Association of polymorphism in the promotor area of the caprine BMPR1B gene with litter size and body measurement traits in Damani goats.

This study aimed to explore polymorphisms in the promoter region of the caprine BMPR1B (Bone morphogenetic protein receptor 1 beta) gene and its association with body measurement and litter size traits in Damani does. A total of 53 blood samples were collected to analyze the association between the BMPR1B gene polymorphism and 11 phenotypic traits in Damani female goats. The results revealed that three novel SNPs were identified in the promoter region of the caprine BMPR1B gene, including g.67 A > C (SNP1), g.170 G > A(SNP2), and g.501A > T (SNP3), among which the SNP1 and SNP2 were significantly (p < 0.05) associated with litter size and body measurement traits in Damani goats. In SNP1 the AC genotype could be used as a marker for litter size, and the CC genotype for body weight in Damani goats. In SNP2, the genotype GG was significantly (p < 0.05) associated with ear and head length. Therefore, we can conclude from the present study, that genetic variants AC and CC of the caprine BMPR1B gene could be used as genetic markers for economic traits through marker-assisted selection for the breed improvement program of the Damani goat.

Morpometric and molecular characterization of Surguli goat through CO1 gene in district Kohat.

The present study was designed with the aim to study morphometric characterization as well as phylogeny and diversity of the local Surguli goat at their breeding tract district Kohat through mitochondrial DNA region, i.e., Cytochrome C Oxidase Subunit One (CO1) gene. Morphometric data and blood samples were collected from thirty (30) pure goats. Morphometric analysis showed that sex had significant effect (p < 0.05) on body weight, body length, hearth girth and horn length while no significant effect (p > 0.05) was observed for other characteristics. The results also indicated that age had significant effect (p < 0.05) on height at rump, ear length, horn length and tail length while no significant effect (p > 0.05) was observed for other characteristics. The phylogenetic analysis through CO1 nucleotide sequences within nucleotide range 1-767 showed nine polymorphic sites segregating into eight haplotypes. The mean intraspecific diversity and mean interspecific diversity were calculated as 0.23 and 2.36%, respectively. Phylogenetic tree revealed that Capra Ibex and native Surguli goat have common ancestors. The morphometric and molecular results obtained from the present study can be exploited as a selection tool for breeding and overall improvement.

Polymorphism and association study of lactoferrin (LF) gene with milk yield, milk composition, and somatic cell count in Beetal goats.

The sequence analysis of PCR product exhibited four novel SNPs in the promoter region of the LF gene at loci g.98T>C, g.143T>A, g.189AC>A, and g.346A>G. Each SNP yielded three genotypes; the genotypes TT (SNP1), AA (SNP3), and GG (SNP4) decreased SCC and increase milk quality traits such as density, protein, and milk yield (P < 0.01). The genotype CC (SNP2) and CA (SNP4) significantly (P < 0.01) decreased the milk quality parameters, while genotypes TC (SNP2) and GG (SNP4) showed significantly (P < 0.01) less SCC and increase lactose % in milk. Furthermore, screening of the LF promoter sequence explored the gain of four TF binding sites at locus g.98T˃C and three TF binding sites at g.346A˃G. However, the loss of four and two TF binding sites was seen at locus g.143T˃A and g.189C˃A, respectively. We can conclude from the present study that the GG, TT, and AA genotype might be utilized as genetic markers in marker-assisted selection for the breed improvement program of Beetal goats.

The efficiency of boric acid as an alternate to NaCl for the preservation of cattle hides.

The fresh buffalo hides (n = 6) were cut into two equal parts and categorized into three equal groups. The first group was treated with 50% NaCl; the second group hides were treated with 5% of boric acid (BA), and the third group hides were with NaCl + BA (10:1). Hair loss was seen at the sample margins of hides treated with 50% NaCl, with a slight odor. In the second group, there was neither hair loss nor a pungent smell was felt. The nitrogen content of the preserved hide was measured at different durations during the experimental period, i.e., 0 h, 24 h on day 7th, and day 14th. The nitrogen level reduced significantly (P < 0.05) from 0 h to day 14th of the experiment in hides treated with 50% of NaCl and 5% of boric acid, while this trend was found non-significant (P > 0.05) in hides treated with the combination of NaCl + BA. At 0 h, the moisture content for 50% of NaCl-treated hides was 64.82 ± 0.38% moisture content for 5% of boric acid was 63.89 ± 0.59, while for the NaCl + BA combination 61.69 ± 1.09 was observed. Moisture content for 50% of NaCl on day 14th was 38.87 ± 0.42; for boric acid, it was 37.76 ± 1.12, and for the combination of both, the moisture content was 34.56 ± 0.41%. A similar decreasing trend of moisture contents was found in hides treated with different preservatives. After 14 days of treatment, the bacterial count for 50% of NaCl was 2 × 109; for boric acid, it was 1 × 109, and for the combination of both, the bacterial count was 3 × 109. The lowest pollution load was observed hides treated with the combination of NaCl + BA (10:1). Total solids (TS) were 21.69 ± 0.57 while total dissolved solids (TDS) were 21.10 ± 0.57, and total suspended solids were 0.60 ± 0.57 mg/l. It is concluded from the present study that boric acid alone or in combination with NaCl efficiently reduced nitrogen content and bacterial count and can reduce water pollution in tannery and hence could be used as a preservative for the hide in the tannery industry.

Effect of dietary inclusion of mealworm frass on growth, hematology, and serum biochemistry of sheep.

Frass is the main component of worm by-product which exhibit anti-microbial and anti-pathogenic properties. In the present study, we assessed the possibility of mealworm frass in sheep feeding regime and evaluated its effect on health and growth performance of sheep. A total of 09 experimental sheep (18-24 months of age) were grouped into three categories (T1, T3, and T3); each group comprised 3 animals including 2 males and 1 female. Group T1 was considered control, group T2 contains 75% commercial feed and 25 mealworm frass, and T3 was 50:50 of commercial feed and mealworm frass. The sheep in group T2 showed average weight gain of 2.9 kg; however, when the dietary inclusion increased up to 50% of mealworm frass or decreased up to 50% of concentrate feed, the average weight gain decreased up to 2.01 kg (group T3). Moreover, the sheep fed with 25% mealworm frass exhibited the lowest feed refusal percentage (6.33%) in total duration of the dietary period (6 weeks). The highest volume of RBC was found in blood collected from sheep fed within group T2 (10.22 1012/L ± 0.34), followed by sheep fed in group T3 (8.96 × 1012/L ± 0.99) (P < 0.05). Significantly (P < 0.05) highest MCV volume in fL (femtoliter) was found in group T2 (32.83 ± 0.44) followed by group T3 (31.23 ± 0.23). The animals in group T3 showed significantly (P < 0.05) highest MCHC volume (40.47 g/dL ± 0.62) followed by group T2 (38.77 ± 0.97). Similar trend was found in MPV (fL); significantly (P < 0.05) highest MPV volume was found in group T3 (12.63 ± 0.09) followed by group T2 (12.53 ± 0.33). Significantly (P < 0.05) high serum phosphorous (P) (6.00 ± 0.29), TG (60.03 ± 3.11), and TP (7.63 g/dL ± 0.23) levels were found in group T3, followed by animals in group T2. We can conclude that inclusion of mealworm frass to replace 25% commercial concentrate feed improved the growth rate and overall health status of the sheep. The present study laid a foundation for the utilization of the mealworm frass (waste product) in ruminant feeding.

Genetic polymorphism of Β-casein gene and its association with milk production and composition in Azi-Kheli buffalo.

The aim of this study was to find out the genetic polymorphism in ß-casein gene CSN2 in Azi-Kheli buffaloes found in district Swat. Blood samples from 250 buffaloes were collected and processed in lab for sequencing to see the genetic polymorphism in CSN2 gene on 67 position of exon7. The ß-casein is a milk second abundant protein having some variants, wherein A1 and A2 are the most common. After performing sequence analysis, it was found that Azi-Kheli buffaloes were homozygous for only A2 type variant. The amino acid change (proline to histadine) on 67 position of exon 7 was not found; however, three other novel SNPs at loci g.20545A > G, g.20570G > A, and g.20693C > A were identified in the study. Amino acid change due to SNPs were found as SNP1, valine > proline; SNP2, leucin > phenylalanine; and SNP3, threonine > valine. Allelic and genotypic frequencies' analysis exhibited that all three SNPs were following the Hardy-Weinberg equilibrium (HWE: P < 0.05). All the three SNPs showed medium PIC value and gene heterozygosity. The SNPs located on different position of exon 7 of CSN2 gene exhibited associations with some of the performance traits and milk composition. Higher daily milk yield of 9.86 ± 0.43 L and the peak milk yield of 13.80 ± 0.60 L were found in response to SNP3 followed by SNP2 and SNP1. The percentage of milk fat and protein was found significantly higher (P ≤ 0.05) in relation to SNP3 followed by SNP2 and SNP1 given as 7.88 ± 0.41, 7.48 ± 0.33, and 7.15 ± 0.48 for fat% and 4.00 ± 0.15, 3.73 ± 0.10 and 3.40 ± 0.10 for protein%. It was concluded that Azi-Kheli buffalo milk contains A2 genetic variant along with other useful novel variants indicating quality milk for human health. Genotypes of SNP3 should be given preference in selection both in indices and nucleotide polymorphism.

Detection of A2A2 genotype of beta casein protein (CSN2) gene in local, exotic and cross bred cattle in Pakistan.

Genetic variants of bovine Beta-casein protein (CSN2) gene especially A1 and A2 are the most important variants in dairy cattle. A1 milk protein is considered as risk factor for different disease and milk intolerance which release Beta-Casomorphin-7 during digestion which is a bioactive opioid but not released from A2 milk protein. This opioid is responsible for several human health problems like Coronary Heart disease, type 1 diabetics, milk intolerance and other neurological disorders. In present study, 360 blood sample were collected from Lohani, Achai, jersey, Holstein Friesian, Achai x jersey, Friesian x Sahiwal and Sahiwal x Friesian from different region of Khyber Pakhtunkhwa (KP) province. The polymerase chain reaction (PCR) amplicons were sequenced for the identification of polymorphism in exon 7 of Beta-casein protein (CSN2) gene. Sequencing analysis explored CSN2 genotype in exon 7 using the Genomic sequence from GenBank (X.71104) g.8101 C > A at codon 67. The allelic and genotypic frequencies of CSN2 gene were analyzed and observed that Holstein Friesian cattle exhibited A1A2 33%, A1A1 50% and A2A2 17%, Jersey cattle show 68% A1A1, 18% A1A2 and 14% A2A2, Sahiwal x Friesian 56% A1A1, 26% A1A2 and 18% A2A2, Jersey × Achai 78% A2A2, 15% A1A2 and 7% A1A1, Achai 100% A2A2 Lohani 100% A2A2. This is a preliminary study, conducted with meager resources, therefore, it is very difficult to make conclusion that which particular breed possess harmful alleles and which breed possess useful alleles of beta-casein gene. Therefore, a comprehensive molecular work is needed to be performed with greater number of samples sequencing.

Bioinformatics analysis and the association of polymorphisms within the caprine GDF9 gene promoter with economically useful traits in Damani goats.

The blood sample from 60 Damani does were collected and genomic DNA was extracted, and DNA integrity were investigated. A 447 bp promoter fragment of the GDF9 gene was amplified and Sanger sequenced for the identification of GDF9 gene polymorphism. Three novel SNPs were identified at positions g. 97(T > A), g. 142 (G > G) and g. 313(C > T) in the promoter region of the caprine GDF9 gene which significantly (P < 0.05) influenced litter size, body measurement, and milk production traits in Damani goats. The genotype CT of SNP1 significantly (P < 0.05) improved litter size, genotype GG of SNP2 significantly (P < 0.05) enhanced milk production, while the genotypes CC of SNP3 significant (P < 0.05) increased body measurement traits in Damani goats. Moreover, in SNP1 loss of 3 transcription factors (TF) binding sites occurred, SNP2 caused loss of two TFs binding sites, and SNP3 caused loss of a single TF binding site. Similarly, SNP1 and SNP2 caused the gain of three new potential TF binding sites, and SNP3 caused gain of two new TF binding sites. It is concluded that caprine GDF9 gene could be used as a candidate gene for litter size, milk production and body measurement traits in Damani goats through marker-assisted selection for future breeding program.

Association among metabolic status, oxidative stress, milk yield, body condition score and reproductive cyclicity in dairy buffaloes.

The aim of this study was to determine the metabolic status, stress and ovarian activity through progesterone profile in dairy buffalo as influenced by post-partum days, body condition score (BCS) and lactation number. A total of 45 dairy buffaloes were involved and divided into three factors based on their BCS (2.5, 3.0 and 3.5, respectively), lactation number (1, 2 and 3, respectively), and post-partum intervals (14, 28, 42 and 56, respectively). Based on findings, from day 14 to day 56 after parturition, blood glucose, cholesterol and triglyceride levels increased significantly (p < .01), while malondialdehyde (MDA) and cortisol levels decreased significantly (p < .05) on day 56 of the trial. With increased BCS levels, milk yield, blood metabolites and progesterone improved significantly (p < .05), whereas stress indicators decreased significantly (p < .01). Blood metabolites, progesterone and milk production were significantly (p < .01) higher and stress indicators (MDA and cortisol) were significantly (p < .01) lower in lactation stage. Buffaloes with a greater lactation stage and growing post-partum stage had better blood metabolite and progesterone concentrations and less stress. It was concluded that better BCS and increased lactation stage have significant impact on milk yield, stress reduction and restoration of ovarian activity in buffaloes during post-partum period.

Transcriptional regulation of adipogenic marker genes for the improvement of intramuscular fat in Qinchuan beef cattle.

The intramuscular fat content plays a crucial role in meat quality traits. Increasing the degree of adipogenesis in beef cattle leads to an increase in the content of intramuscular fat. Adipogenesis a complex biochemical process which is under firm genetic control. Over the last three decades, the Qinchuan beef cattle have been extensively studied for the improvement of meat production and quality traits. In this study, we reviewed the literature regarding adipogenesis and intramuscular fat deposition. Then, we summarized the research conducted on the transcriptional regulation of key adipogenic marker genes, and also reviewed the roles of adipogenic marker genes in adipogenesis of Qinchuan beef cattle. This review will elaborate our understanding regarding transcriptional regulation which is a vital physiological process regulated by a cascade of transcription factors (TFs), key target marker genes, and regulatory proteins. This synergistic action of TFs and target genes ensures the accurate and diverse transmission of the genetic information for the accomplishment of central physiological processes. This information will provide an insight into the transcriptional regulation of the adipogenic marker genes and its role in bovine adipogenesis for the breed improvement programs especially for the trait of intramuscular fat deposition.

Mitochondrial phylogenetic and diversity analysis in Azi-Kheli buffalo.

NOVELTY STATEMENT: The present study was conducted for the first time in Pakistan to investigate Cytochrome C Oxidase Subunit 1 (CO1) gene and full-length Displacement Loop (D-loop) region of mitochondrial DNA in Azi-Kheli buffalo breed native to northern hilly areas of Khyber Pakhtunkhwa Province of Pakistan. The present study was designed to investigate phylogeny and diversity in Azi-Kheli buffalo, through two mitochondrial DNA regions, i.e., Cytochrome C Oxidase Subunit-I (CO1) and Displacement Loop (D-loop) region. Thirty (30) blood samples were taken from Azi-Kheli pure breed animals from original breeding tract, i.e., Khwazakhela, Swat. Polymerase chain reactions using gene-specific primers were carried out for amplifying 709-bp region of CO1 gene and 1159-bp region of D-Loop for identification, phylogeny, and diversity in Azi-Kheli buffalo, respectively. The sequences of CO1 gene revealed four (04) haplotypes, whereas D-loop sequences revealed five (05) haplotypes. Mean interspecific diversity with related species was 2.56%, and mean intraspecific diversity within Azi-Kheli buffalo was 0.25%, estimated via Kimura-2 parameter. Phylogenetic tree (maximum likelihood) revealed clustering of Azi-Kheli haplotypes with river buffalo and is distinct from swamp buffalo and other related species of genus Bubalus. Mean haplotype and nucleotide diversity of D-loop were Hd = 0.9601 ± SD = 0.096 and π = 0.01208 ± SD = 0.00182, respectively. Phylogenetic tree (neighbor-joining) revealed two main clades, i.e., river buffalo and swamp buffalo clade. The haplotypes of Azi-Kheli clustered with haplotypes of different river buffalo breeds at different positions. The current study suggests that Azi-Kheli has common origin with other river buffalo breeds; hence, it is river buffalo which harbors high genetic diversity.

Corrigendum: Bovine Pre-adipocyte Adipogenesis Is Regulated by bta-miR-150 Through mTOR Signaling.

[This corrects the article DOI: 10.3389/fgene.2021.636550.].

RNA-Seq Reveals Function of Bta-miR-149-5p in the Regulation of Bovine Adipocyte Differentiation.

Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R-BTA-373813 receptor CXCR2 binding ligands CXCL1 to 7, R-BTA-373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R-BTA-210991 basigin interactions, R-BTA-380108 chemokine receptors binding chemokines, R-BTA-445704 calcium binding caldesmon, and R-BTA-5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly (p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis.

Bovine Pre-adipocyte Adipogenesis Is Regulated by bta-miR-150 Through mTOR Signaling.

Micro RNA (miR) are recognized for their important roles in biological processes, particularly in regulatory componentization. Among the miR, miR-150 has been the focus of intense scrutiny, mostly due to its role in malignant tumors. A comparison between steer and bull adipose tissues identified bta-miR-150 as one of the nine downregulated miRNAs, although its function remains unknown (GEO:GSE75063). The present study aimed to further characterize the role of bta-miR-150 in cattle. bta-miR-150 has a negative regulatory effect on the differentiation of bovine adipocytes and promotes proliferation. Overexpression of bta-miR-150 can promote mRNA and protein expression of the marker genes CDK1, CDK2, and PCNA, increase the number of EdU-stained cells, promote adipocyte proliferation, inhibit adipocyte differentiation, and reduce lipid droplet formation. Results of RNA-seq and WGCNA analyses showed that the mammalian target of the rapamycin signaling pathway, which plays a major regulatory role, is dysregulated by the overexpression and inhibition of miR-150. We found that the target gene of bta-miR-150 is AKT1 and that bta-miR-150 affects AKT1 phosphorylation levels. These results showed that bta-miR-150 plays a role in adipogenic differentiation and might therefore have applications in the beef industry.

Genetic variants in the TORC2 gene promoter and their association with body measurement and carcass quality traits in Qinchuan cattle.

The TORC2 gene is responsible for nutrient metabolism, gluconeogenesis, myogenesis and adipogenesis through the PI3K-Akt, AMPK, glucagon and insulin resistance signaling pathways. Sequencing of PCR amplicons explored three novel SNPs at loci g.16534694G>A, g.16535011C>T, and g.16535044A>T in the promoter region of the TORC2 gene in the Qinchuan breed of cattle. Allelic and genotypic frequencies of these SNPs deviated from Hardy-Weinberg equilibrium (HWE) (P < 0.05). SNP1 genotype GG, SNP2 genotype CT and SNP3 genotype AT showed significantly (P <0.05) larger body measurement and improved carcass quality traits. Haplotype H1 (GCA) showed significantly (p<0.01) higher transcriptional activity (51.44%) followed by H4 (ATT) (34.13%) in bovine preadipocytes. The diplotypes HI-H3 (GG-CC-AT), H1-H2 (GG-CT-AT) and H3-H4 (GA-CT-TT) showed significant (P<0.01) associations with body measurement and improved carcass quality traits. Analysis of the relative mRNA expression level of the TORC2 gene in different tissues within two different age groups revealed a significant increase (P<0.01) in liver, small intestine, muscle and fat tissues with growth from calf stage to adult stage. We can conclude that variants mapped within TORC2 can be used in marker-assisted selection for carcass quality and body measurement traits in breed improvement programs of Qinchuan cattle.

Bioinformatics analysis and transcriptional regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes.

Intramuscular fat is the an important factor that defines meat quality; however, enhancing its deposition without increasing the other three adipose depots (subcutaneous, visceral, and intermuscular fat) is a challenge for animal science and the meat industry. The TORC1 is a key regulator of adipogenesis and its regulation in bovine intramuscular preadipocytes has not been studied. The TORC1 is a member of the gene family that codes for a binding proteins which regulate transcription of cAMP which, is a key regulator of adipogenesis. In the present study, expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes. Bioinformatics tools were applied to characterize TORC1. To investigate the molecular mechanism of bovine TORC1 gene regulation, we cloned a 1008 bp of the 5'UTR regulatory region into a luciferase reporter vector. Different fragments were amplified using 5'UTR unidirectional deletion of the TORC1 promoter. Site directed mutation, dual luciferase reporter assay, RNAi interference and DNA-protein interaction (EMSA) were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes. The core promoter region of the TORC1 gene was identified at a location -410 to -155 bp upstream of transcription start site. Different vectors were constructed through serial deletion of the 5'UTR flanking region and in combination with site directed mutations and transcription interference through siRNA or shRNA, two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. The core promoter region of the TORC1 gene, spanning from -410 to -155 bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.

Postpartum endocrine activities, metabolic attributes and milk yield are influenced by thermal stress in crossbred dairy cows.

This study was conducted on 30 freshly parturated multiparous crossbred dairy cows possessing three levels of Holstein Frisian genetic makeup (62.5, 75.0, and 87.5%). Data on temperature humidity index (THI) were classified into comfortable (≤ 71), mild stress (72-79), moderate stress (80-89), and stressful (≥90) zone. Results showed that serum cortisol concentration increased significantly (P < 0.05) in cows during stressful condition irrespective of genetic makeup compared to the other zones. Daily milk yield (DMY) was significantly (P < 0.05) lower in cows during stressful condition. Triglyceride was significantly higher in cows with genetic makeup 87.5% compared to the others, while total serum protein was significantly (P < 0.05) higher in cows during both moderate and stressful conditions. The mean concentration of cortisol and protein increased linearly from comfort to the stressful condition, while mean serum triglyceride, glucose, progesterone (P4), and luteinizing hormone (LH) decreased by moving from comfort to stressful conditions. Results also indicated that higher cortisol level in higher grade crossbred cows was adversely associated with LH concentration and milk yield under thermal stress conditions. Greater triglyceride in high-grade crossbred (87.5%) cows indicates higher fat mobilization reflecting a negative energy balance. We concluded that heat stress increased blood cortisol and protein, and reduced milk yield in dairy cows irresptive of the genetic makeup. In addition, there was no significant difference in blood metabolites and daily milk yield in the different levels of genetic makeup cows.