The world's first cloned swamp buffalo (Bubalus bubalis) derived from adult ear skin fibroblast has been reported. Donor fibroblast cells were produced from biopsies taken from adult male ear skin and in vitro matured oocytes obtained from a slaughterhouse were used as cytoplasts. A total of 39 blastocysts and 19 morulae fresh embryos were transferred into 12 recipient buffaloes. Progesterone assays indicated establishment of pregnancy in 10 of the 12 buffaloes (83.3%) after 45 days, with six animals still pregnant at 3 months. One recipient maintained pregnancy to term and naturally delivered a 40 kg male calf after 326 days of gestation. DNA analysis showed that the cloned calf was genetically identical to the donor cells. Genotype analyses, using 12 buffalo microsatellite markers, confirmed that the cloned calf was derived from the donor cell lines. In conclusion, the present study reports, for the first time, the establishment of pregnancy and birth of the first cloned Thai swamp buffalo derived from adult ear skin fibroblast cells.

Studies on in vivo digestion, rates of passages, metabolism of nitrogen, urinary purine derivative excretion and blood metabolites were carried out in Thai Brahman cattle and Thai swamp buffaloes (16 months old). The animals were fed mixed diets based on pineapple (Ananas comusus) waste silage containing urea-N (NPN) and true protein from a concentrate (TP). The Brahman cattle (310 +/- 15 kg) were heavier than the swamp buffaloes (195 +/- 9.4 kg) and had higher dry matter (DM), organic matter (OM) and neutral detergent fiber (NDF) intakes when compared on the basis of their metabolic body weight (BW(0.75)), but these intakes did not differ significantly when the diets of each animal species were compared. The total tract, apparent digestibilities of dry matter (DM) and organic matter (OM) were not significantly different between the animal species when comparing the two types of diets. The NDF digestibility was significantly (P < 0.01) decreased in both animal species when fed the TP diet, but was significantly (P < 0.01) greater in cattle than in swamp buffaloes. The passage rate digesta k(1) (P < 0.01) and the passage rate digesta k(2) (through the caecum and proximal colon) (P < 0.03) were significantly slower, and the total mean retention time (TMRT) (P < 0.01) was significantly longer in swamp buffaloes when compared to Brahman cattle, but the transit time (TT) showed no difference (P = 0.07) between the animal species or the diets. The N intakes were not different in both animal species and diets, but urine-N was greater (P < 0.05) in Brahman cattle than that in swamp buffaloes. Urine N and digestibility of N were significantly (P < 0.04) higher in animals fed the NPN diet than those fed the TP diet. Urinary purine derivatives (PD) and the creatinine (Cr) excretion of swamp buffaloes were significantly (P < 0.01) lower than those in Brahman cattle. Plasma urea-N (BUN) concentration was significantly (P < 0.01) higher in swamp buffaloes than that in Brahman cattle, but plasma glucose and insulin concentrations were significantly (P < 0.01) higher in Brahman cattle than in swamp buffaloes. The concentrations of non-esterified fatty acids (NEFA) were not significantly (P > 0.05) different in animals fed different diets. The present study demonstrated that Brahman cattle were better in fiber digestibility than swamp buffaloes at utilizing pineapple waste silage with both N sources.

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This paper aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. Oocytes were matured and fertilized in vitro, and they were fixed at various time points after IVF. At 6 h after IVF, 44.4% matured oocytes were penetrated by spermatozoa. Partial ZP digestion, however, did not improve fertilization rate compared to control (P > .05). At 12 h after IVF, the fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the paternal chromatin continued to decondense. A sperm aster was observed radiating from the base of the decondensing sperm head. At 18 h after IVF, most presumptive zygotes had reached the pronuclear stage. The sperm aster was concurrently enlarged to assist the migration and apposition of pronuclei. Cell cleavage was facilitated by microfilaments and firstly observed by 30 h after IVF. In conclusion, the cytoskeleton actively involves with the process of fertilization and cleavage in swamp buffalo oocytes. The centrosomal material is paternally inherited. Fertilization failure is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate.

ABSTRACT:Methods have been devised for detecting polymorphisms in the D-loop regions using the PCR followed by restriction enzyme digestion to reveal restriction fragment length polymorphism. Total DNA were extracted from the white blood cell of swamp buffaloes. Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of local swamp buffalo from Lamphayaklang Livestock Research and Breeding Station in Thailand were analyzed using three enzymes (Ava lI, Bam H I and Hae III) which recognize 4 and 6 nucleotides. Among the 49 animals were analyzed, Ava II, BamH I and Hae III revealed different polymorphisms. Ava II showed two types of cleavage pattern. BamH I showed two type of cleavage pattern. And Hae III showed four types of cleavage pattern. The polymorphic fragments patterns for these three restriction endonuclease (Ava II-BamH I-Hae III) revealed five types of D-loop mtDNA in all the animals examined. These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes. These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes.