Evidence of novel Treponema phylotypes implicated in contagious ovine digital dermatitis and association of treponemes with major lameness causing foot pathogens.

In this study 269 swabs collected from 254 ovine foot lesions and 15 apparently healthy ovine feet were screened by PCR for the presence of major lameness causing foot pathogens viz. Treponema species, D. nodosus, F. necrophorum and T. pyogenes with the presumption that ovine foot lesion positive for Treponema species alone or in association with other three pathogens were categorized as contagious ovine digital dermatitis (CODD). While samples positive for D. nodosus alone or its combination with F. necrophorum and T. pyogenes were considered as footrot (FR) and samples in which F. necrophorum or T. pyogenes was found either alone or in combination were considered as interdigital dermatitis (ID). The overall occurrence of Treponema sp. in ovine foot lesions was 48.0%, and ranged from 33 to 58%. In Treponema positive samples D. nodosus, F. necrophorum and T. pyogenes were present in 34 (27.4%), 66 (54.4%) and 84 (68.5%) in contrast to Treponema negative samples in which these were present in 15 (11.1%), 20 (14.12%) and 17 (12.6%) samples, respectively. The data signifies that Treponema sp. are significantly associated with these foot pathogens and their different combinations with Treponema sp. influence the severity of CODD lesion. The identification of Treponema phylotypes was done by sequencing the 16S rRNA gene fragment of ten representative samples. Out of ten sequences, four (Trep-2, Trep-4, Trep-7 and Trep-10) were identical to Treponema sp. phylotype 1 (PT1) that belongs to phylogroup T. refringens-like, one sequence (Trep-1) was genetically close (90% sequence homology) to Treponema brennaborense while five sequences (Trep-3, Trep-5, Trep-6, Trep-8 and Trep-9) matched with uncultured bacterium clones of treponemes forming separate monophyletic group in phylogenetic tree and could represent new digital dermatitis phylogroup presently containing five ovine specific phylotypes. This is the first report on the presence of Treponema phylotypes other than three digital dermatitis (DD) Treponema phylogroups viz. T. phagedenis-like, T. medium/T. vincentii-like, and T. pedis-like that are frequently detected in CODD lesions. Metagenomic analysis of two representative samples revealed the abundance of genus Treponema in CODD lesion while this genus was absent in swab collected from clinically healthy foot suggesting that it might play primary role in producing CODD. These findings may further aid in understanding the etiopathogenesis of CODD and could help to develop appropriate treatment and mitigation strategies to combat the disease.

In vitro Assessment of Tris Egg Yolk and Soybean Lecithin Based Extenders for Cryopreservation of Crossbred Ram Semen.

BACKGROUND: The replacement of egg yolk with alternative plant-derived soybean lecithin is gaining interest in both animal and human sperm cryopreservation owing to biosecurity issues with egg yolk based extenders. OBJECTIVE: To evaluate the comparative effect of egg yolk and soyabean lecithin based extenders on the quality of cryopreserved crossbred ram semen. METHODS: Pooled ejaculates (total ejaculates = 36) were divided into two aliquots and extended with Tris egg yolk extender (Tris extender) and soybean lecithin based commercial extender (Ovixcell) RESULTS: Among the two extenders, Ovixcell showed better sperm quality both at the pre-freeze (Sperm motility) and post-thaw stages. Lower malondialdehyde (MDA) level (nmol/mL) was observed in Ovixcell as compared to Tris extender. Both sperm quality and MDA level decreased significantly (P < 0.05) from pre-freeze to post-thaw in both the extenders. CONCLUSION: The findings of the present study indicate that Ovixcell is a comparable alternative to Tris extender for the cryopreservation of crossbred ram semen.

Comparison of efficiency of in vitro cloned sheep embryo production by conventional somatic cell nuclear transfer and handmade cloning technique.

Conventional somatic cell nuclear transfer (SCNT) technique of in vitro production of cloned embryos involves use of costly and complicated micromanipulators. Handmade cloning (HMC) technique has been applied as efficient and cost-effective alternative in many livestock species. The aim of the present study was to compare the efficiency of in vitro production and in vitro development of cloned sheep embryos by the two techniques. Cloned embryos were produced by conventional SCNT using micromanipulator apparatus and by HMC technique. Enucleation efficiency and efficiency of fusion with somatic cell (nucleus donor) were compared. Cleavage percentage was observed on day 2 of in vitro culture (IVC), and morula and blastocyst percentages were calculated on day 7 of IVC. Higher enucleation efficiency (96.98 ± 1.01 vs. 93.62 ± 1.03; p > .05) as well as fusion efficiency was obtained with HMC technique than with conventional SCNT (96.26 ± 1.34 vs. 92.63 ± 0.70, p < .05); 181 cloned sheep embryos were produced in vitro by conventional SCNT and 92 by HMC. Cleavage percentage observed on day 2 of in vitro culture was higher in HMC than SCNT (66.92 ± 3.72 vs. 55.97 ± 2.5, respectively, p < .05). Morula percentage obtained was higher in SCNT than HMC (44.12 ± 2.93 vs. 30.43 ± 6.79, respectively, p < .05), whereas blastocyst percentage obtained by HMC was higher (12.46 ± 4.96) than SCNT (5.31 ± 2.25; p > .05). It was inferred that HMC technique provides a cost-effective and efficient method of in vitro production of cloned sheep embryos with a comparatively simpler technique with a possibility of automation. Efficiency of cloned embryo production could be improved further by propagating and standardizing this technique.

Open pulled straw vitrification and slow freezing of sheep IVF embryos using different cryoprotectants.

The aim of the present study was to evaluate the post-thaw survival and hatching rates of sheep blastocysts using different cryoprotectants. In Experiment 1, Day 6 sheep embryos were cryopreserved by a slow freezing protocol using 10% ethylene glycol (EG), 10% dimethyl sulfoxide (DMSO) or a mixture of 5% EG and 5% DMSO. Hatching rates were higher in the 10% EG group than in the 10% DMSO or EG + DMSO groups (30% vs 18% and 20%, respectively). In Experiment 2, embryos were cryopreserved by open pulled straw (OPS) vitrification using either 33% EG, 33% DMSO or a mixture of 16.5% EG + 16.5% DMSO. Re-expansion and hatching rates in the EG + DMSO group (79.16% and 52.74%, respectively) were higher than those in the EG group (64.28% and 30.02%, respectively), whereas the outcomes for the DMSO group were the lowest (45.18% and 8.6%, respectively). In Experiment 3, embryos were cryopreserved by OPS vitrification using either 40% EG, 40% DMSO or a mixture of 20% EG + 20% DMSO. Re-expansion and hatching rates were highest in the EG group than in the EG + DMSO and DMSO groups (92.16% vs 76.30% and 55.84% re-expansion, respectively; and 65.78% vs 45.55% and 14.46% hatching, respectively). In conclusion, OPS vitrification was found to be more efficient for cryopreservation of in vitro-developed sheep embryos than traditional freezing.

Comparison of slow freezing and vitrification on ovine immature oocytes.

BACKGROUND: Immature oocytes are more sensitive to cold injury than mature oocytes. OBJECTIVE: The study was to evaluate the post thaw normal oocytes, cleavage and blastocyst rates of ovine cumulus oocyte complexes (COC's) using different cryoprotectants by slow freezing and Open pulled straw (OPS) vitrification. METHODS: In five replicates, abattoir derived COC's were collected and distributed into three groups. In Experiment 1, COC's were cryopreserved by a slow freezing protocol using 10% concentration of ethylene glycol (EG), 10% dimethyl sulphoxide (DMSO) or 5% EG and 5% DMSO mixture. In Experiment 2 and 3 embryos were cryopreserved by OPS vitrification using either 33% or 40% (EG, DMSO or an equal mixture of EG and DMSO mixture. Normal oocytes post thaw were in vitro matured and parthenogenetically activated. RESULTS: Although, there was no difference in the number of post thaw normal oocytes between the groups, cleavage and blastocyst rates were higher in 10% slow freezing group than any of the vitrified groups. CONCLUSION: The study demonstrates better cryopreservation of ovine COC's by controlled slow freezing than OPS vitrification.

Buffalo (Bubalus bubalis) ES cell-like cells are capable of in vitro skeletal myogenic differentiation.

When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.

Bio-Inspired Untethered Robot-Sensor Platform for Minimally Invasive Biomedical Sensing.

Conventional catheter- or probe-based in vivo biomedical sensing is uncomfortable, inconvenient, and sometimes infeasible for long-term monitoring. Existing implantable sensors often require an invasive procedure for sensor placement. Untethered soft robots with the capability to deliver the sensor to the desired monitoring point hold great promise for minimally invasive biomedical sensing. Inspired by the locomotion modes of snakes, we present here a soft kirigami robot for sensor deployment and real-time wireless sensing. The locomotion mechanism of the soft robot is achieved by kirigami patterns that offer asymmetric tribological properties that mimic the skin of the snake. The robot exhibits good deployability, excellent load capacity (up to 150 times its own weight), high-speed locomotion (0.25 body length per step), and wide environmental adaptability with multimodal movements (obstacle crossing, locomotion in wet and dry conditions, climbing, and inverted crawling). When integrated with passive sensors, the versatile soft robot can locomote inside the human body, deliver the passive sensor to the desired location, and hold the sensor in place for real-time monitoring in a minimally invasive manner. The proof-of-concept prototype demonstrates that the platform can perform real-time impedance monitoring for the diagnosis of gastroesophageal reflux disease.

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PURPOSE: The aim of the present study is to compare the ability of homologous and heterologous embryonic fibroblast feeder layers to support isolation and proliferation of buffalo ES-like cells generated from hatched and expanded blastocysts produced by in vitro fertilization and characterization of derived cells through expression of pluripotent markers. METHODS: Embryonic stem cells were derived from hatched and expanded blastocysts through intact blastocyst culture and enzymatic method respectively and compared for proliferation rate on homologous (buffalo) and heterologous feeder layers (goat and sheep). RESULTS: A total of 69 hatched and 83 expanded blastocysts were used for isolation of inner cell masses which were seeded on buffalo, goat and sheep embryonic feeder layers. Following seeding, attachment rate, primary colony formation rate and survival to maximum number of passages were observed to be higher on homologous feeder layers. CONCLUSIONS: Upon comparison of different feeder layer cells for derivation and maintenance of buffalo ES-like cells from hatched and expanded blastocysts, buffalo embryonic fibroblast cells were able to provide a better environment for maintaining pluripotency in culture conditions.

Activation of zona-free buffalo (Bubalus bubalis) oocytes by chemical or electrical stimulation, and subsequent parthenogenetic embryo development.

Parthenogenetic activation using zona-free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona-free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6-dimethylaminopurine (6-DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona-free oocytes when compared to zona-intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona-free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6-DMAP resulted in the highest blastocyst rate.

Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens.

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin-C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages strongly expressed stage-specific embryonic antigen (SSEA)-4 but lacked expressions of SSEA-1 and SSEA-3. Putative ES cells also expressed tumour rejection antigen (TRA)-1-60, TRA-1-81 and Oct4. Whereas in all early embryonic stages, TRA-1-60 was observed only in the periplasmic space, and TRA-1-81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency-related surface antigen phenotype, which resembles that of the ICM.

Sexual dimorphism and geographical variance: their impact on the reliability of the antilingula as a landmark in human mandibular surgery.

The intraoral vertical ramus osteotomy (IVRO) is an orthognathic procedure that is used to correct dentofacial abnormalities, and is performed by approaching the lateral aspect of the mandibular ramus. This approach, however, precludes visualisation of the inferior alveolar nerve (IAN) on the medial side, thereby placing it at risk of iatrogenic damage. The antilingula, a bony prominence on the lateral mandibular ramus, has been proposed as a landmark for prediction of the IAN's location during IVRO. The current study aimed to evaluate the variation in incidence and position of the antilingula, and therefore to determine its suitability as a surgical landmark during IVRO. The study included 480 dry hemimandibles from eight geographical populations from the Duckworth Collection in Cambridge. Skulls were sexed by visual analysis of dimorphic traits. Positional relations were determined through the digitisation of nine anatomical landmarks. The antilingula was identified in all specimens. No significant difference was identified in the positional relation between the antilingula and mandibular foramen between sexes, but multiple differences were identified in this relation between geographical populations. Our data showed that, irrespective of geographical variation, an osteotomy performed 8mm posterior to the antilingula would avoid the mandibular foramen in 98.8% of cases.

Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis).

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.

Differential vulnerability of oculomotor, facial, and hypoglossal nuclei in G86R superoxide dismutase transgenic mice.

In recent years, several mouse models of amyotrophic lateral sclerosis (ALS) have been developed. One, caused by a G86R mutation in the superoxide dismutase-1 (SOD-1) gene associated with familial ALS, has been subjected to extensive quantitative analyses in the spinal cord. However, the human form of ALS includes pathology elsewhere in the nervous system. In the present study, analyses were extended to three motor nuclei in the brainstem. Mutant mice and control littermates were evaluated daily, and mutants, along with their littermate controls, were killed when they were severely affected. Brains were removed after perfusion and processed for Nissl staining, the samples were randomized, and the investigators were blinded to their genetic status. Stereologic methods were used to estimate the number of neurons, mean neuronal volumes, and nuclear volume in three brainstem motor nuclei known to be differentially involved in the human form of the disease, the oculomotor, facial, and hypoglossal nuclei. In the facial nucleus, neuron number consistently declined (48%), an effect that was correlated with disease severity. The nuclear volume of the facial nucleus was smaller in the SOD-1 mutant mice (45.7% difference from control mice) and correlated significantly with neuron number. The oculomotor and hypoglossal nuclei showed less extreme involvement (<10% neuronal loss overall), with a trend toward fewer neurons in the hypoglossal nucleus of animals with severe facial nucleus involvement. In the oculomotor nucleus, neuronal loss was seen only once in five mice, associated with very severe disease. There was no significant change in the volume of individual neurons in any of these three nuclei in any transgenic mouse. These results suggest that different brainstem motor nuclei are differentially affected in this SOD-1 mutant model of ALS. The relatively moderate and late involvement of the hypoglossal nucleus indicates that, although the general patterns of neuronal pathology match closely those seen in ALS patients, some differences exist in this transgenic model compared with the progression of the disease in humans. However, these patterns of cellular vulnerability may provide clues for understanding the differential susceptibility of neural structures in ALS and other neurodegenerative diseases.

Semiquantitative determination of ajmaline by the weisz ring-oven technique.

Ajmaline has been determined semiquantitatively with p-dimethylaminobenzaldehyde as reagent, with an error of +/-2%.

Cytologic, histologic and clinical correlations of precancerous and cancerous oral lesions in 57,518 industrial workers of Gujarat, India.

A group of 57,518 industrial workers of Gujarat, India were screened for oral cancerous and precancerous oral lesions between 1967 and 1971. All subjects were 35 years of age or older and 95 per cent were males. The 27,841 oral lesions found were examined by cytologic scrapings and 13,230 were biopsied. Also, all lesions were followed clinically to assure a malignancy was not overlooked. There were 51 oral cancers diagnosed (.18% of the lesions and .09% of the entire study group). Computing all the cytologic smears, there was an overall accuracy exceeding 99 per cent. When assessing just the malignancies, the accuracy decreased to 80.4 per cent. Since there was a high degree of clinical suspicion on the part of the screeners, only one unsuspected cancer was discovered by cytology. There were four false positive interpretations; and 53 other specimens classified as "suspicious" subsequently were shown to be benign. Although exfoliative cytology has proved useful in assessing oral lesions as an adjunct to biopsy, the low frequency of oral cancer limits the value of this technique as a screening modality. Most false negatives have been associated with leukoplakic (hyperkeratotic) lesions. Therefore, in a persistent oral lesion, even though a cytologic scraping may not be suspicious or characteristic of malignancy, a biopsy should still be strongly considered.

In vitro development of goat-sheep and goat-goat zona-free cloned embryos in different culture media.

The gradual decline in the genetic diversity of farm animals has threatened their survival and risk of their extinction has increased many fold in the recent past. Endangered species could be rescued using interspecies embryo production. The objective of this study was to investigate the effect of three different culture media on the development of Handmade cloned intraspecies (goat-goat) and interspecies (goat-sheep) embryo reconstructs. Research vitro cleave media (RVCL) yielded higher cleavage and morula-blastocyst development in intraspecies and interspecies nuclear transfer groups compared with G1.G2 and modified synthetic oviductal fluid (mSOFaaci). Cleavage frequency of intraspecies cloned embryos in RVCL, mSOFaaci, and G1.G2 did not differ significantly (87.12%, 82.45%, and 92.52%, respectively). However, the morula/blastocyst frequency in RVCL was greater in mSOFaaci and G1.G2 (51.18% vs. 38.28% vs. 36.50%, respectively). Cleavage and morula/blastocyst frequency in interspecies cloned embryos was greater in RVCL than in mSOFaaci and G1.G2 (76.14% and 42.3% vs. 65.9% and 38.3% vs. 58.56% and 33.1%, respectively). Goat oocytes were parthenogenetically activated and cultured in RVCL, mSOFaaci, and G1.G2 and kept as control. Cleavage and morula/blastocyst frequency in this group was greater in RVCL than in mSOFaaci and G1.G2 (89.66% and 65.26% vs. 85.44% and 48.05% vs. 86.58% and 42.06%, respectively). Conclusively, the results suggest that not only can the interspecies embryos of goat be produced using sheep oocytes as donor cytoplast but also the percentages can be improved by using RVCL media for culturing of the embryos.

Perioperative use of continuous renal replacement therapy for orthotopic liver transplantation.

PURPOSE: We present a retrospective study describing the perioperative use of continuous renal replacement therapy (CRRT) for orthotopic liver transplantation (OLT). MATERIALS AND METHODS: We retrospectively reviewed the clinical course of patients who underwent OLT with the perioperative use of CRRT. The following variables were recorded: Gender, age, indication for transplantation, time when CRRT was initiated, postoperative need for CRRT, and the patient and organ (liver, kidneys) outcome up to 1 year after transplantation. RESULTS: Among 105 patients who underwent OLT from 2006 to 2010; we used CRRT in 12 cases (11.4%) perioperatively, including 9 (8.3%) patients intraoperatively. Perioperative CRRT was employed for volume, electrolyte, and/or pH management. All patients who underwent CRRT perioperatively were alive at 1 month, 10 (83.3%), at 3 and 6 months and 9 (75%) at 1 year after OLT. Only 1 surviving patient (8.3%) required renal replacement therapy at 1 month after surgery. Renal replacement therapy was not required in any surviving patient up to 12 months posttransplantation. CONCLUSION: Perioperative and especially intraoperative use of CRRT therapy can potentially improve the outcomes of patients undergoing OLT.

Effect of post-fusion holding time, orientation and position of somatic cell-cytoplasts during electrofusion on the development of handmade cloned embryos in buffalo (Bubalus bubalis).

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.

Production of interspecies handmade cloned embryos by nuclear transfer of cattle, goat and rat fibroblasts to buffalo (Bubalus bubalis) oocytes.

The possibility of producing interspecies handmade cloned (iHMC) embryos by nuclear transfer from donor cells of cattle, goat and rat using buffalo oocytes as recipient cytoplasts was explored. Zona-free buffalo oocytes were enucleated by protrusion cone-guided bisection with a microblade. After electrofusion with somatic cells, reconstructed oocytes were activated by calcimycin A23187, treated with 6-dimethylaminopurine and were cultured in K-RVCL-50® medium for 8 days. Although the cleavage rate was not significantly different when buffalo, cattle, goat or rat cells were used as donor nuclei (74.6 ± 3.8, 82.8 ± 5.3, 86.0 ± 4.9 and 82.3 ± 3.6%, respectively), the blastocyst rate was significantly higher (P<0.01) for buffalo (51.4 ± 2.6) than for cattle (3.5 ± 1.0) or the goat (2.2 ± 0.9), whereas none of the embryos crossed the 32-cell stage when rat cells were used. However, the total cell number was similar for buffalo-buffalo (175.0 ± 5.07) and cattle-buffalo embryos (178.0 ± 11.84). Following transfer of 3 buffalo-buffalo embryos each to 6 recipients, 3 were found to be pregnant, though the pregnancies were not carried to full term. These results suggest that interspecies blastocyst stage embryos can be produced by iHMC using buffalo cytoplasts and differentiated somatic cells from cattle and goat and that the source of donor nucleus affects the developmental competence of interspecies embryos.