Species identification of Malayan Gaur, Kedah-Kelantan and Bali cattle using polymerase chain reaction-restricted fragment length polymorphism.

Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme digestion. The amplification, using both specific and universal primers, produced a 154- and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.

The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers.

Bali cattle is a domestic cattle breed that can be found in Malaysia. It is a domestic cattle that was purely derived from a domestication event in Banteng (Bos javanicus) around 3,500 BC in Indonesia. This research was conducted to portray the phylogenetic relationships of the Bali cattle with other cattle species in Malaysia based on maternal and paternal lineage. We analyzed the cytochrome c oxidase I (COI) mitochondrial gene and SRY of Y chromosome obtained from five species of the Bos genus (B. javanicus, Bos gaurus, Bos indicus, Bos taurus, and Bos grunniens). The water buffalo (Bubalus bubalis) was used as an outgroup. The phylogenetic relationships were observed by employing several algorithms: Neighbor-Joining (PAUP version 4.0), Maximum parsimony (PAUP version 4.0) and Bayesian inference (MrBayes 3.1). Results from the maternal data showed that the Bali cattle formed a monophyletic clade, and together with the B. gaurus clade formed a wild cattle clade. Results were supported by high bootstrap and posterior probability values together with genetic distance data. For the paternal lineage, the sequence variation is low (with parsimony informative characters: 2/660) resulting an unresolved Neighbor-Joining tree. However, Bali cattle and other domestic cattle appear in two monophyletic clades distinct from yak, gaur and selembu. This study expresses the potential of the COI gene in portraying the phylogenetic relationships between several Bos species which is important for conservation efforts especially in decision making since cattle is highly bred and hybrid breeds are often formed. Genetic conservation for this high quality beef cattle breed is important by maintaining its genetic characters to prevent extinction or even decreased the genetic quality.

Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA.

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/µL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 µM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 µM primers for Cyt b and COI, 0.3 µM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

Phylogenetic relationships of Malayan gaur with other species of the genus Bos based on cytochrome b gene DNA sequences.

The Malayan gaur (Bos gaurus hubbacki) is one of the three subspecies of gaurs that can be found in Malaysia. We examined the phylogenetic relationships of this subspecies with other species of the genus Bos (B. javanicus, B. indicus, B. taurus, and B. grunniens). The sequence of a key gene, cytochrome b, was compared among 20 Bos species and the bongo antelope, used as an outgroup. Phylogenetic reconstruction was employed using neighbor joining and maximum parsimony in PAUP and Bayesian inference in MrBayes 3.1. All tree topologies indicated that the Malayan gaur is in its own monophyletic clade, distinct from other species of the genus Bos. We also found significant branching differences in the tree topologies between wild and domestic cattle.

Cryotop no desenvolvimento de oócitos bovinos imaturos vitrificados / Cryotop and development of vitrified immature bovine oocytes

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10 percent DMSO + 10 percent ethylene glycol (EG) for 30-45sec and 2: 20 percent DMSO + 20 percent EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58 percent respectively. Vitrified oocytes using OPS and EMG showed 26 and 32 percent; and 35 and 46 percent of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes.

Avaliou-se a eficácia de diferentes dispositivos de congelamento (envasamento em palhetas (EP), microscopia eletrônica de grade (MEG) e Cryotop) para vitrificação de ovócitos imaturos de bovinos. Para tal, foram determinados o corpo polar, a metáfase II (MII), a viabilidade e as subsequentes taxas de desenvolvimento. Foram utilizados somente ovócitos com quatro ou cinco camadas de células do cumulus. Os ovócitos foram equilibrados em duas soluções de vitrificação - 1: DMSO (10 por cento) + etilenoglicol (EG; 10 por cento) por 30 a 45 segundos e 2: DMSO (20 por cento) + EG (20 por cento) + sacarose (0,5M) por 25 segundos -, transferidos para os dispositivos de congelamento e mantidos, por 10 dias, em nitrogênio líquido. Imediatamente após serem retirados do nitrogênio, os ovócitos foram removidos dos dispositivos e processados para maturação, fertilização e cultivo in vitro. Os ovócitos vitrificados com o Cryotop apresentaram as maiores taxas de extrusão do corpo polar (CP) e de maturidade nuclear (MII), 41 e 58 por cento, respectivamente. Para os ovócitos vitrificados com EP e MEG, as taxas de CP e as de MII foram, respectivamente, de 26 e 32 por cento e de 35 e 46 por cento. As taxas de viabilidade não diferiram entre os grupos Cryotop e EMG. Os ovócitos vitrificados com Cryotop apresentaram as maiores taxas de clivagem e de blastocisto. Para todas as variáveis estudadas, as taxas para os ovócitos vitrificados foram significativamente menores do que as do grupo-controle (P<0,05). Os resultados deste estudo mostraram a superioridade do dispositivo Cryotop para vitrificação de ovócitos imaturos de bovinos.

Optimal Culture Conditions for Mycelial Growth of Lignosus rhinocerus.

Lignosus rhinocerus is a macrofungus that belongs to Polyporaceae and is native to tropical regions. This highly priced mushroom has been used as folk medicine to treat diseases by indigenous people. As a preliminary study to develop a culture method for edible mushrooms, the cultural characteristics of L. rhinocerus were investigated in a range of culture media under different environmental conditions. Mycelial growth of this mushroom was compared on culture media composed of various carbon and nitrogen sources in addition to C/N ratios. The optimal conditions for mycelial growth were 30℃ at pH 6 and 7. Rapid mycelial growth of L. rhinocerus was observed on glucose-peptone and yeast extract peptone dextrose media. Carbon and nitrogen sources promoting mycelial growth of L. rhinocerus were glucose and potassium nitrate, respectively. The optimum C/N ratio was approximately 10 : 1 using 2% glucose supplemented as a carbon source in the basal media.

Successful cryopreservation of mouse blastocysts using a new vitrification solution.

Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54.2 and one-step, 45.0 versus 60.0%, P > 0.05). These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification.

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.

Effects of various cryoprotectants on the survival of mouse embryos cryopreserved by the quick freezing method.

The effects of concentrations of glycerol, ethylene glycol or dimethylsulphoxide (DMSO) in the presence of either 0.25 M lactose or sucrose on the post-thaw survival of mouse quickly-frozen compacted morulae were studied. In this method, the embryos were directly frozen in liquid nitrogen (LN2) vapor at approximately -170 degrees C for 2 min before being plunged into LN2. High survival rates of frozen-thawed embryos were obtained when the freezing medium contained 3 M ethylene glycol with either 0.25 M lactose or sucrose (76.5 and 70.2%, respectively). When the embryos were frozen in glycerol, significantly high survival was obtained with 3 M glycerol + 0.25 M sucrose (73.5%, P less than 0.001). However, a freezing medium containing DMSO with either sugar gave lower survival rates. At a higher concentration of 4 M, ethylene glycol with 0.25 M lactose gave significantly higher survival rate than glycerol or DMSO (P less than 0.05). Significantly higher rates were obtained at 2 M with all 3 cryoprotectants when the freezing medium contained lactose rather than sucrose (P less than 0.05). This study showed that glycerol and ethylene glycol were effective cryoprotectants in the quick freezing of mouse embryos, while DMSO was less effective. In addition, the protective effects of these cryoprotectants are affected by their concentrations and the type of sugar used.