Spatio-temporal expression of polyphenol oxidase unveils the dynamics of L-DOPA accumulation in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is a winter season grain legume and a rich source of the anti-parkinson drug, L-3,4-dihydroxyphenylalanine (L-DOPA). The biosynthesis of L-DOPA in plants is not uniform and remains largely unexplored. While the hydroxylase activities of Tyrosine Hydroxylase (TH), the Cytochrome P450 (CYP450) class of enzymes, and Polyphenol Oxidases (PPOs) on tyrosine substrate have been reported in plants, only the roles of PPOs in L-DOPA biosynthesis have been recently established in velvet bean (Mucuna pruriens). To understand the differential accumulation of L-DOPA in different tissues of faba bean, profiling of L-Tyrosine, L-DOPA, Tyramine, and Dopamine in different tissues was performed. Differential accumulation of L-DOPA depended on tissue type and maturity. Furthermore, dopamine biosynthesis through L-DOPA from L-Tyr was confirmed in faba bean. The expression analysis of PPOs in leaf and flower tissues revealed the selective induction of only four (HePPO-2, HePPO-7, HePPO-8b, and HePPO-10) out of ten genes encoding different PPOs mined from the faba bean genome. Higher accumulation of L-DOPA in young leaves and flower buds than in mature leaves and flowers was accompanied by significantly higher expression of HePPO-10 and HePPO-7, respectively. The role of various transcription factors contributing to such metabolite dynamics was also predicted. Further exploration of this mechanism using a multi-omics approach can provide meaningful insight and pave the way for enhancing L-DOPA content in crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01449-2.

Variation in immuno-reproductive milieu of testis in Clarias magur from pre-spawning to spawning phase: An indication towards non-canonical role of immune elements in testes.

Immune mechanisms are major players in ensuring the normal functioning of testicular functions. However, apart from their role in active defence against pathogens, prior studies have also suggested a possibility for reproduction-related (non-immune) functions of certain immune elements. This study employs a comparative transcriptomics approach followed by network analysis for tracking the variation in the immuno-reproductive milieu of Clarias magur testis in spawning versus pre-spawning phase. The results show a significant modulation of both reproduction and immune-relevant genes in spawning versus pre-spawning phase. The functional enrichment of the upregulated reproduction-relevant gene network also shows immune-related biological processes which indicates a probability of involvement of these candidates in spermatogenesis-related events for switching from pre-spawning to spawning phase. The upregulated immune network is highly dense with 40 hubs, 10 cluster sub-networks and 142 functionally enriched pathways in comparison to its downregulated counterpart with only 5 hubs, 1 cluster and 1 enriched pathway. These findings indicate that the synchronisation in modulation of both reproductive and immune-related factors is critical for progression of testicular events guiding the switch from pre-spawning to spawning phase. The reproductive phase-dependent variation in plasma sex steroid levels and the selected genes for quantitative PCR also corroborated this hypothesis. The study also serves as a preliminary screening step for probable immune candidates that may be involved in reproductive functions of testis in addition to defence.

Genetic diversity in mitochondrial DNA D-loop region of indigenous pig breeds of India.

India is home for at least 18 indigenous pig breeds; however, the genetic diversity of Indian pig, Sus scrofa domesticus, population is poorly known. Here, the hypervariable region (HVR) of mitochondrial DNA D-loop (~487 bp) of 214 pigs representing five indigenous and three exotic breeds was sequenced and analysed with reference sequences from other countries. A total of 54 segregating sites among the sequences revealed 56 different haplotypes. Two, 11, eight, seven and six haplotypes were identified with some haplotype sharing in indigenous breeds: Doom, Ghungroo, Mali, Niang-Megha and Tenyi-Vo, respectively. Population pairwise differences (PhiST) (0.409) were found significant (P<0.001), and variance within breeds (59.1%) was more than that of among breeds (40.9%). Similar topology was noted in phylogeny and median-joining network. Indian domestic pigs from this study were found to possess unique and highly differentiated haplotypes on network analysis. The diverse haplotypes and phylogenetic lineages identified here is the first report on Indian pig breeds that need to be further explored by complete mitochondrial DNA sequencing and analysis. These findings provide indicative insights for conservation and optimum utilization of the porcine genetic resources.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

Evaluation of candidate genes related to litter traits in Indian pig breeds.

Improvement in litter traits is the key to profitable pig farming that directly enhances the economic standing of the farmers in developing countries. The present study aimed to explore oestrogen receptor (ESR), epidermal growth factor (EGF), follicle-stimulating hormone beta subunit (FSHß), prolactin receptor (PRLR) and retinol-binding protein 4 (RBP4) genes as possible candidate genetic markers for litter traits in indigenous pigs of India. The breeds included in the study were Ghungroo, Mali, Niang Megha and Tenyi Vo, and the reproductive traits considered were litter size at birth (LSB), number born alive (NBA), litter weight at birth (LWB), litter size at weaning (LSW) and litter weight at weaning (LWW) at their first parity. PCR-RFLP and primer-based mutation detection methods were used to identify polymorphism, and associations between the genotypes and the traits were analysed using a general linear model. The Ghungroo pigs recorded the best litter performances among the breeds (p < .05, LWB p < .01). Different alleles and genotypes of the genes under study were detected. Short interspersed nuclear element (SINE) -/- genotype of FSHß revealed significantly higher litter traits (p < .05, LSB p < .01). The LWW was also found to be significantly influenced by ESR BB and AB, EGF AB and BB, and PRLR CC genotypes (p < .05). Although we did not find statistically significant and consistently superior litter traits with respect to different genotypes of other studied genes than genotype SINE -/- of the FSHß, PRLR CC genotype demonstrated superior performances for all the litter traits. Our study revealed the FSHß as a potential candidate genetic marker for litter traits in indigenous pig breeds of India.

Dietary supplementation of n-3 polyunsaturated fatty acid alters endometrial expression of genes involved in prostaglandin biosynthetic pathway in breeding sows (Sus scrofa).

The present investigation was designed to study the effect of dietary supplementation of omega-3 (n-3) PUFA on endometrial expression of fertility-related genes in breeding sows. Sixteen crossbred sows were randomized to receive diets containing 4% (wt/wt) flaxseed oil as n-3 PUFA source (TRT group) or iso-nitrogenous, iso-caloric standard control diet (CON group), starting from the first day of estrus up to 40 days and were artificially bred on the second estrus. Endometrial samples were collected during days 10-11 and 15-16 post-mating for studying relative expression profile of candidate genes viz. Prostaglandin F Synthase (PGFS), microsomal Prostaglandin E Synthase-1 (mPGES-1) and Carbonyl Reductase-1 (CBR-1) using quantitative Real-Time PCR. Expression level of mPGES-1 gene transcript was 2.1-fold higher (P < 0.05) during 10-11 days of pregnancy and 1.4-fold higher (P > 0.05) during 15-16 days of pregnancy in TRT group as compared to CON group. Relative expression of PGFS gene transcript was significantly lower (P < 0.05) during 10-11 days of pregnancy in TRT group while there was no significant effect (P > 0.05) of dietary supplementation during 15-16 days of pregnancy. Endometrial mRNA level of CBR1 was significantly lower (P < 0.05) with 3.93-fold decrease in TRT group during 10-11 days of pregnancy whereas 2.82-fold reduction in expression (P > 0.05) was observed subsequently during 15-16 days of pregnancy as compared to CON group. Collectively, these results indicate that dietary n-3 PUFA supplementation can modulate gene expression of key enzymes in prostaglandin biosynthetic pathway during early gestation, which in turn might have beneficial impact on overall reproductive response in breeding sows. These findings partly support strategic dietary supplementation of plant-based source of n-3 PUFA with an aim to improve overall reproductive performance in sows.

Steroid and metabolic hormonal profile of porcine serum vis-à-vis ovarian follicular fluid.

AIM: This study was conducted to understand whether serum level of the steroid and metabolic hormones may be indicative of their level in ovarian follicular fluid (FF) in porcine, and its influence on fertility. MATERIALS AND METHODS: Ovaries from pigs (n=32) of two genetic groups, namely, native (Ghungroo; n=16) and crossbred (Hampshire × Ghungroo; n=16) were collected. Both the genetic groups comprised gilts (n=8) and sows (n=8), and sows were in luteal phase of estrus cycle. FF was aspirated from small, medium and large follicles, and centrifuged for the collection of supernatant for further analysis. Blood samples were collected from the same animals, and serum was separated. Hormones, namely, cortisol, T3, T4 and testosterone were estimated by radioimmunoassay. Two-way ANOVA was used for analysis of data considering genetic background (native or crossbred), stage of reproductive life (gilt or sow), and source of sample (serum or FF) as fixed effects. RESULTS: It was observed that all the hormones except cortisol differed significantly (p<0.01) based on genetic background. Stage of reproductive life and source of sample did not affect the studied hormonal level. Within the genetic groups, stage of reproductive life influenced T3 (p<0.01), cortisol (p<0.05) and testosterone (p<0.01) level in crossbred pigs as compared to T3 (p<0.01) only in native pigs. The level of T3 in serum, as well as FF, was higher (p<0.01) in Ghungroo gilts compared to sows. However, a reverse of this was observed in the case of crossbred pigs. The level of cortisol (p<0.05) and testosterone (p<0.01) was higher in crossbred sows than gilts in both serum and FF. CONCLUSION: The study revealed that serum level of the steroid and metabolic hormones is indicative of their level in the ovarian FF. Further, varying level of steroid and metabolic hormones in pigs based on genetic background may be due to variation in body size, rate of energy metabolism and stage of (re)productive life.

Microsatellite and Mitochondrial Diversity Analysis of Native Pigs of Indo-Burma Biodiversity Hotspot.

Assessment of genetic diversity in indigenous animals is an important and essential task for animal genetic improvement studies as well as conservation decision-making. The genetic diversity and evolutionary relationships among geographically and phenotypically distinct three pig breeds/types native to Indo-Burma and Eastern Himalayan global biodiversity hotspots were determined by genotyping with a panel of 22 ISAG recommended microsatellite loci as well as sequencing partial MTRNR1gene. The mean number of alleles per locus, effective number of alleles and observed heterozygosity were found to be 11.27 ± 0.85, 5.29 ± 0.34, and 0.795 ± 0.01, respectively. The moderate FST value (0.115 ± 0.01) indicated a fair degree of genetic differentiation among the native breeds. The Nei's unbiased genetic identity estimates indicated less genetic distance (0.2909) between Niang Megha and Tenyi Vo pigs than the both individually with Ghoongroo breed. The divergence time was also estimated from the microsatellite analysis. Analysis of MTRNR1gene revealed distinct clustering of native Indian pigs with Chinese pigs over European pigs. The study revealed the abundance of genetic variation within native Indian pigs and their relationships as well as genetic distances.

A comparative study of SINE insertion together with a mutation in the first intron of follicle stimulating hormone beta gene in indigenous pigs of India.

The Follicle stimulating hormone beta polypeptide (FSHß) gene has been investigated as a candidate gene for litter size because of its role in maturation of small and medium follicles into ovulating large follicles. Genes coding follicle stimulating hormone have been found to have significant effects on litter size in pigs. The aim of the study was to investigate the polymorphism of the first intron of FSH-ß gene present in Ghungroo, Niang Megha and Nagaland local pigs of India. PCR based short interspersed nuclear elements (SINE) detection method and PCR-RFLP were carried out to identify the polymorphism in FSHß gene. Significantly higher frequencies of SINE-/- and SINE+/- genotypes were observed in Ghungroo pigs as compared to Niang Megha and Nagaland local pigs. PCR-RFLP study also showed higher frequency of T allele in all the three breeds even though there existed difference in genotypes among breeds. Association studies clearly demonstrated that SINE-/- genotypes have significantly higher litter size and weight at birth as well as at weaning. SINE -/- genotypes have higher number of live births too. Moderately high D' value indicates that haplotypic pattern within the two loci can be utilized for marker assisted selection for litter traits in pigs.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis).

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the ß(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Identification of novel allelic variants of integrin beta 2 (ITGB2) gene and screening for Bubaline leukocyte adhesion deficiency syndrome in Indian water buffaloes (Bubalus bubalis).

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).