CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy.

Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.

Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Evaluation of thermo-adaptability between Tharparkar (Bos indicus) and crossbred (Bos indicus X Bos taurus) calves in a controlled environment.

The global warming driven climate change has increased the susceptibility of livestock around the globe to heat stress (HS), which reduces animal productivity and threatens the sustainability of marginal farmers. The objective of this investigation was to evaluate thermo-adaptability between Tharparkar calves (TC), an indigenous milch breed of India and crossbred calves (CC) during induced heat stress in controlled environment. For this purpose, 12 apparently healthy male calves (six in each group) aged 5-6 months, were selected. The experiment was conducted at physiologically comfortable temperature (25 °C), moderate HS (31 °C) and severe HS (37 °C) for 21 days each in a psychrometric chamber. In each experimental day, the calves were exposed to 6 h of heat. There were 7 days of acclimatization period before experiment and 10 days of recovery period at ambient temperature between each 21 day exposure period. During experimental period, the blood was collected at 1st, 6th, 11th, 16th, 21st day and among ten-day recovery period the blood was collected at 5th day. Physiological responses, serum electrolytes, metabolic enzymes profiles, antioxidant capacity, oxidative stress status and general endocrine milieu were studied. Relative mRNA expression study of Heat Shock Protein (HSP) 70, HSP90, induced Nitric Oxide Synthase (iNOS) and endothelial NOS (eNOS) were carried out by qPCR. There was significant (p < 0.05) change in the displacement in rectal temperature, respiration rate, serum alanine aminotransferase level between two breeds at moderate and severe HS. Similar change was observed in total antioxidant capacity, superoxide dismutase, and endocrinological parameters. The comparatively lower mRNA expression of HSP70 and higher expression of HSP90 in TC than CC point the better thermo-adaptability of the same. The results of the experiment indicated that TC are more thermo-adaptable than CC at different modality of stress in controlled temperature conditions.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Transcriptome profiling of different developmental stages of corpus luteum during the estrous cycle in pigs.

To better understand the molecular basis of corpus luteum (CL) development and function RNA-Seq was utilized to identify differentially expressed genes (DEGs) in porcine CL during different physiological stages of the estrous cycle viz. early (EL), mid (ML), late (LL) and regressed (R) luteal. Stage wise comparisons obtained 717 (EL vs. ML), 568 (EL vs. LL), 527 (EL vs. R), 786 (ML vs. LL), 474 (ML vs. R) and 534 (LL vs. R) DEGs with log2(FC) ≥1 and p < 0.05. The process of angiogenesis, steroidogenesis, signal transduction, translation, cell proliferation and tissue remodelling were significantly (p < 0.05) enriched in EL, ML and LL stages, where as apoptosis was most active in regressed stage. Pathway analysis revealed that most annotated genes were associated with lipid metabolism, translation, immune and endocrine system pathways depicting intra-luteal control of diverse CL function. The network analysis identified genes AR, FOS, CDKN1A, which were likely the novel hub genes regulating CL physiology.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Modulation of granulosa cell function via CRISPR-Cas fuelled editing of BMPR-IB gene in goats (Capra hircus).

BMPs are multifunctional growth factors implicated in regulating the ovarian function as key intra-ovarian factors. Biological effects of BMPs are mediated through binding with membrane bound receptors like BMPR-IB and initiating downstream Smad signaling pathway. FecB mutation, regarded as a loss of function mutation in the BMPR-IB gene was identified in certain sheep breeds having high fecundity. Similar type of fecundity genes in goats have not been discovered so far. Hence, the current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in goats. The BMPR-IB gene was knocked out using CRISPR-Cas technology in granulosa cells and cultured in vitro with BMP-4 stimulation for three different durations In addition, the FecB mutation was introduced in the BMPR-IB gene applying Easi-CRISPR followed by BMP-4/7 stimulation for 72 h. Steroidogenesis and cell viability were studied to explore the granulosa cell function on BMPR-IB gene modulation. BMPRs were found to be expressed stage specifically in granulosa cells of goats. Higher transcriptional abundance of R-Smads, LHR and FSHR indicating sensitisation of Smad signaling and increased gonadotropin sensitivity along with a significant reduction in the cell proliferation and viability was observed in granulosa cells upon BMPR-IB modulation. The inhibitory action of BMP-4/7 on P4 secretion was abolished in both KO and KI cells. Altogether, the study has revealed an altered Smad signaling, steroidogenesis and cell viability upon modulation of BMPR-IB gene in granulosa cells similar to that are documented in sheep breeds carrying the FecB mutation.

Análise do resultado clínico-funcional do tratamento das fraturas-luxações da articulação de Lisfranc / Analysis of clinical and functional results from the treatment of Lisfranc joint fracture-dislocations

Objetivo: Analisar o resultado clínico-funcional do tratamento das fraturas-Iuxações agudas da articulação tarsometatarsal (Lisfranc) e estabelecer os fatores prognósticos, a médio prazo, destas lesões. Material e métodos: Vinte e um pacientes (21 pés), todos vítimas de traumatismos no médio pé com fratura-Iuxação de Lisfranc, foram reavaliados após tempo médio de seguimento de 71 meses. Doze pacientes eram do sexo masculino (57 por cento) e nove do feminino (43 por cento). A média de idade, no momento do trauma, foi de 26 anos (variando de cinco a 48 anos). Os traumas causados por alta energia foram os mais freqüentes, ocorrendo em cerca de 86 por cento dos casos. A redução aberta, seguida da fixação interna, foi empregada em 17 pés (81 por cento) e o tratamento incruento, em quatro (19 por cento). Imobilização com bota gessada foi utilizada durante 12 semanas e, em seguida, iniciou-se fisioterapia. Resultados: A pontuação média, segundo a escala AOFAS para médio pé, foi de 83 pontos (variando de 53 a 100). A dor residual foi o sintoma mais freqüente, presente em 12 dos 21 pés (58 por cento), com intensidade leve em seis dos pés (29 por cento) e moderada ou grave nos outros seis pés (29 por cento). Os resultados considerados insatisfatórios foram associados às fraturas expostas, à fratura das duas colunas do pé, às fraturas desviadas do cubóide e a seqüela de síndrome compartimental. A influência negativa da lesão de Lisfranc nas demais articulações foi evidenciada pela redução significativa na amplitude de movimento (perda de 20 por cento no tornozelo, 22 por cento na subtalar, 8 por cento na tarsometatarsal, 37 por cento na metatarsofalangiana do hálux e 18 por cento na interfalângica do hálux). Conclusões: O resultado clínico-funcional do tratamento das fraturasluxações de Lisfranc, a médio prazo, foi satisfatório em 72 por cento dos casos avaliados. Os fatores de mau prognóstico, associados aos resultados insatisfatórios, foram: fraturas expostas, síndrome compartimental, esmagamento do osso cubóide e as fraturas envolvendo as duas colunas do pé. Após fratura-Iuxação de Lisfranc, espera-se significativa redução da amplitude de movimento nas demais articulações do tornozelo e nas demais articulações do pé