Association and expression study of SEPW1 and JAML as preliminary candidate genes related to lamb odor and flavor.

The current study aimed to identify the SEPW1 and JAML genes in lamb as candidate genes related to lamb odor and flavor. The polymorphism study showed that the SEPW1 gene was polymorphic at the BanI restriction site with three genotypes (AA, AG, and GG), whereas the JAML gene was monomorphic at HhaI with genotype (GG). The association of SEPW1 between genotype and lamb odor and flavor (BCFAs and skatole) was analyzed using GLM (General Linear Model). MNA (4-methylnonanoic) was significantly associated (p < 0.05) with lamb odor and flavor. AA genotype has a lower level of MNA than AG and GG, while MOA (4-methyloctanoic), EOA(4-ethyloctanoic), MI (3-methylindole) and MP (3-methylphenol) was not significantly associated with lamb odor and flavor (p > 0.05). Furthermore, to analyze the mRNA expression of SEPW1 in liver tissues, the lambs were divided into three groups based on the genotypes AA, AG, and GG, however, mRNA expression was not differentially expressed between AA, AG, and GG (p > 0.05). These results will enhance the understanding of the functions of SEPW1 gene relation to odor and flavor traits and will shed light on the polymorphism of SEPW1 gene in lamb as a candidate gene for reducing MNA in lamb.

Microphthalmia-associated transcription factor mutations are associated with white-spotted coat color in swamp buffalo.

A candidate gene analysis of the microphthalmia-associated transcription factor (MITF) gene was used in an attempt to identify the genetic basis for a white-spotted coat color phenotype in the Asian swamp buffalo (Bubalus bubalis carabanensis). Ninety-three buffaloes-32 solid, 38 spotted and 23 white individuals-were Sanger-sequenced for all MITF exons as well as highly conserved intronic and flanking regions. MITF cDNA representing skin and iris tissue from six spotted, nine solid and one white buffaloes was also Sanger-sequenced to confirm detected mutations. Two independent loss-of-function mutations, a premature stop codon (c.328C>T, p.Arg110*) and a donor splice-site mutation (c.840+2T>A, p.Glu281_Leu282Ins8), both of which cause white-spotted coat color in swamp buffaloes, were identified. The nonsense mutation leads to a premature stop codon in exon 3, and likely removal of the resulting mRNA via nonsense-mediated decay pathway, whereas the donor splice-site mutation leads to aberrant splicing of exon 8 that encodes part of a highly conserved region of MITF. The resulting insertion of eight amino acid residues is expected to perturb the leucine zipper part in the basic helix-loop-helix leucine zipper (bHLH-Zip) domain and will most likely influence dimerization and DNA binding capacity. Electrophoretic mobility shift assay was performed using mutant and wild-type MITF proteins and showed that the mutant MITF protein resulting from the splice-site mutation decreased in vitro DNA binding capacity compared to wild-type MITF. White-spotted buffalo bulls are sacrificed in funeral ceremonies in Tana Toraja, Indonesia, because they are considered holy, and our results show that genetic variation causes a tie to the cultural use of these buffaloes.

Three types of polymorphisms in exon 14 in porcine Mx1 gene.

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1c deletion is associated with variation in resistance to the myxovirus family in the pig.

Development of a simple, portable carbon dioxide incubator for in vitro production of bovine embryos.

The objective of this study was to develop a simple and portable CO2 incubator using effervescent granules (EG) and to examine the effect of negative and positive air pressure for in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocytes. In experiment 1, cumulus-oocyte complexes (COCs) were matured (22 h), fertilized (5 h) and cultured (7 days) using 0.25, 0.5 or 1.0 g of EG per 0.6 l added to maintain an optimum level of CO2 (approximately 3, 6 or 12%, respectively) for in vitro production of embryos. Control oocytes, zygotes and embryos were cultured in a standard CO2 incubator. The blastocyst production rates observed on Days 7 to 9 after insemination were 20.5+/-4.2%, 18.5+/-3.9% and 28.7+/-5.1% for the 0.25 g EG, 0.5 g EG treatments and control, respectively. These rates were significantly higher (P < 0.05) than that of the 1.0 g EG treatment (8.7+/-2.6%). The number of cells in the inner cell mass (ICM) and trophectoderm (TE) produced from blastocysts using the control procedure were 40.8+/-2.9 and 81.2+/-5.3, respectively, and were higher (P < 0.05) compared to the 0.50 g EG (34.6+/-2.9 and 66.8+/-5.7) and 1.0 g EG treatments (33.4+/-3.4 and 67.2+/-7.3). In experiment 2, COCs were placed in a small box with 0.25 g of EG so that the effects on IVM, IVF and IVC of positive or negative air pressure could be compared. The blastocyst production rate observed in the negative air pressure treatment (29.6+/-4.6%) was higher (P < 0.01) than that of the positive air pressure treatment (6.2+/-1.5%) or the normal treatment pressure (P < 0.05; 18.7+/-4.2%) but did not differ from that of the control (30.7+/-4.4%). These results indicate that this simple type of incubator with negative air pressure can be successfully used for in vitro production of bovine embryos and could be used at the field level.

Effects of centrifugation and lipid removal on the cryopreservation of in vitro produced bovine embryos at the eight-cell stage.

The effects of intracellular lipid polarization and lipid removal treatments on the postthawed in vitro development of frozen bovine embryos at the 8-cell stage were studied. As the first step, bovine presumptive zygotes were centrifuged at 16,000 g for 20 min for the cytoplasmic lipid polarization and their lipid layers were removed by micromanipulation in order to examine the influence of these treatments on the developmental capacity of bovine zygotes. As the second step, bovine embryos developed to the 8-cell stage following centrifugation treatment at various forces (8000, 12,000, and 16,000 g) or lipid removal treatment at the 1-cell stage were frozen in 1.8 M ethylene glycol + 0.05 M trehalose supplemented with 5% polyvinylpyrrolidone in a one-step procedure. There were no significant differences among the control (nontreatment), lipid-polarized, and lipid-removed groups with respect to the developmental capacity of fresh nonfrozen zygotes (experiment 1). The rates of survival and development to the blastocyst of frozen-thawed 8-cell embryos increased slightly with increasing force of centrifugation (experiment 2). The rate of development into blastocysts of the frozen-thawed 8-cell embryos was significantly higher in the groups that underwent centrifugation (at 16,000 g for 20 min; P < 0.05) or lipid removal (P < 0.01) treatments than the control (intact) group. However, there were no significant differences among the groups with respect to the rate of development to the expanded/hatched blastocyst stage. In addition, the mean cell numbers of embryos developed into blastocysts (day 8) derived from frozen-thawed 8-cell embryos tended to be low in the centrifugation and lipid removal groups compared to the controls (experiment 3). These results suggest that although the centrifugation with or without lipid removal treatments has no detrimental effects on the developmental capacity of bovine zygotes, the freezing tolerance of bovine 8-cell embryos was not improved by these treatments.

Morphological classification of the ovaries in relation to the subsequent oocyte quality for IVF-produced bovine embryos.

Although some inferences have been made regarding the morphological aspects of the ovaries in relation to the subsequent oocyte developmental competence in an in vitro system, the influence of ovarian morphology, taken as a pair, has yet to be demonstrated. The present study addresses this limitation. Forty pairs of ovaries from 5 morphological classes were examined to determine whether their characteristics could influence oocyte yield and developmental competence in vitro. An ovary was designated as bearing a corpus luteum (CL) with a dominant follicle (DF) a cyst (CY) or none of these structures (NO). Thus, the paired classes considered in this study consisted of 1) CL-NO 2) CL-DF 3) CL + DF-NO 4) NO-DF and 5) NO-NO. Comparisons were made among the members of 3 subgroups CL, NO and DF. Within the CL-subgroup, the pairs of CL-NO ovaries resulted in higher (P < 0.01) number of oocytes, cleavage rates and blastocyst formation per ovary than in the other categories (CL + DF-NO and CL-DF), with the latter being superior (P < 0.01) to that of CL + DF-NO in terms of cleavage rates. In the NO-subgroup, NO-CL pairs yielded higher (P < 0.01) rates of oocyte recovery and cleavage than the NO-DF pairs, and the latter was inferior (P < 0.05) to that of NO-NO ovaries for the 2 indices. Further, blastocyst rates from the NO-CL pairs was higher (P < 0.01) compared with those of NO-CL + DF, NO-DF, and NO-NO groups. And, in the DF-subgroup, the DF-CL pairs gave a higher (P < 0.05) oocyte yield and cleavage rate (P < 0.01) than the pairs of DF-NO ovaries but not significantly different in blastocyst formation. The overall oocyte recovery, cleavage and blastocyst rates for the 5 classes were, in a decreasing order CL-NO; NO-NO; CL-DF; CL + DF-NO; and DF-NO. Our results suggest that the morphological classification of ovarian pairs could be a useful means for predicting the developmental competence of oocytes in vitro, and that the presence of a dominant follicle in either one or both ovaries of a pair has a negative effect on the IVF-produced bovine embryos.

The effect of sperm-oocyte incubation time on in vitro embryo development using sperm from a tetraparental chimeric bull.

The present study was designed as 5 x 4 factorial to investigate the effects of using sperm from 5 bulls, and varied sperm-oocyte incubation times (5, 10, 15 and 20 h) on the fertilization, cleavage rates and blastocyst formation on an in vitro bovine embryo production system. The bulls included a tetraparental Chimera, its sires (Japanese Black and Limousin), its maternal grand-sires (Japanese Brown and Holstein). The proportion of polyspermy, 2-pronuclei formation, fertilization, cleavage and development to blastocyst were affected (P < 0.001) by the duration of sperm-oocyte incubation, as well as by the interaction between bulls and their corresponding sperm-oocyte incubation time. Blastocyst rate observed after 5 h in oocytes inseminated with Chimera, Japanese Black and Limousin were higher (p < 0.05) than those observed at 20 h incubation. The proportion of blastocysts from oocytes inseminated with Japanese Black observed at 10 h of incubation did not differ from that of Chimera, but both were higher (p < 0.05) than those observed for the Limousin, Japanese Brown and Holstein sires. The present study showed that there was an effect by the duration of sperm-oocyte incubation on in vitro embryo development. The optimal time of sperm-oocyte incubation for the Chimera was similar to that of its sires (Japanese Black and Limousin) but differed from its maternal grand-sires (Japanese Brown and Holstein). The fertilization rates for the sperm from the Holstein bull increased up to 15 h suggesting that this might be the only bull that would benefit from a long incubation period for insemination.

Fertility of sperm from a tetraparental chimeric bull.

The purpose of the present study was to examine the ability of a tetraparental Chimera in producing IVF embryos. Cumulus oocytes complexes (COCs) were matured in vitro for 22 h. Frozen-thawed sperm of a Chimera (CH), as well as Japanese Black (JB), Limousin (L), Japanese Brown (JBr), Holstein (H) bulls were used for IVF. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h incubation was higher (P < 0.01) with the Chimera (CH) than with the Holstein and in Japanese Brown bulls, but did not differ from Japanese Black and Limousin bull sperm (79.0%, 71.2%, 72.5%, 57.8% and 57.0% for CH, JB, L, JBr and H sperm, respectively). Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera (O-CH) sperm were higher (P < 0.05) than with Japanese Brown (O-JBr) and (P < 0.01) than with Holstein (O-H) sperm, but did not differ from Japanese Black (O-JB) and Limousin (O-L) sperm (36/44, 81.8%; 28/35, 80.0%; 25/36, 69.4%; 19/43, 44.2% and 6/33, 18.2% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The cleavage rates of IVM oocytes inseminated with Chimera sperm were also higher (P < 0.001) than in Holstein, (P < 0.01) Japanese Brown and (P < 0.05) Limousin, but did not differ from Japanese Black sperm (181/239, 75.7%; 123/171, 71.9%; 108/186, 58.1%; 80/196, 40.8% and 30/186, 16.1% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The blastocyst rates of IVM oocytes inseminated with sperm were higher (P < 0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black (69/181, 38.1%; 48/123, 39.0%; 27/108, 25.0%; 7/30, 23.3% and 16/80, 17.8% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). Three findings suggested the sperm from this tetraparental Chimeric bull were able to be used for producing bovine IVF embryos.

Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro.

The objective of this study was to evaluate in vitro fertilization and cleavage rates of frozen-thawed bovine oocytes at the germinal vesicle (GV) stage. In mouse oocytes, spindle microtubule reorganization after GV breakdown is particularly sensitive to cold and readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. The effects of various permeating cryoprotective agents [1.8 M ethylene glycol (EG), 1.3 M ethylene glycol monomethyl ether (EME), and 1.6 M 1,2-propanediol (PROH)] and different concentrations of trehalose (T) and polyvinylpyrrolidone (PVP) on post-thaw developmental capacity were examined. When bovine GV oocytes were frozen slowly in mixtures of 1.8 M EG plus 5% PVP and 0.05 M T, almost 80% developed to metaphase II; 22.2% degenerated after in vitro maturation, and none of those that had been cryopreserved underwent parthenogenetic activation. The total fertilization rate was higher (P < 0.05) for oocytes frozen in a mixture of 1.8 M EG plus 0.05 M T or 0.1 M T than in a mixture of 1.8 M EG with or without 0.2 M T; however, there was no difference in the number of normally fertilized or polyspermic oocytes that had been frozen in various cryoprotective solutions. No significant difference was observed in subsequent development using EG, EME, and PROH for GV oocytes. The addition of 0.05 or 0.1 M trehalose to the freezing solution yielded significantly better cleavage and blastocyst rates than the solutions containing 0.2 M or no trehalose. For unfrozen controls, GV oocytes yielded significantly higher (P < 0.01) cleavage and blastocyst rates compared with frozen-thawed GV oocytes. It was found that 5% PVP had a beneficial effect compared with 10 or 20% concentrations for the development of blastocysts. Transfer of six blastocysts derived from frozen-thawed GV oocytes into three recipient heifers resulted in three pregnancies and the birth of one set of twins and one singleton calf.

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration.

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).

Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone.

In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5 degrees C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20 degrees C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified-warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.

The influence of polyvinylpyrrolidone on freezing of bovine IVF blastocysts following biopsy.

A study was conducted to develop a better freezing protocol for in vitro developed biopsied bovine blastocysts. Biopsied blastocysts were exposed to 1.8 M ethylene glycol (EG) + 0.05 M trehalose (T) and different concentration (5, 10, and 20%) of polyvinylpyrrolidone (PVP). Exposure to the solutions alone did not affect their in vitro development (Experiment 1). Experiments 2, 3, and 4 tested the viability of biopsied blastocysts cryopreserved in 1.8 M EG + different concentrations of T (0, 0.05, 0.1, and 0.3 M), 1.8 M EG + different concentrations of PVP (0, 5, 10, and 20%), and 1.8 M EG + 0.05 M T + different concentrations of PVP (0, 5, 10, and 20%), respectively. The proportion of biopsied blastocysts that reexpanded following cryopreservation in 1.8 M EG + 0.05 M T (38.5%) and 1.8 M EG + 0.1 M T (36.1%) was significantly (P < 0.05) higher than the proportion that reexpanded in 1.8 M EG + 0.3 M T (13.9%) (Experiment 2). The viability and the percentage of embryos that developed to > 250 microns in diameter in the 5, 10, and 20% PVP groups (77.8 and 50.0%, 78.1 and 43.8%, 76.9 and 65.4%, respectively) were significantly higher than those that developed cryopreserved without PVP (55.1 and 20.7%) (Experiment 3). Optimum development of in vitro culture of frozen-thawed biopsied blastocysts was obtained using 1.8 M EG + 0.05 M T and 20% PVP. Analysis of blastocysts > 250 microns in diameter showed that the number of ICM cells of biopsied blastocysts cryopreserved in 1.8 M EG + 0.05 M T with or without PVP was not different from the number of unfrozen biopsied blastocysts. These results indicate that PVP has some beneficial effect on freezing of biopsied bovine blastocysts.

Development in vitro and in vivo of aggregated parthenogenetic bovine embryos.

Mature bovine oocytes were activated with 7% ethanol followed by cytochalasin B or D treatment. Most oocytes extruded a second polar body and formed one pronucleus when treated with 7% ethanol alone [35/43 (81%)]. With ethanol followed by cytochalasin B or D, overall activation frequency was 70% (309/441), with activated oocytes containing two pronuclei. The cleavage rate was not significantly different between treatment with ethanol alone and ethanol followed by 5 micrograms mL-1 cytochalasin B, but it was significantly lower than in fertilized oocytes (P < 0.01). However, the blastocyst production rate was significantly different (P < 0.01) among the treatments. The incidence of parthenogenetic embryos with normal (diploid) complements and with chromosome anomalies (2N/4N) was 68% (17/25) and 32% (8/25) respectively, and this was not affected by cryopreservation treatment. The longitudinal diameter of aggregated-four embryos cultured in vitro was greater (P < 0.01) than aggregated-two or single embryos. One of the aggregated-four parthenogenetic embryos was further cultured in vitro and developed up to Day 27 after activation, with a diameter of 2980 microns. The aggregated-four parthenogenetic embryos were transferred to five recipients. The oestrus was prolonged in three recipients and they returned to oestrus on Day 57, 62 and 67 after the previous oestrus. These results indicate that aggregating parthenogenetic embryos can prolong their survival in vitro and in vivo.