古田 洋樹

It has been investigated that transgenic chicken can be produced by various methods by introducing an exogenous gene into chicken Primordial Germ Cells (PGCs). Blood containing PGCs was collected from the blood vessels of embryos at stages 12-15. An exogenous gene encoding Green Fluorescent Protein (GFP) was introduced into the PGCs by lipofection and electroporation. Electroporation was carried out under 200 V/25 μF, 200 V/200 μF and 200 V/950 μF. GFP expression was observed in PGCs. Efficiency of introduction into PGCs was low. Electroporation was superior to lipofection for the introduction of the exogenous gene into chicken PGCs. PGCs treated for GFP introduction by electroporation were injected into blood vessels of recipient embryos at stages 12-15. After incubation until stage 26, GFP bands were obtained from gonads of embryos, thereby demonstrating that treated PGCs had migrated to the gonad. © Medwell Journals, 2013.

In this study, 714 cows from 26 dairy herds were reclassified as healthy or mastitic cows on the basis of long-term somatic cell count (SCC) in milk. Cows with more than three consecutive lactation records of SCC from the first or second to fifth lactation, were selected, and their BoLA-DRB3 (DRB3) alleles were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Cows with an SCC of < 200 000 cells/mL in all monthly records were classified as healthy (n = 91). Cows with an SCC of > 300 000 cells/mL in two consecutive tests or four non-consecutive tests or cows with an SCC of > 500 000 cells/mL in any one test during lactation, regardless of parity, were classified as mastitic (n = 201). Mastitic cows (n = 153) from another 40 herds were considered to be infected if bacteriological testing revealed mastitis pathogens in milk. Their DRB3 alleles were identified using PCR-sequence-based typing (PCR-SBT). The differences in DRB3 allelic frequencies between healthy cows and cows with various degrees of mastitis were re-investigated. Moreover, the associations of various amino acid motifs in DRB3 alleles with resistance or susceptibility to mastitis pathogens were re-examined. DRB3.2*8(DRB3*1201) and DRB3.2*16(DRB3*1501) alleles were found to be associated with susceptibility, while DRB3.2*22(DRB3*1101), DRB3.2*23(DRB3*2703), and DRB3.2*24(DRB3*0101) alleles were found to be associated with resistance.

Diagnosis of corpus luteum (CL) function by rectal palpation (RP) has been widely used for recipient selection of embryo transfer (ET), a technology essential for genetic improvements in cattle. To examine the accuracy of RP diagnosis method, the relationship between RP-based CL function and reproductive performance was compared in this study. In Experiment 1, CL of Holstein heifers on day 7 after estrus was classified into functional or hypoplastic by RP, and the results were compared with ultrasonographic (US) images and plasma progesterone (P4) levels. As a result, heifers with functional CL judged by RP had a mean maximum CL diameter of 20.1 +/- 3.1 mm on US and a mean P4 concentration of 8.1 +/- 2.3 ng/mL. These values were significantly greater than those of heifers with hypoplastic CL (12.4 +/- 5.4 mm, 4.0 +/- 2.8 ng/mL) (P < 0.001). In Experiment 2, the length of the estrus cycle was examined between functional CL and hypoplastic CL. The rate of heifers with a normal estrus cycle length with 1825 days was significantly lower with hypoplastic CL than with functional CL (16/24 vs. 43/46, P < 0.01). In Experiment 3, 543 inseminated heifers were similarly classified by CL function by RP 7 days after estrus. The heifers with functional CL showed higher pregnancy rate compared with the heifers with hypoplastic CL (75.2 vs. 47.9%, P < 0.0001). Finally, the CL function of 66 heifers was examined by RP on day 7 post-estrus, and ET was performed in 49 (74.2%) heifers with functional CL. As a result, 27 (55.1%) of them became pregnant. Taken together, these results reconfirm that RP on day 7 after estrus is useful for selection of heifers with functional CL.

This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 mu g GnRH (gonadotropin-releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 mu g GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 13 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.

Three experiments were conducted to develop methods to control the amount of water loss and to evaluate the metabolic effects of water condition in the White Leghorn breeder eggs during incubation. One hundred twenty, 54, and 90 julia strain White Leghorn breeder eggs were incubated at 37.8 degrees C, 60% RH in experiments 1, 2, and 3. In experiment 1, eggs were drilled with various bore diameters of 0, 0.5, 1, 2, 3, 4, and 5 mm on the blunt end of the eggshell. In experiment 2, 4 x 4 mm(2) windows were cut into the eggs or the eggs were drilled with 5 holes of bore diameter 2 mm on the blunt end of eggshell. In experiment 3, eggs were drilled with 1, 3, 5, and 7 holes of diameter 2 mm on the blunt end of eggshell. Eggs were treated on d 3 of each experiment and the amount of water loss was recorded on d 19 of incubation. Embryo growth was evaluated in experiments 2 and 3. In addition, the livers of embryos were collected in the 0-, 1-, 3-, and 5-hole treatment groups after weighing eggs to determine 3-hydroxy acyl coenzyme A dehydrogenase activity. In experiment 1, although higher water loss was observed in all windowed eggs than in control, there were no differences in amount of water loss among all bore diameters. Accordingly, that was not successful to control amount of water loss. In experiment 2, higher water loss was observed in drilled eggs at the same levels in windowed eggs as in control. Drilling holes was a more useful treatment to control amount of water loss on incubated eggs than windowing. In experiment 3, amount of water loss increased linearly with increasing number of holes on the blunt end of eggshell. Hepatic 3-hydroxy acyl coenzyme A dehydrogenase activity increased with increasing the number of drilled holes.

It was proved that an exogenous gene was successfully transferred to chicken embryo and Primordial Germ Cells (PGCs) by the injection of the blastoderm. In this experiment, an exogenous gene, Lac Z constructs encoding Escherichia coli beta-galactosidase was introduced into chicken blastoderm (stage X) just ovoposition using lipofection reagent. After injection of gene the eggs were incubated in routine manner until developmental stage 12-15. Survival rate of the treated embryo was 35.6%. Lac Z expression was mosaic manner and low efficiency. However, it was obtained Lac Z specific band from chicken PGCs by PCR method.

Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12-15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes, identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method, with resistance and susceptibility to mastitis caused by Streptococci, coagulase-negative Staphylococci, Escherichia coli and Staphylococcus aureus was investigated. Blood samples for DNA extraction were collected from 170 Holstein cows (129 mastitis and 41 healthy cows) from 5 districts in Chiba prefecture, Japan. Susceptibility or resistance to the mastitis-causing pathogens was thought to vary by the presence of amino acid substitutions at the 9, 11, 13, and 30 positions. DRB3*0101 and DRB3*1501 had amino acid motifs of Glu9, Ser11, Ser13, and Tyr30, and they were considered to have susceptibility to all 4 mastitis pathogens. In contrast, DRB3*1101 and DRB3*1401 had amino acid motifs of Gln9, His11, Gly13, and His30 in these positions, and they also had Val86, so these alleles were considered to have resistance to Streptococcal and coagulase-negative Staphylococcal mastitis. However, in the case of Escherichia coli mastitis, amino acid substitutions at the 9, 11, 13, and 30 positions had little effect, but rather substitutions at the 47, 67 positions of pocket 7, and at the 71, 74 positions of pocket 4, Tyr47, Ile67, Ala71, and Ala74, were associated with resistance. This motif was present in DRB3*1201.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101, *1501, *1201, and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101, *1501, *1201, and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli, and DRB3*1501 might be associated with susceptibility to Escherichia coli. However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci, and DRB3*1201, with resistance to Streptococci, Escherichia coli, and Staphylococcus aureus.

Holstein Cows (n = 702) from 26 dairy herds in the Tama area of Tokyo, Japan were examined for polymorphisms of the BoLA-DRB3 allele using a PCR-RFLP method. Twenty alleles were observed and allelic frequencies ranged from < 1 % to 20.3%. Nine alleles (DRB3.2*24, *16, *8, *23, *22 *3, *11, *10 and *7 in order) constituted 90.0% of all alleles. Somatic cell counts (SCC) were used to classify healthy (group 1), mastitis (group 2) and suspected (group 3) cows. Frequencies of DRB3.2*11 and DRB3.2*23 were slightly higher in group 1 than in group 2, whereas, frequencies of DRB3.2*8 and DRB3.2*16 were slightly higher in group 2 than in group 1. However, none of the differences in frequencies between the two groups were statistically significant. For combinations of alleles, frequencies of DRB3.2*8/*23 (P<0.1) and DRB3.2*16/*24/ (P < 0.05) were significantly higher in group 2 than in group 1, and their odds ratios were 2.1, 2.5, respectively. However, there were no significant differences between genotypes in their effects for SCC. On the other hand, frequency of DRB3.2*23/*23 including combinations of DRB3.2*23 with minor alleles was significantly higher in group 1 than in group 2 (P < 0.01), and the odds ratio was 0.3. Therefore, it was considered that mastitis resistance or susceptibility of cows may vary with the combination of BoLA-DRB3 alleles.

To study behavioral responses to sweet taste stimuli in young chickens, White leghorn chicks were given mixed-solutions of quinine and saccharin or glycine for 10 minutes after being deprived of water for 6 h. The consumption of quinine alone solution was significantly lower than water control (p<0.05), whereas saccharin (1.0 mM) or glycine (0.5 M) produced a recovery from taste aversion of quinine to approximately 82-83% of baseline (control) level. Our results suggest that chicks seem to sense sweetness and this short-term test may be a useful tool to evaluate taste sense for chicks.

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12-15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.

Primordial germ cells (PGCs) of white leghorn Maria line hens embryos as a donor were transferred white leghorn Laura line hens embryos as a recipient. During stage 12 to 15 of incubating fertilized eggs, donor PGCs which were taken out from blood vessels of donor embryos, were transferred into blood vessels of recipient embryos at the same stage. At 15 days of incubation, survival rate of the treated embryos was 35.7% when the PGCs were transferred into the same sex of donor and recipient embryos (homo-sexual) and 38.8% into antagonistic sex ones (hetero-sexual) respectively. Although the gonad of embryos which were transferred PCGs homo-sexually were developed normally, morphological and histological abnormalities were formed in the gonad derived with hetero-sexually transfer. The abnormalities of gonad were observed in 8 out of 33 samples transferred PCGs from male to female and 12 out of 49 from female to male respectively. Size of male right gonad was degenerated and flat sexual cords were observed. The gonads of germ line chimric chicken was detected donor derived DNA polymorphism to genetic diagnosis by LEI92 of microsatellite marker. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.

To obtain transgenic mouse embryos by in vitro fertilization transgenic mouse sperm were produced by electroporation using green fluorescent protein (GFP) gene as a marker. The sperm was cultured in medium GFP gene. Electoroporation was carried out under 200 V-1 mu F, 200V-25 mu F and 200V-50 mu F. The survival rate of sperm decreased by each treatment. Although the DNA transfer into sperm mediated by electroporation was very easy, efficiency of introduction into embryos was very low. Sperm mediated gene transfer may be inferior to microinjection.

A foreign gene (Green Fluorescent Protein; GFP) was injected into the germinal crescent region (GCR) of chicken embryos (stage 9-11) to produce germ-line transgenic chickens. After 3 days of injection, 21 manipulated embryos (19.4%) had the expression of GFP gene in embryonic tissues. The presence of injected gene was recognized in the gonads (95.5%), blood (35.0%) and sperm cells (14.3%) when examined by PCR analysis. In addition, in the progeny test, the fertility (42.9%) of the cross breed between DNA treated chickens was lower than that (88.0%) of the cross breed between the treated chickens and the normal ones. The expression of GFP gene in the progeny embryos (F-0) was not confirmed under a fluorescent microscope. The presence of GFP gene in the progenies was approximately 80% for the hybrid between treated chickens and approximately 29% for the hybrid between the treated chicken and normal ones. In this experiment, on the other hand, some of the treated chicken showed abnormal characteristics such as low fertility and production of normal egg. Collectively, the present study suggests the possibility of successful production of transgenic chicken by means of the introduction of foreign gene into the developing chicken embryos.

The present experiment was designed to elucidate whether transgenic offspring may be produced via germline chimeric chicken. Firstly, gem-dine chimeric chickens were produced via inter-embryonic transfer of primordial germ cells (PGCs) using Rhode Island Red (RIR) chickens and White Leghorn (WH) hens as donor and recipient birds. The PGCs were introduced into the early stage of developmental embryos by using green fluorescent protein (GFP) or Miw Z as marker genes. The introduction of exogenous genes was conducted to blatoderm or germinal crescent of the embryos with lipofection or electroporation methods. Secondly, the PGCs obtained from the donor embryos (RIR) were injected into embryonic blood vessels of the recipient embryos (WL). The expression of exogenous genes GFP and Miw Z transferred from the RIR embryos was confirmed in germinal ridge of recipient WL embryos. The survival rate of the recipient embryos at stage 24-26 was ranged from 38% to 50%. The results from the present experiments indicate that transgenic offspring could be produced via the PGCs obtained from germline chimeric chickens.

Aim: To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. Methods: The DT40 cells incorporated with exogenous gene ( lacZ constructs encoding Escherichia coli beta -galactosidase: beta -gal) were introduced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial 1) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of beta -galactosidase and polymerase chain reaction (PCR) analysis. Results: The survival rates of the manipulated embryos incubated for 3 days (stage 18 -20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively. The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos. Conclusion: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.

It was proved that an exogenous gene was successfully transferred to chicken embryos by the injection of the gene into blastoderm of freshly oviposited fertilized eggs. In this study, green fluorescent protein (GFP) was used as a marker gene. The GFP dissolved in DOTAP Liposomal Transfection Reagent was introduced into the blastoderm by the method of lipofection or electroporation. After the injection of gene, the eggs were incubated in routine manner until developmental stage 11 to 15. Survival rate of the treated embryos at the stage 11 was 46 per cent (27/59) for the lipofection and 49 per cent (81/166) for the electroporation, respectively. Expression percentages of the GFP for the treated eggs were 74 per cent (20/27) for the lipofection and 5 per cent (4/81) for the electroporation, respectively. The GFP was observed in several sites of embryonic tissues and in some of the primordial germ cells (PGCs) in the former method only. Thus lipofection method was considered slightly superior to the electroporation method for the introduction of exogenous gene to chicken embryos.

Aim: To evaluate the characteristics of semen produced by one- and two-years old White Italian ganders during the entire reproductive season, in order to clarify whether the young ganders are responsible for a low fertility rate in young geese. Methods: Males were kept individually in cages under natural light. Semen was collected by dorso-abdominal massage three times a week and routine examination was performed. Results: The mean ejaculate volume (2.1 and 1.6 mL, respectively) and sperm concentration (323 and 281 x 10(6)/mL, respectively) in one-year-old ganders were higher than those of two-year-old ones. The percentages viable spermatozoa of one- and two-year-old ganders were similar (91.4 and 92.3%, respectively), but the percentage of normally formed viable spermatozoa was significantly higher in the older ganders than in the younger (47.8 and 42.9%, respectively, P <0.05). Conclusion: The semina from one or two-year-old Ganders were similar in regard to volume, sperm density and sperm motility, but the percentage of normally formed viable spermatozoa, which is critical for fertilization, was significantly higher in the older ganders. It appears that the ganders an responsible for the low fertility rate in young geese.

Primordial germ cells (PGCs) of White Leghorn chicken embryos as a donor were transferred to Rhode Island Red chicken embryos as a recipient. At 48-50 h (stage 13-15) of incubation of fertilized eggs. donor PGCs, which were taken out From blood vessels of donor embryos, were injected into blood vessels of recipient embryos. Sex of the treated embryos was determined after the transfer of PGCs using remaining blood samples. In the present experiments, survival rate of the treated embryos was 33.3% far homo-sexual and 35.4% for hetero-sexual transfers of PGCs, respectively, when determined at 17 days of incubation. In this study, most of the treated embryos could not survive more than 18 days of incubation, though the reason for that was not clarified in the present work. The gonads removed from embryos that died after 18 days of incubation and the organs from newly hatched chicks were examined for morphological and histological features. The gonads removed from the embryos with home-sexual transfer of PGCs showed normal development in appearance. On the contrary, some (35.3%) of the embryos with hetero-sexual transfer of PGCs possessed abnormal gonads similar to ovotestis by histological observation. In cases where the gonads developed to be normal organs (64.7%) the sex of embryos was the same as recipient ones. The present results suggest that hetero-sexual transfer of the PGCs may bring about the possibility of development of the embryos bearing sexually different gametes, spermatogonia or oogonia.

Aim: This study was designed to investigate the effect of busulfan treatment on the proliferation of chicken primordial germ cells (PGCs) in vivo, focusing on the preferential settlement of PGCs onto the germinal ridges of chicken embryos. Methods: Busulfan (250 ng/egg) was injected into the egg white of freshly oviposited fertilized eggs, which were then incubated. Embryonic development and viability were examined, and exogenous PGCs collected from embryonic blood vessels were injected into the germinal crescent region of recipient embryos. The number of PGCs resided onto germinal ridges of the right and left sides were compared. Results: Busulfan had a slight harmful effect on the embryo viability and the PGCs proliferation. The number of PGCs resided onto the left side of germinal ridges was slightly higher as compared with the right side. Conclusion: Busulfan suppressed the viability of embryos and the proliferation of endogenous PGCs in the recipient embryos. However, the number of exogenous PGCs proliferated was higher in embryos treated with busulfan than those without busulfan. Data also suggest the possibility of a preferential residence of PGCs toward the left side of the germinal crescent region as compared with the right, which may be due toa more advanced functional development of the left gonad than the right. © 1999, Asian Journal of Andrology.