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Psoriasis is an inflammatory skin disease with an estimated heritability of around 70%. Previous GWASs have detected several risk loci for psoriasis. To further improve the understanding of the genetic risk factors impacting the disease, we conducted a discovery GWAS in FinnGen and a subsequent replication and meta-analysis with data from the Estonian Biobank and the UK Biobank; the study sample included 925,649 individuals (22,659 cases and 902,990 controls), the largest sample for psoriasis yet. In addition, we conducted downstream analyses to find out more about psoriasis' cross-trait genetic correlations and causal relationships. We report 6 risk loci, which, to our knowledge, are previously unreported, most of which harbor genes related to NF-κB signaling pathway and overall immunity. Genetic correlations highlight the relationship between psoriasis and smoking, higher body weight, and lower education level. In addition, we report causal relationships between psoriasis and mood symptoms as well as 2-directioned causal relationship between psoriasis and lower education level. Our results provide further knowledge on psoriasis risk factors, which may be useful in the development of future treatment strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Coronary artery calcium (CAC) is validated for risk prediction among middle-aged adults, but there is limited research exploring implications of CAC among older adults. We used data from the Atherosclerosis Risk in Communities (ARIC) study to evaluate the association of CAC with domains of healthy and unhealthy aging in adults aged ≥75 years., Methods: We included 2,290 participants aged ≥75 years free of known coronary heart disease who underwent CAC scoring at study visit 7. We examined the cross-sectional association of CAC = 0, 1-999 (reference), and ≥1000 with seven domains of aging: cognitive function, hearing, ankle-brachial index (ABI), pulse-wave velocity (PWV), forced vital capacity (FVC), physical functioning, and grip strength., Results: The mean age was 80.5 ± 4.3 years, 38.6% male, and 77.7% White. 10.3% had CAC = 0 and 19.2% had CAC≥1000. Individuals with CAC = 0 had the lowest while those with CAC≥1000 had the highest proportion with dementia (2% vs 8%), hearing impairment (46% vs 67%), low ABI (3% vs 18%), high PWV (27% vs 41%), reduced FVC (34% vs 42%), impaired grip strength (66% vs 74%), and mean composite abnormal aging score (2.6 vs 3.7). Participants with CAC = 0 were less likely to have abnormal ABI (aOR:0.15, 95%CI:0.07-0.34), high PWV (aOR:0.57, 95%CI:0.41-0.80), and reduced FVC (aOR:0.69, 95%CI:0.50-0.96). Conversely, participants with CAC≥1000 were more likely to have low ABI (aOR:1.74, 95%CI:1.27-2.39), high PWV (aOR:1.52, 95%CI:1.15-2.00), impaired physical functioning (aOR:1.35, 95%CI:1.05-1.73), and impaired grip strength (aOR:1.46, 95%CI:1.08-1.99)., Conclusions: Our findings highlight CAC as a simple measure broadly associated with biological aging, with clinical and research implications for estimating the physical and physiological aging trajectory of older individuals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael J. Blaha has grants funded by NIH, FDA, AHA, Bayer, Novo Nordisk, Amgen. He is on advisory boards for Amgen, Novartis, Novo Nordisk, Bayer, Roche, Inozyme, Kaleido, 89Bio; and consults for Emocha health and Kowa. The other authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Hearing loss may contribute to frailty through cognitive and physical decline, but population-based evidence using validated measures remains scarce. We investigated the association of hearing loss with phenotypic frailty and its individual components and explored the potential protective role of hearing aid use., Design: Cross-sectional study of community-dwelling older adults at visit 6 (2016-2017) of the Atherosclerosis Risk in Communities (ARIC) study, a cohort study of older adults from 4 U.S. communities (Washington County, MD; Forsyth County, NC; Jackson, MS; and Minneapolis, MN)., Setting and Participants: Population-based study of 3179 participants (mean age = 79.2 years, 58.9% female)., Methods: Pure-tone audiometry at 0.5-4 kHz was used to assess unaided hearing, and the better-hearing ear's pure-tone average was categorized as follows: no [≤25 dB hearing level (HL)], mild (26-40 dB HL), and moderate or greater (>40 dB HL) hearing loss. Hearing aid use was self-reported. The Fried/physical frailty phenotype was used to categorize frailty status (robust, pre-frail, or frail). Multivariable multinomial and logistic regression models were used to study the association of hearing loss/hearing aid use with frailty status and individual frailty components, respectively., Results: In our sample, 40% had mild and 27% had moderate or greater hearing loss (12% and 55% reported hearing aid use, respectively). Moderate or greater hearing loss was associated with greater odds of being pre-frail [odds ratio (OR), 1.25; 95% CI, 1.01-1.57] and frail (OR, 1.62; 95% CI, 1.06-2.47) vs robust, and greater odds of having slow gait, low physical activity, and exhaustion, compared with no hearing loss. Among those with hearing loss (>25 dB HL), compared with hearing aid users, nonusers had greater odds of being frail vs robust, and having unintentional weight loss, slow gait, and low physical activity., Conclusions and Implications: Hearing loss is associated with pre-frailty and frailty. Longitudinal studies are warranted to establish if hearing aid use may prevent or delay frailty onset., (Copyright © 2023 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

It is unclear whether women have higher brain tau pathology. The objective of this study was to examine whether women have higher tau burden than men, and whether tau differences are independent of amyloid β (Aβ) burden. We conducted a cross-sectional analysis of a multiethnic sample of 252 nondemented late middle-aged (mean age: 64.1 years) adults with tau and amyloid Positron Emission Tomography (PET) data. Tau burden was measured as global standardized uptake value ratio (SUVR) in the middle/inferior temporal gyri and medial temporal cortex with 18 F-MK-6240 PET. Aβ was measured as global SUVR with 18 F-Florbetaben PET. Women had higher middle/inferior temporal gyri tau SUVR compared to men. However, no sex differences in the medial temporal cortex were observed. Women had higher brain Aβ SUVR compared to men. Continuous Aβ SUVR was positively correlated with medial temporal cortex and middle/inferior temporal gyri tau SUVR. However, there was no evidence of effect modification by Aβ SUVR on sex and tau. Compared with men, women in late middle age show higher tau burden, independent of Aβ., (Copyright © 2021. Published by Elsevier Inc.)

Metabolic syndrome (MetS) is associated with dementia, but it is unclear whether MetS is related to Alzheimer's disease (AD). We investigated the association of MetS with brain amyloid, a key AD feature, and neurodegeneration. A community-based sample of 350 middle-aged Hispanics in New York City had cerebral amyloid β (Aβ) burden ascertained with 18 F-Florbetaben positron emission tomography. Neurodegeneration was ascertained as cortical thickness in AD signature regions from 3T brain MRI. MetS and its components (glucose, blood pressure, triglycerides, high-density lipoprotein, adiposity) were defined using the National Institutes of Health criteria. Neither the presence of MetS nor the MetS score was associated with Aβ or neurodegeneration. Among the MetS components, elevated glucose was associated with lower Aβ burden, and this association was not explained by diabetes treatment. Glucose and triglycerides were related to smaller cortical thickness. Our findings suggest that MetS as an arbitrary measure of aggregate metabolic and vascular risk does not capture the risk of AD neuropathology in late middle age and that other approaches to measure the aggregate risk should be examined., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A functional canonical WNT signaling pathway exists in preimplantation embryos and inhibits embryonic development. Recent studies suggest that this pathway is over-expressed in nuclear transferred (NT), compared to IVF embryos. The present study investigated the effects of Dickkopf-1 (DKK1), an inhibitor of canonical WNT signaling pathway and colony stimulating factor-2 (CSF2), an embryokine, on the developmental competence, quality, gene expression and live birth rate of NT buffalo embryos produced by Hand-made cloning (HMC). Following supplementation of the in vitro culture medium on day 5 with DKK1 (100 ng/mL), CSF2 (10 ng/mL), DKK1+CSF2 or no supplementation (control), the blastocyst rate was higher (P < 0.05) with DKK1 and DKK1+CSF2 (42.6 ± 1.4% and 46.6 ± 0.9%, respectively) than with CSF2 or controls (40.6 ± 1.3% and 39.0 ± 1.3%, respectively). The apoptotic index of the blastocysts was lower (P < 0.05) for DKK1, CSF2 and DKK1+CSF2 groups (3.44 ± 0.14, 3.39 ± 0.11 and 3.11 ± 0.22, respectively) compared to controls (6.64 ± 0.25), and was similar to that of the IVF blastocysts (3.67 ± 0.18). Although the total cell number was similar for the DKK1, CSF2, DKK1+CSF2 and control groups (200.4 ± 3.05, 196.4 ± 3.73, 204.7 ± 3.71 and 205 ± 4.03, respectively), the inner cell mass:trophectoderm cell number ratio of DKK1, CSF2 and DKK1+CSF2 groups (0.21 ± 0.01, 0.17 ± 0.01 and 0.22 ± 0.02, respectively) was higher (P < 0.05) than controls (0.13 ± 0.01) and was similar to that of IVF blastocysts (0.19 ± 0.01). Treatment with DKK1 or CSF2 or both increased (P < 0.05) the expression level of OCT4, NANOG,SOX2, GATA6, BCL2, PTEN, P53, FGF4, GLUT1 and IFN-τ, and decreased that of C-MYC, CDX2, CASPASE, DNMT3a, TCF7 and LEF1 in blastocysts, compared to controls. Transfer of DKK1-treated embryos to 13 recipients resulted in 4 pregnancies (30.8%; 2 live births, one abortion and one currently at 9 months of pregnancy) whereas, transfer of DKK1+CSF2-treated embryos to 16 recipients, resulted in 4 pregnancies (25.0%), all of which resulted in live births. No pregnancy was obtained after transfer of control and CSF-treated embryos to 12 and 16 recipients, respectively. These results suggest that DKK1 treatment of NT embryos increases the blastocyst, conception and live birth rate, and improves their quality whereas, CSF2 treatment, does not affect the blastocyst, conception and live birth rate despite improvement in embryo quality., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ± 4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ± 0.25%) and lipofectamine 3000 (15.57 ± 0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo β-lactoglobulin (buBLG) promoter and terminator buBLG 3'UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ± 1.8% vs 48.7 ± 2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ± 0.9, 3.8 ± 0.5 and 1.8 ± 0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ± 17.0 and 137.2 ± 7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Application of cloning technology on a wide scale is severely limited by the very low live birth rate obtained with cloned embryos. Embryo quality is an important factor which affects the conception and live birth rate of cloned embryos. microRNA-21 (miR-21) has been implicated in the regulation of apoptosis and the expression level of several important genes which control apoptosis. We examined the effects of treatment of reconstructed buffalo embryos, produced by Hand-made cloning, with miR-21 mimic on developmental competence, quality and gene expression of cloned embryos. Expression level of miR-21, which increased from 2-cell to 8-cell stage and then decreased at the blastocyst stage, showed a similar pattern in cloned and IVF embryos. It was lower in cloned than in IVF embryos at 2-, 4- and 8-cell (P < 0.001) and blastocyst (P < 0.05) stages but not at morula stage. Treatment of reconstructed embryos with miR-21 mimic for 1 h after 1 h of electrofusion, increased (P < 0.05) the total cell number (251.3 ± 10.7 vs 181.5 ± 2.13). Blastocysts produced from miR-21-treated reconstructed embryos had lower (P < 0.05) apoptotic index than controls and IVF blastocysts (2.01 ± 0.17, 5.46 ± 0.26 and 4.19 ± 0.15, respectively). The treatment also improved the inner cell mass:trophectoderm cell number ratio of blastocysts than in controls (0.21 ± 0.01 vs 0.11 ± 0.003) to values observed in IVF blastocysts (0.20 ± 0.008). However, miR-21 mimic treatment did not affect the blastocyst rate, which was similar for treatment, control and negative control groups (36.58 ± 3.64, 36.58 ± 3.64 and 32.2 ± 2.86%, respectively). miR-21 mimic treatment increased (P < 0.01) the expression level of apoptosis- (BCL2 and PTEN), pluripotency- (OCT4 and SOX2) and development-related genes (GLUT1, FGF4 and P53), but not that of CASPASE3 than in untreated controls in blastocysts. These results suggest that treatment of reconstructed embryos with miR-21 mimic improves blastocyst quality, reduces apoptosis and alters gene expression without improving the blastocyst rate., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures obtained in monolayer differentiation had a big nucleus and a surrounding ZP4 coat, the unique attributes of a female gamete. These oocyte-like structures, in extended cultures, showed embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence. This, as per our knowledge, is first such study in higher mammals, especially farm animals, and assumes significance for its potential use in transgenic animal production, elite animal conservation and propagation, augmentation of reproductive performance in poor breeding buffalo species, and as a model for understanding human germ cell formation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus-oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus-oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus-oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.

We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many pathologic and physiological responses including embryonic development. This study investigated the effect of supplementation of IVC medium with an ER stress inducer, tunicamycin (TM), and an inhibitor, tauroursodeoxycholic acid (TUDCA), on the developmental competence, apoptosis, and gene expression in buffalo embryos produced by IVF. Treatment of presumed zygotes with TM resulted in a significant (P < 0.01) decrease in the blastocyst rate, whereas TUDCA supplementation did not improve the blastocyst development rate. Further, presence of TUDCA could not ameliorate the adverse effects of TM in terms of the blastocyst rate in combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) the apoptotic index and reduced the total cell number, whereas TUDCA did not affect them significantly. However, TUDCA reduced the extent of TM-mediated apoptosis during combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) and TUDCA treatment decreased (P < 0.01) the expression level of ER chaperones, GRP78 and GRP94. In the combined TM + TUDCA treatment, TUDCA decreased their expression level compared to that in the controls. A similar pattern was observed in the case of proapoptotic gene BAX. We did not find any significant difference in the expression level of BCl-XL, BID, P53, and CASPASE 3 after TM and TUDCA supplementation. In conclusion, our study reported that TM induces ER stress in buffalo embryos produced in vitro resulting in a decrease in the blastocyst rate and an increase in the level of apoptosis and that these actions are mediated by modulating the expression of apoptosis-related genes and ER chaperones. Tauroursodeoxycholic acid did not improve the developmental potential of buffalo embryos; however, it attenuated the TM-induced apoptosis by downregulating BAX and ER chaperones., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study compared the cloning efficiency of donor cells of fibroblast and epithelial origin isolated from ear skin of a wild buffalo (Bubalus arnee) and used with cytoplasts from domestic buffalo (Bubalus bubalis) in interspecies SCNT by hand-made cloning. The cleavage (93.0 ± 2.8% vs. 85.6 ± 2.4%) and blastocyst rates (50.6 ± 4.0% vs. 20.5 ± 2.6%) were higher (P < 0.05) for fibroblasts than those for epithelial cells, whereas the total cell number (490 ± 42 and 492 ± 95, respectively) and apoptotic index (2.3 ± 0.3 and 2.5 ± 0.6, respectively) of blastocysts were similar. The global level of H3K18ac and H3K27me3 was lower (P < 0.05) in fibroblasts than that in epithelial cells. The global level of H3K18ac was higher (P < 0.05) in fibroblast than that in epithelial cell-derived blastocysts, whereas that of H3K27me3 was similar between the two groups. The expression level of HDAC1, DNMT1, DNMT3a, and P53 was higher (P < 0.05) in fibroblasts than that in epithelial cells; that of CASPASE3 showed an opposite pattern (P < 0.001), whereas CASPASE7 expression level was similar in the two groups. In the embryos, the expression level of HDAC1, DNMT3a, and CDX2 was lower (P < 0.05) in fibroblast than that in epithelial cell-derived blastocysts; that of NANOG showed an opposite pattern (P < 0.05), whereas that of OCT4 was similar between the two groups. In conclusion, donor cells of fibroblast origin are easier to reprogram than those of epithelial origin in interspecies SCNT, and cloning efficiency, epigenetic status, and gene expression pattern vary among cells having different origin although they may be from the same tissue., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Trichostatin A (TSA), a histone deacetylase inhibitor, has been used to improve nuclear reprogramming in somatic cell nuclear transfer embryos. However, the molecular mechanism of TSA for the improvement of the pre- and postimplantation embryonic development is unknown. In the present study, we investigated mechanism of cell cycle arrest caused by TSA and also determined embryo quality and gene expression in cloned bovine embryos produced from TSA-treated donor cells compared with embryos produced by in vitro fertilization or parthenogenetic activation. We observed that, 50 nM TSA-treated cells were synchronized at G0/G1 stage with concomitant decrease in the proportion of these cells in the S stage of the cell cycle, which was also supported by significant changes in cell morphology and decreased proliferation (P<0.05). Measurement of relative expression using real-time polymerase chain reaction of a some cell cycle-related genes and microRNAs in treated donor cells showed decreased expression of HDAC1, DNMT1, P53, CYC E1, and CDK4 and increased expression of DNMT3a, CDKN1A, CDK2, CDK3, miR-15a, miR-16, and miR-34a (P<0.05). No change in the relative expression of miR-449a was noticed. Trichostatin A treatment of donor cells significantly improved both cleavage and blastocyst rate (P<0.05) compared with the control embryos, also apoptotic index in treated cloned blastocysts was significantly decreased compared with the nontreated blastocysts (P<0.05) and was at the level of IVF counterpart. Relative expression of HDAC1 and DNMT3a was significantly lower in treated cloned and parthenogenetic embryos than that of nontreated and IVF counterpart, whereas in case of P53, expression level between treated and IVF embryos was similar, which was significantly lower than nontreated cloned and parthenogenetic embryos. In conclusion, our data suggested that TSA improves yield and quality of cloned bovine embryos by modulating the expression of G0/G1 cell cycle stage-related microRNA in donor cells, which support that TSA might be great cell cycle synchronizer apart from potent epigenetic modulator in cloning research in future., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

DNA methylation is an important mechanism by which gene transcription and hence cellular function are regulated. Here, we provide detailed functional genome-wide methylome maps of 5 primary peripheral blood leukocyte subsets including T cells, B cells, monocytes/macrophages, and neutrophils obtained from healthy individuals. A comparison of these methylomes revealed highly specific cell-lineage and cell-subset methylation profiles. DNA hypomethylation is known to be permissive for gene expression and we identified cell-subset-specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels suggesting these HMRs may regulate corresponding cell functions. Single-nucleotide polymorphisms associated with immune-mediated disease in genome-wide association studies preferentially localized to these cell-specific regulatory HMRs, offering insight into pathogenesis by highlighting cell subsets in which specific epigenetic changes may drive disease. Our data provide a valuable reference tool for researchers aiming to investigate the role of DNA methylation in regulating primary leukocyte function in health and immune-mediated disease.

The present study was conducted primarily to optimize electrofusion conditions for efficient production of zona-free nuclear transfer embryos in buffalos (Bubalus bubalis). We found that 4V AC current for proper triplet alignment and single step fusion method, using a single DC pulse of 3.36 kV/cm for 4-μs duration, produced the most convincing results for efficient reconstitution of zona-free cloned embryos. Lysis rate was very high (84.28 ± 2.59%) when triplets were in physical contact with negative electrode after applying DC current, however, cleavage rate and blastocyst rate were found to be similar when the triplets were not in physical contact with either positive or negative electrodes or when they were in physical contact with the positive electrode. Significant improvement in blastocyst production was observed when the somatic cell faced the positive electrode than when it faced the negative electrode (39.17 ± 2.74% vs. 25.91 ± 2.00%, respectively) during electrofusion. Similarly, the blastocyst rate (52.0 ± 3.4%) was found to be significantly higher when reconstructed embryos were activated 6 h post electrofusion as compared to 0, 2, 4 and 8 h (16.04 ± 6.3%; 18.36 ± 1.4%; 22.44 ± 3.7% and 30.02 ± 4.6%, respectively). This study establishes the application of zona-free nuclear transfer procedures for the production of handmade cloned buffalo embryos through optimization of electrofusion parameters and post fusion holding time for enhancing their preimplantation development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate the effect of scriptaid, a histone deacetylase inhibitor, on the developmental competence of Handmade cloned (HMC) and parthenogenetically activated (PA) buffalo embryos. Scriptaid treatment was given for 10 h immediately after reconstruction of HMC and PA embryos. Treatment of HMC embryos with 500 or 1000 nmol/L scriptaid significantly increased (P<0.01) the cleavage rate (91.3±2.8 and 91.9±2.0%, respectively) than that of controls (80.7±2.4%). Blastocyst rate was significantly higher (P<0.01) following treatment with 1000 nmol/L (54.1±5.0%) than that with 500 nmol/L scriptaid (42.6±2.9%) which, in turn, was higher (P<0.01) than that of the controls (38.0±2.6%). Also, HMC embryos treated with 500 or 1000 nmol/L scriptaid had higher (P<0.01) cell number (339.9±1.4 and 343.4±2.4, respectively) than the untreated embryos (150.7±2.0). In contrast, in zona-free PA embryos, the cleavage and blastocyst rate was significantly lower (P<0.01) following treatment with 500 (46.3±1.0 and 22.0±1.4%, respectively) or 1000 nmol/L scriptaid (46.2±1.9 and 16.6±1.3%, respectively) than that of untreated embryos (60.4±3.2 and 29.5±2.1%, respectively). Transfer of HMC embryos that had been untreated (N=13) or treated with 1000 nmol/L scriptaid (N=15) to synchronized recipients resulted in one pregnancy each. In conclusion, these results demonstrate that scriptaid treatment improves the developmental potential of HMC buffalo embryos, but compromises that of zona-free PA embryos., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The increasing use of whole-genome array screening has revealed the important role of DNA copy-number variations in the pathogenesis of neurodevelopmental disorders and several recurrent genomic disorders have been defined during recent years. However, some variants considered to be pathogenic have also been observed in phenotypically normal individuals. This underlines the importance of further characterization of genomic variants with potentially variable expressivity in both patient and general population cohorts to clarify their phenotypic consequence. In this study whole-genome SNP arrays were used to investigate genomic rearrangements in 77 Estonian families with idiopathic mental retardation. In addition to this family-based approach, phenotype and genotype data from a cohort of 1000 individuals in the general population were used for accurate interpretation of aberrations found in mental retardation patients. Relevant structural aberrations were detected in 18 of the families analyzed (23%). Fifteen of those were in genomic regions where clinical significance has previously been established. In 3 families, 4 novel aberrations associated with intellectual disability were detected in chromosome regions 2p25.1-p24.3, 3p12.1-p11.2, 7p21.2-p21.1 and Xq28. Carriers of imbalances in 15q13.3, 16p11.2 and Xp22.31 were identified among reference individuals, affirming the variable phenotypic consequence of rare variants in some genomic regions considered as pathogenic., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A(260/280)), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2)., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Human identification systems such as criminal databases, forensic DNA testing and genetic genealogy require reliable and cost-effective genotyping of autosomal, mitochondrial and Y chromosome markers from different biological materials, including venous blood and saliva. Although many such assays are available, few systems are capable of simultaneously detecting all three targets in a single reaction. Employing the APEX-2 principle, we have characterized a novel 124-plex assay, using specific primer extension, universal primer amplification and single base extension on an oligonucleotide array. The assay has been designed for simultaneous genotyping of SNPs from the single copy loci (46 autosomal and 29 Y chromosomal markers) side by side with SNPs from the mitochondrial genome (49 markers) that appears in up to thousands of copies per cell in certain tissue types. All the autosomal SNPs (from the SNPforID Consortium) included in the multiplex assay are unlinked and are distributed widely across autosomes, enabling genetic fingerprints to be distinguished. Mitochondrial DNA and Y chromosome polymorphisms that define haplogroups common in European populations are included to allow for maternity and paternity testing and for the analysis of genetic genealogies. After assay optimization we estimated the accuracy (99.83%) and call rate (99.66%) of the protocol on 17 mother-father-child/children families and five internal control DNAs. In addition, 79 unrelated Estonian and Swedish DNA samples were genotyped and the accuracy of mtDNA and Y chromosome haplogroup inference by the multiplex method was assessed using conventional genotyping methods and direct sequencing.

Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0+/-1.8% (35/145), 33.0+/-8.0% (56/163), 21.0+/-9.3% (29/133) and 49.6+/-1.9% (77/154), respectively. Blastocyst rates were higher (P<0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.

Oocytes from abattoir-derived bubaline (Bubalus bubalis) ovaries were subjected to IVM and IVF; the objective was to develop a pronuclear DNA microinjection technique to produce embryos expressing green fluorescent protein (GFP). The largest proportion (61.2%) of zygotes in which one (1 PN) or two pronuclei (2 PN) were visible was when centrifugation (14,000 x g for 15 min) was done 16 h after insemination. Centrifugation had no adverse effects on cleavage rate, development to morulae/blastocysts, and total cell number of embryos. Piercing the pronuclear but not the plasma membrane reduced (P<0.05) cleavage rate (44.0% vs. 51.0%), without affecting subsequent development. Following microinjection of a GFP-DNA construct, cleavage rate (55.9, 38.9, and 30.9%) and proportion of cleaved embryos that developed to morulae (39.9, 25.6, and 15.5%) and blastocyst stages (22.4, 13.4, and 2.8%) were higher (P<0.05) for non-injected controls than for those injected with buffer alone, which, in turn, were higher (P<0.05) than for those injected with buffer containing 5 microg/mL DNA. The cleavage rate (39.2% vs. 34.8%) and proportion of cleaved embryos that developed to morulae/blastocysts (37.5% vs. 10.9%) were higher (P<0.05) for microinjected zygotes with 2 PN than for those with 1 PN. The cleavage rate and the proportion of cleaved embryos that developed to morulae and blastocysts were higher (P<0.05) following culture of microinjected zygotes in mCR2aa medium (40.7, 32.7, and 9.1%, respectively) compared to those for mSOFaa (33.3, 26.0, and 6.5%, respectively) or after culture in TCM-199+co-culture with buffalo oviductal epithelial cells (31.2, 25.0, and 4.5%, respectively). The proportion of embryos expressing GFP was higher (P<0.01) for 2 PN than for 1 PN zygotes (15.9% vs. 13.7%). Thirty-five embryos expressed GFP; the proportion of mosaic embryos (62.8%) was higher (P<0.01) than of embryos in which all blastomeres expressed GFP (37.2%); eight and two of those embryos developed to the morula and blastocyst stages, respectively.

The rapid advancement of high-resolution DNA copy number assessment methods revealed the significant contribution of submicroscopic genetic imbalances to abnormal phenotypes, including mental retardation. In order to detect submicroscopic genetic imbalances, we have screened 20 families with X-linked mental retardation (XLMR) using a chromosome X-specific array-MAPH platform with median resolution of 238kb. Among the 20 families, 18 were experimental, as they were not previously screened with any microarray method, and two were blind controls with known aberrations, as they were previously screened by array-CGH. This study presents the first clinical application of chromosome X-specific array-MAPH methodology. The screening of 20 affected males from 20 unrelated XLMR families resulted in the detection of an unknown deletion, spanning a region of 7-23kb. Family studies and population screening demonstrated that the detected deletion is an unknown rare copy number variant. One of the control samples, carrying approximately 6-Mb duplication was correctly identified, moreover it was found to be interrupted by a previously unknown 19kb region of normal copy number. The second control 50kb deletion was not identified, as this particular region was not covered by array-MAPH probes. This study demonstrates that the chromosome X-specific array-MAPH platform is a valuable tool for screening patients with XLMR, or other X-linked disorders, and emerges the need for introducing new high-resolution screening methods for the detection of genetic imbalances.

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.

Ten buffalo were superovulated by administration of 8 doses of FSH in a descending schedule spread over 4 d (5.5/5.5, 4.5/4.5, 3.5/3.5 and 2.5/2.5 mL, i.m.; total dose of 64 AU in 32 mL) beginning on Day 10 of an unstimulated estrous cycle, and 30 and 20 mg Lutalyse was given alongwith the 5th and 6th injections of FSH, respectively, to induce luteolysis. The number of corpora lutea (CL) was determined on 6 d post estrus. The ovaries were examined daily by ultrasonography from Day -5 to Day 5 (Day 0 = day of start of superovulation). The animals were retrospectively classified into 2 groups depending upon the presence (n = 4) or absence of a dominant follicle (n = 6). The mean diameter of the largest follicle (F1) increased from 8.25 +/- 0.48 mm on Day -5 to 10.75 +/- 0.25 mm on Day 0 in the dominant group, whereas in the nondominant group the F1 follicle exhibited a progressive decrease from 9.00 +/- 0.45 mm to 7.00 +/- 0.65 mm during the same period, the difference in profiles between the 2 groups was significant (P = 0.042). The profile of the diameter of the second largest follicle (F2) and the difference in diameters between largest and second largest follicles (F1-F2) were not significantly different between the 2 groups. The profile of mean number of large (> or = 10 mm diameter), but not small (2 to 5 mm diameter) or medium (6 to 9 mm diameter) follicles differed significantly (P = 0.001) between the 2 groups from Day -5 to Day 5 (P = 0.030). The number of CL was not significantly different between nondominant (4.00 +/- 0.97) and dominant groups (3.25 +/- 1.31). The number of CL was positively correlated (P < 0.01) with the number of medium follicles and the total number of follicles on the day of initiation of superovulation, but not with follicles of any size category or total number of follicles on any previous day. The results of this study indicate that following the use of morphological criteria based on the size of the largest follicle alone, the superovulation response is not affected by the presence of a dominant follicle at the initiation of superovulation in buffalo.

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day -13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at -48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day -12 to Day -4, every 24 h from Day -4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days -2, -1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 +/- 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.

The present study investigated the peripheral plasma inhibin levels in relation to 1) the stage of estrous cycle and the effect of climatic variations. Blood samples were collected from cyclic buffalo (n=5) once daily for 32 consecutive days during the tropical hot humid (summer) and cold (winter) seasons. Estrus was recorded by parading a vasectomized bull as well as by plasma progesterone determination. In the winter season, peripheral inhibin concentrations which were lowest (0.35 +/- 0.02 ng/ml) during the mid-luteal phase of estrous cycle (Day 6 to Day 14, Day 0 = day of estrus) increased significantly (P < 0.02) to 0.47 +/- 0.04 ng/ml during the late luteal phase (Day -4 to Day -2) and then further to 0.52 +/- 0.03 ng/ml (P< 0.02) during the periestrus phase (Day -1 to Day 1). Inhibin concentrations then decreased significantly (P < 0.02) to 0.40 +/- 0.03 ng/ml during the early luteal phase (Day 2 to Day 5). In the summer season the differences in peripheral inhibin concentrations among different phases of estrous cycle were found to be nonsignificant. A comparison of the circulating inhibin concentrations between the two seasons indicated that inhibin concentrations were significantly higher in the late luteal phase (P < 0.01) and periestrus phase (P < 0.05) during the winter season compared with corresponding periods during the summer season. The present study suggests that peripheral inhibin concentrations change in the estrous cycle during cooler breeding season and that environmental heat stress can cause a reduction in peripheral inhibin concentrations.

This study examined the effect of gestation on the hypophyseal responsiveness of buffalo to GnRH-induced LH and FSH release. Peripheral plasma LH and FSH concentrations were measured at 1 h before and upto 6 h after administration of GnRH (1 ug/kg body weight) or saline at Days 60, 150 and 240 of gestation in 2 groups of buffalo (n = 4 each). Basal LH concentrations did not vary at the 3 stages of gestation, while basal FSH concentrations exhibited a significant reduction (P < 0.05) from Day 60 to Day 150 of gestation. There was a significant reduction in the total LH (P < 0.05) and FSH (P < 0.01) released in response to GnRH from Day 60 to Day 240 of gestation. The duration of LH and FSH peaks and the time to attain peak concentration was not affected by the stage of gestation. The results of the present study point to a progressive decline in LH and FSH release responses to GnRH during the advancement of gestation in the buffalo.

A sensitive, specific RIA was validated and used for measurement of peripheral plasma immunoreactive inhibin (irinhibin) levels during the estrous cycle in Murrah buffalo. The RIA employed an 125-I iodinated inhibin as tracer and an antiserum against dimeric inhibin. The procedure had a sensitivity of 16 pg/tube, and the nonspecific effects of buffalo plasma were compensated for by including 200 ul bullock plasma in the standards. Separation of free and bound inhibin was affected by the use of a second antibody and precipitation with polyethylene glycol. Blood samples were collected once daily for 30 d from Murrah buffalo (n = 6) during the hot month of July. Cyclic activity and estrus were confirmed by plasma progesterone determination. Peripheral plasma concentrations of ir-inhibin fluctuated between 0.40 +/- 0.07 and 0.67 +/- 0.13 ng/ml during the estrous cycle in buffalo. During the same period, plasma progesterone levels increased from 0.21 +/- 0.01 ng/ml at Day 0 to a peak of 3.30 +/- 0.72 ng/ml on Day 13, declining sharply by Day -5. Ir-inhibin levels exhibited an increase during the follicular phase, with the maximum concentration of 0.65 +/- 0.01 ng/ml occuring on the day of estrus, a decline thereafter, and no pattern during the luteal phase. The differences, however, were not statistically significant throughout the estrous cycle.

The objective of this study was to investigate the hypophysial responsiveness to GnRH at different intervals post partum in Murrah buffalo. Plasma LH and FSH levels were measured at 1 h before and upto 6 h subsequent to the administration of GnRH (1 ug/kg body weight) or saline on Days 2, 20 and 35 post partum in 2 groups of buffalo (n=4 each). Plasma progesterone levels were measured in samples collected once daily from Day 3 to Day 46 post partum. Pretreatment basal LH levels exhibited a progressive increase from Day 2 through Day 35 post partum, while the basal FSH levels increased only until Day 20 post partum. Following a highly subdued LH response to GnRH on Day 2 post partum, a 408% increase (P < 0.01) was observed in the total LH released in response to GnRH on Day 20 post partum, followed by a 20% reduction (non-significant) over Days 20 to 35 post partum. The interval from parturition was highly correlated with total LH released (r = 0.711, P < 0.01). Unlike LH, a substantial amount of FSH was released following GnRH treatment on Day 2 post partum, which was not significantly different from the FSH response on Days 20 and 35 post partum. The LH and FSH response to GnRH was not significantly different between animals in which luteal activity resumed and in those which showed no luteal activity post partum. While pointing to a dramatic enhancement in the hypophysial responsiveness to GnRH between Days 2 and 20 post partum, these results suggest that pituitary responsiveness to GnRH does not appear to be the limiting factor for resumption of estrous cycles by Day 35 post partum in Murrah buffalo.