Quantitative trait loci analysis for growth and carcass traits in a half-sib family of purebred Japanese Black (Wagyu) cattle.

We used a half-sib family of purebred Japanese Black (Wagyu) cattle to locate economically important quantitative trait loci. The family was composed of 348 fattened steers, 236 of which were genotyped for 342 microsatellite markers spanning 2,664 cM of 29 bovine autosomes. The genome scan revealed evidence of 15 significant QTL (<5% chromosome-wise level) affecting growth and carcass traits. Of the 15 QTL, six QTL were significant at the 5% experiment-wise level and were located in bovine chromosomes (BTA) 4, 5, and 14. We analyzed these three chromosomes in more detail in the 348 steers, with an average marker interval of 1.2 cM. The second scan revealed that the same haplotype of the BTA 4 region (52 to 67 cM) positively affected LM area and marbling. We confirmed the QTL for carcass yield estimate on BTA 5 in the region of 45 to 54 cM. Five growth-related QTL located on BTA 14, including slaughter and carcass weights, were positively affected by the same region of the haplotype of BTA 14 (29-51 cM). These data should provide a useful reference for further marker-assisted selection in the family and positional cloning research. The research indicates that progeny design with moderate genotyping efforts is a powerful method for detecting QTL in a purebred half-sib family.

Perturbation of estradiol-feedback control of luteinizing hormone secretion by immunoneutralization induces development of follicular cysts in cattle.

We used immunoneutralization of endogenous estradiol to investigate deficiencies in the estradiol-feedback regulation of LH secretion as a primary cause of follicular cysts in cattle. Twenty-one cows in the prostaglandin (PG) F(2alpha)-induced follicular phase were assigned to receive either 100 ml of estradiol antiserum produced in a castrated male goat (n = 11, immunized group) or the same amount of castrated male goat serum (n = 10, control group). The time of injection of the sera was designated as 0 h and Day 0. Five cows in each group were assigned to subgroups in which we determined the effects of estradiol immunization on LH secretion and follicular growth during the periovulatory period. The remaining six estradiol-immunized cows were subjected to long-term analyses of follicular growth and hormonal profiles, including evaluation of pulsatile secretion of LH. The remaining five control cows were used to determine pulsatile secretion of LH on Day 0 (follicular phase) and Day 14 (midluteal phase). The control cows exhibited a preovulatory LH surge within 48 h after injection of the control serum, followed by ovulation of the dominant follicle that had developed during the PGF(2alpha)-induced follicular phase. In contrast, the LH surge was not detected after treatment with estradiol antiserum. None of the 11 estradiol-immunized cows had ovulation of the dominant follicle, which had emerged before estradiol immunization and enlarged to more than 20 mm in diameter by Day 10. Long-term observation of the six immunized cows revealed that five had multiple follicular waves, with maximum follicular sizes of 20-45 mm at 10- to 30-day intervals for more than 50 days. The sixth cow experienced twin ovulations of the initial persistent follicles on Day 18. The LH pulse frequency in the five immunized cows that showed the long-term turnover of cystic follicles ranged from 0.81 +/- 0.13 to 0.97 +/- 0.09 pulses/h during the experiment, significantly (P < 0.05) higher than that in the midluteal phase of the control cows (0.23 +/- 0.07). The mean LH concentration in the immunized cows was also generally higher than that in the luteal phase of the control cows. However, the LH pulse and mean concentration of LH after immunization were similar to those in the follicular phase of the control cows. Plasma concentrations of total inhibin increased (P < 0.01) concomitant with the emergence of cystic follicles and remained high during the growth of cystic follicles, whereas FSH concentrations were inversely correlated with total inhibin concentrations. In conclusion, neutralization of endogenous estradiol resulted in suppression of the preovulatory LH surge but a normal range of basal LH secretion, and this circumstance led to an anovulatory situation similar to that observed with naturally occurring follicular cysts. These findings provide evidence that lack of LH surge because of dysfunction in the positive-feedback regulation of LH secretion by estradiol can be the initial factor inducing formation of follicular cysts.

Restoring ovulation in beef donor cows with ovarian cysts by progesterone-releasing intravaginal silastic devices.

The objective of this study was to determine the efficacy of a progesterone-releasing intravaginal silastic device (Controlled Internal Drug Release: CIDR) for inducing ovulation in beef cows with persistent ovarian cysts. Fifteen cows with cysts and abnormal cycles for over 40 days were randomly assigned to receive either a single CIDR (CIDR group, n=9), or a CIDR containing no progesterone (blank CIDR) (BLANK group, n=6) for about 14 days. Determination of plasma progesterone levels at the beginning of CIDR treatment indicated 4 of 6 BLANK cows with non-luteinized cysts and 5 of 9 CIDR cows with non-luteinized cysts. In 5 of 6 BLANK cows, one follicular wave appeared and newly emerged dominant follicles increased in size up to 20 mm in diameter and persisted during the experiment, while one cow experienced estrus with spontaneous ovulation. In contrast, during CIDR treatment, 2 or 3 waves, in which dominant follicles were from 7 to 15 mm in diameter, appeared approximately at 7-day intervals. Within 3 days after CIDR removal, estrous behavior was detected followed by ovulation of the dominant follicle in the last wave. All CIDR cows resumed normal cyclicity with 2 follicular waves for over 2 months. Insertion of a CIDR caused a rapid increase of about 2 ng/mL in plasma progesterone. The levels were greater than 1.3 ng/mL until removal of a CIDR, then dropped under 0.3 ng/mL. Concentrations of plasma estradiol in BLANK cows increased during growth of the cystic follicles, with high levels greater than 10 pg/mL for over 10 days. In 4 of 5 cows with non-luteinized cysts, with high plasma estradiol on the day of CIDR insertion, CIDR treatment resulted in rapid decline of estradiol levels. During placement of the CIDR, estradiol levels showed no increase in the growth phase of a newly appeared dominant follicle. After CIDR removal, however, estradiol significantly increased associated with the growth of ovulatory follicles in all 9 cows. A transient increase in plasma FSH levels preceded detection of each follicular or cyst wave in both BLANK and CIDR cows. Pulse frequency and mean concentration of LH in cows with non-luteinized cysts showed values corresponding to those in normal follicular phase. However, throughout CIDR treatment, these parameters reduced to levels found in the normal luteal phase. In cows with luteinized cysts, parameters of LH secretion were as low as in the normal luteal phase before and during CIDR treatment, then increased significantly after CIDR removal. Present results indicate that treatment with CIDR proved effective in restoring ovulation and reestablishing normal cyclicity in beef donor cows with cysts persistent for a long period. The CIDR reduced and maintained LH secretion at normal luteal levels, thereby, inducing atresia of estrogen-active cysts and preventing formation of cysts from the newly emerged follicles.

Chediak-Higashi syndrome mutation and genetic testing in Japanese black cattle (Wagyu).

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disorder that affects several species including mice, humans, and cattle. Evidence based on clinical characteristics and somatic cell genetics suggests that mutations in a common gene cause CHS in the three species. The CHS locus on human chromosome 1 and mouse chromosome 13 encodes a lysosomal trafficking regulator formerly known as LYST, now known as CHS1, and is defective in CHS patients and beige mice, respectively. We have mapped the CHS locus to the proximal region of bovine chromosome 28 by linkage analysis using microsatellite markers previously mapped to this chromosome. Furthermore, we have identified a missense A:T-->G:C mutation that results in replacement of a histidine with an arginine residue at codon 2015 of the CHS1 gene. This mutation is the most likely cause of CHS in Wagyu cattle. In addition, we describe quick, inexpensive, PCR based tests that will permit elimination of the CHS mutation from Wagyu breeding herds.

Six cloned calves produced from adult fibroblast cells after long-term culture.

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as "gene knock-out" by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10-15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10-12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells.

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF, nuclear transfer, and parthenogenetic activation.

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus.

Usefulness of hepatitis C virus RNA counts by second generation HCV bDNA-probe in chronic hepatitis C based on the HCV genotype.

Detection of hepatitis C virus (HCV) RNA by a second generation (ver 2) HCV bDNA-probe method (bDNA-probe) was compared with detection by the first generation (ver 1) assay. The two assays were performed simultaneously with the same serum samples of HCV genotypes 1b, 2a, 2b, 3a, and 3b. The positive rates with ver 1 were 82% for HCV genotype 1b (type 1b), 57.6% for HCV genotype 2a (type 2a), 75.0% for HCV genotype 2b (type 2b), 55.6% for HCV genotype 3a (type 3a), and 93.8% for HCV genotype 3b (type 3b). The positive rates with ver 2 were 95.0% for type 1b, 93.9% for type 2a, 83.3% for type 2b, 100% for type 3a, and 93.8% for type 3b. With Fisher's exact test, the detection rate for type 2a was significantly higher (P = 0.001) with ver 2 than with ver 1. We obtained regression lines using the HCV counts measured by bDNA-probe on the y axis and the HCV counts obtained by an HCV reverse transcriptase (RT)-competitive polymerase chain reaction method (competitive PCR) on the x axis. The gradients for types 1b, 2a, and 3b were greater with ver 2 compared to ver 1. The gradients for types 2a and 3b were the highest: for type 2a, y = 0.135x + 0.6 with ver 1 and y = 0.248x + 0.1 with ver 2; for type 3b, y = 0.366x + 0.1 with ver 1 and y = 0.727x + 0.3 for ver 2. In addition, HCV-RNA counts for all the genotypes tested in this study were significantly higher with ver 2 than with ver 1. Hence, we conclude that ver 2 of the bDNA-probe measures HCV-RNA counts closer to those obtained with competitive PCR than the ver 1 assay.

Characterization of 42 highly polymorphic bovine microsatellite markers.

We have isolated 42 highly polymorphic microsatellite (GT/CA)n markers from Japanese black cattle Wagyu (Bos taurus). Forty-one of the markers were assigned to bovine autosomes with lod scores > 6, through linkage analyses performed on the International Bovine Reference Family Panel (IBRP). The remaining marker showed X-linked inheritance. These markers exhibited an average heterozygosity value of 0.67 with between four and 17 alleles on the IBRP.