate (%) was found significantly (P = 0.000) greater with the 1 ng/ml of IL-7 supplementation (69.5) than control (60.0). In contrast, the maturation rate was reduced significantly (P = 0.000) with the 2 (47.1), 5 (39.2) and 10 ng/ml (39.1) of IL-7 as compared to the control. The level of intracellular ROS in the matured COC was found considerably higher with the 5 ng/ml of IL-7 followed by 1 ng/ml of IL-7 and control. It was evident that in the presence of superoxide dismutase-inhibitor, 1 ng/ml of IL-7 did not stimulate oocyte maturation. In contrast, oocyte maturation was improved with 5 ng/ml of IL-7 supplementation in the presence of NADPH-oxidase-inhibitor. IL-7 supplementation influenced gene expression in COC in a dose and time dependant manner. The expression of genes related to ROS production and apoptosis were upregulated and the genes associated with antioxidant mechanisms were downregulated noticeably with the supplementation of 5 ng/ml of IL-7. In conclusion, IL-7 at low concentration was beneficial for oocyte maturation, which was likely mediated through the favourable level of intracellular ROS and antioxidant mechanisms. In contrast, the detrimental effects of greater IL-7 concentrations on oocyte maturation were possibly arbitrated through the ROS-mediated oxidative stress, compromised antioxidant mechanism and stimulated apoptotic signalling.

Evaluation of in vitro ruminal fermentation of ensiled fruit byproducts and their potential for feed use.

OBJECTIVE: Ensiling of tannin-rich fruit byproducts (FB) involves quantitative and qualitative changes in the tannins, which would consequently change the rumen fermentation characteristics. This study aimed to evaluate whether ensiled FBs are effective in mitigating methane emission from ruminants by conducting in vitro assessments. METHODS: Fruit byproducts (grape pomace, wild grape pomace, and persimmon skin) were collected and subjected to four-week ensiling by Lactobacillus buchneri inoculant. A defined feed component with or without FB samples (both fresh and ensiled material) were subjected to in vitro anaerobic culturing using rumen fluid sampled from beef cattle, and the fermentation parameters and microbial populations were monitored. RESULTS: Reduced methane production and a proportional change in total volatile fatty acids (especially enhanced propionate proportion) was noted in bottles containing the FBs compared with that in the control (without FB). In addition, we found lower gene copy number of archaeal 16S rRNA and considerably higher levels of one of the major fibrolytic bacteria (Fibrobacter succinogenes) in the bottles containing FBs than in the control, particularly, when it was included in a forage-based feed. However, in the following cultivation experiment, we observed that FBs failed to exhibit a significant difference in methane production with or without polyethylene glycol, implying that tannins in the FBs may not be responsible for the mitigation of methane generation. CONCLUSION: The results of the in vitro cultivation experiments indicated that not only the composition but also ensiling of FBs affected rumen fermentation patterns and the degree of methane generation. This is primarily because of the compositional changes in the fibrous fraction during ensiling as well as the presence of readily fermented substrates, whereas tannins in these FBs seemed to have little effect on the ruminal fermentation kinetics.

Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications.

Nutraceuticals are gaining importance owing to their potential applications in numerous sectors including food and feed industries. Among the emerging nutraceuticals, d-tagatose occupies a significant niche because of its low calorific value, antidiabetic property and growth promoting effects on beneficial gut bacteria. As d-tagatose is present in minute quantities in naturally occurring food substances, it is produced mainly by chemical or biological means. Recently, attempts were made for bio-production of d-tagatose using l-arabinose isomerase enzyme to overcome the challenges of chemical process of production. Applications of d-tagatose for maintaining health and wellbeing are increasing due to growing consumer awareness and apprehension against modern therapeutic agents. This review outlines the current status on d-tagatose, particularly its production, properties, biological role, applications, and the future perspectives.

Methane mitigation potential of phyto-sources from Northeast India and their effect on rumen fermentation characteristics and protozoa in vitro.

AIM: The aim of the study was to explore the anti-methanogenic potential of phyto-sources from Northeast region of the country and assess the effect on rumen fermentation characteristics and protozoa for their likely inclusion in animal diet to reduce methane emission. MATERIALS AND METHODS: Twenty phyto-sources were collected from Northeast state, Assam, during March to April 2014. Phyto-sources were analyzed for their tannin content followed by screening for methane mitigation potential using in vitro system. The effect of tannin on methane production and other fermentation parameters was confirmed by attenuating the effect of tannin with polyethylene glycol (PEG)-6000 addition. About 200 mg dried phyto-source samples were incubated for 24 h in vitro, and volume of gas produced was recorded. The gas sample was analyzed on gas chromatograph for the proportion of methane in the sample. The effect of phyto-sources on rumen fermentation characteristics and protozoal population was determined using standard methodologies. RESULTS: Results from studies demonstrated that Litchi chinensis, Melastoma malabathricum, Lagerstroemia speciosa, Terminalia chebula, and Syzygium cumini produced comparatively less methane, while Christella parasitica, Leucas linifolia, Citrus grandis, and Aquilaria malaccensis produced relatively more methane during in vitro incubation. An increase (p<0.05) in gas and methane production from the phyto-sources was observed when incubated with PEG-6000. Entodinimorphs were prominent ciliates irrespective of the phyto-sources, while holotrichs represented only small fraction of protozoa. An increase (p<0.05) in total protozoa, entodinimorphs, and holotrichs was noted when PEG-6000 added to the basal substrate. Our study confirmed variable impact of phyto-sources on total volatile fatty acid production and ammonia-N. CONCLUSION: It may be concluded that L. chinensis, M. malabathricum, L. speciosa, S. cumini, and T. chebula are having potent methane suppressing properties as observed in vitro in 24 h. These leaves could be supplemented in the animal diet for reducing methane emission; however, in vivo trials are warranted to confirm the methane inhibitory action and optimize the level of supplementation.

In Silico evaluation and identification of fungi capable of producing endo-inulinase enzyme.

The enzyme endo-inulinase hydrolyzes inulin to short chain fructooligosaccharides (FOS) that are potential prebiotics with many health promoting benefits. Although the raw materials for inulin production are inexpensive and readily available, commercial production of FOS from inulin is limited due to inadequate availability of the enzyme source. This study aimed to identify the fungi capable of producing endo-inulinase based on the in silico analysis of proteins retrieved from non-redundant protein sequence database. The endo-inulinase of Aspergillus ficuum was used as reference sequence. The amino acid sequences with >90% sequence coverage, belonging to different fungi were retrieved from the database and used for constructing three-dimensional (3D) protein models using SWISS-MODEL and Bagheerath H. The 3D models of comparable quality as that of the reference endo-inulinase were selected based on QMEAN Z score. The selected models were evaluated and validated for different structural and functional qualities using Pro-Q, ProSA, PSN-QA, VERIFY-3D, PROCHECK, PROTSAV metaserver, STRAP, molecular docking, and molecular dynamic simulation analyses. A total of 230 proteins belonging to 53 fungal species exhibited sequence coverage >90%. Sixty one protein sequences with >60% sequence identity were modeled as endo-inulinase with higher QMEAN Z Score. The evaluations and validations of these 61 selected models for different structural and functional qualities revealed that 60 models belonging to 22 fungal species exhibited native like structure and unique motifs and residues as that of the reference endo-inulinase. Further, these models also exhibited similar kind of interaction between the active site around the conserved glutamate residue and substrate as that of the reference endo-inulinase. In conclusion, based on the current study, 22 fungal species could be identified as endo-inulinase producer. Nevertheless, further biological assessment of their capability for producing endo-inulinase is imminent if they are to be used for commercial endo-inulinase production for application in FOS industry.

Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase.

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL-1 after 5 days of growth at 30°C (0.019 g·mL-1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

Current status of sperm functional genomics and its diagnostic potential of fertility in bovine (Bos taurus).

With artificial insemination (AI) and other precision dependent assisted reproductive technologies (ART) being followed in large scale in human and animal reproduction, assessing semen quality and fertilizability is under continuous scrutiny. Various tests have been developed to predict semen quality, but so far no single, highly reliable test is available. In this regard, transcriptomic profiling of spermatozoa assumes significance as it carries the information about spermatogenesis, sperm function, and paternal roles in post-fertilization events. Human spermatozoal transcriptome profiling has been carried out on a large number of individuals to predict the semen quality. A study in human indicated that the outcome of some idiopathic couples seeking reproductive care could be helped using transcriptomic profiling of spermatozoa. Such studies have a direct impact on the bovine dairy industry, wherein AI is practiced. Limited studies in bovine spermatozoal transcriptome profiling have revealed that the spermatozoa contain various classes of RNA, like in human. Approximately 13,000 bovine genes yield a series of spermatozoal transcripts, of which most are fragmented in nature. Their abundance is indicative of the timing of events associated with spermatogenesis, e.g., PRM1, IGF1, BMP2; sperm function, TSSK6, CRISP, HSFY2; fertility, UBE2D3, Integrin-ß, LDC-1; and embryonic development, miR34c-5p, BCL2L11, BRCA1. The most abundant translated bovine transcripts are BSP3 and SPATA18, and are involved in regulation of germ cell development and the maintenance of chromatin integrity during spermatogenesis respectively. The presence of transcripts associated with placental development, e.g., placental associated glycoproteins (PAGs) have suggested their possible influence beyond early embryonic development. Changes in transcript levels like RPL31 and PRKCE that increase, and PRM1 that decreases, during cryopreservation need to be defined in order to optimize cryopreservation and fertility yield. Spermatozoal transcriptome profiling with validation studies are warranted in large numbers of animals to elucidate their significance for selecting fertile bulls for the breeding program. Abbreviations: AI: artificial insemination; BSE: breeding soundness evaluation; cfs-mRNA: cell-free seminal mRNA; piRNA: PIWI-interacting RNA; tRNA: transfer RNA; fg: femtogram; TPM: transcripts per million reads; RPKM: reads per kilobase million; rRNA: ribosomal RNA; mt-RNA: mitochondrial RNA; lncRNA: long non-coding RNA; sncRNA: small noncoding RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; miRNA: microRNA; snaR: small NF90-associated RNAs; SINES: short interspersed nuclear elements; LINES: long interspersed nuclear elements; MER: medium reiterated sequence; F1 offspring: filial 1 offspring; PAGs: placental associated glycoproteins; TCP: Transcription factor T complex protein; BSP3: bovine seminal plasma protein 3; SCNT: somatic cell nuclear transfer; qPCR: quantitative (real-time) polymerase chain reaction; SSH: suppression subtractive hybridization; SNP: single nucleotide polymorphism; 2-DE: 2 dimensional gel electrophoresis; LC-MS/MS: liquid chromatography-tandem mass spectrometry.

Temporal expression of cumulus cell marker genes during in vitro maturation and oocyte developmental competence.

PURPOSE: Cumulus cells (CC) play important roles in oocyte development and cumulus expressed genes can be used as markers for oocyte quality. This study aimed to investigate temporal changes in the expression of cumulus marker genes during oocyte maturation as possible biomarkers of embryo developmental competence in ovine. METHODS: Gene expression was assessed in the CC of the BCB+ (developmentally competent) and BCB- (developmentally poor) oocytes at 0, 12, and 24 h of in vitro maturation (IVM). Further, the association between the temporal cumulus gene expression and in vitro oocyte and embryo development was assessed. RESULTS: The maturation and blastocyst formation rates were found significantly greater for the BCB+ than the BCB- oocytes. At the 0 h of IVM, a significant upregulation in the expression of PTGS2, STAR, SDC2, LHR, FGF2, BCL2, IL7RA, HSPA1A, and IFNT was observed in the CC of the poor (BCB-) as compared to the competent (BCB+) oocytes. In contrast, it was observed that as maturation progressed, the cumulus expression of most of the favorable genes was reduced and was found significantly downregulated at the completion of IVM in the poor as compared to the competent oocytes. CONCLUSIONS: The study revealed noticeable differences in the cumulus gene expression profile at different stages of IVM between ovine oocytes of differential developmental ability. The results indicated that the loss of cumulus gene expression along the maturation period in the poor oocytes was related to their intrinsic poor quality in the ovarian follicle.