Single-Step Genomic Prediction of Superovulatory Response Traits in Japanese Black Donor Cows.

We assessed the performance of single-step genomic prediction of breeding values for superovulatory response traits in Japanese Black donor cows. A total of 25,332 records of the total number of embryos and oocytes (TNE) and the number of good embryos (NGE) per flush for 1874 Japanese Black donor cows were collected during 2008 and 2022. Genotype information on 36,426 autosomal single-nucleotide polymorphisms (SNPs) for 575 out of the 1,874 cows was used. Breeding values were predicted exploiting a two-trait repeatability animal model. Two genetic relationship matrices were used, one based on pedigree information (A matrix) and the other considering both pedigree and SNP marker genotype information (H matrix). Estimated heritabilities of TNE and NGE were 0.18 and 0.11, respectively, when using the H matrix, which were both slightly lower than when using the A matrix (0.26 for TNE and 0.16 for NGE). Estimated genetic correlations between the traits were 0.61 and 0.66 when using H and A matrices, respectively. When the variance components were the same in breeding value prediction, the mean reliability was greater when using the H matrix than when using the A matrix. This advantage seems more prominent for cows with low reliability when using the A matrix. The results imply that introducing single-step genomic prediction could boost the rate of genetic improvement of superovulatory response traits, but efforts should be made to maintain genetic diversity when performing selection.

Bayesian estimation of genetic parameters for superovulatory response traits in Japanese Black donor cows using count data models.

We estimated genetic parameters for two in vivo embryo production-related superovulatory response traits-total number of embryos and oocytes (TNE) and number of good embryos (NGE)-in Japanese Black donor cows through Bayesian count regression analysis. We used 20,257 records of superovulation treatments from 1546 Japanese Black cows, with 1102 (5.4%) zero-count records for TNE and 3533 (17.4%) for NGE. Two generalized mixed linear models (MLMs; repeatability animal models)-Poisson (POI) and zero-inflated Poisson (ZIP) regression models-were fitted to the untransformed phenotypic records. A Gaussian MLM was also fitted to untransformed phenotypic records (GAU), natural log-transformed records (LOG), and records with Anscombe's variance stabilizing transformation (ANS). The estimated heritabilities and repeatabilities of TNE were 0.30 and 0.43 by POI, 0.35 and 0.47 by ZIP, 0.27 and 0.36 by GAU, 0.21 and 0.31 by LOG, and 0.24 and 0.35 by ANS, respectively. Those of NGE were 0.29 and 0.36 by POI, 0.31 and 0.40 by ZIP, 0.18 and 0.25 by GAU, 0.19 and 0.24 by LOG, and 0.20 and 0.25 by ANS, respectively. Under the ZIP, the estimated heritabilities and repeatabilities of the probability of zero counts were 0.43 and 0.71 for TNE and 0.42 and 0.51 for NGE, respectively, and the rank correlations between estimated breeding values of the 1546 donor cows for superovulation response and those for the probability of zero count were around -0.40 for TNE and -0.50 for NGE.

Genetic relationship between superovulatory response traits and carcass traits in Japanese Black cattle.

We estimated the genetic correlations between superovulatory response traits and carcass traits in Japanese Black cattle. As regards the superovulatory response traits in cows, we analyzed the phenotypic records of the total number of embryos and oocytes (TNE) and the number of good embryos (NGE) collected from 1532 donors between 2008 and 2018. As regards the carcass traits in fattened animals, we analyzed the phenotypic records for cold carcass weight, rib eye area, rib thickness, subcutaneous fat thickness, estimated yield percent, and marbling score for 1448 progenies derived from 596 donors and slaughtered between 2004 and 2020. Variance components were estimated using single-trait and two-trait animal models and the restricted maximum likelihood approach. The estimated genetic correlations with the carcass traits ranged from -0.05 to 0.04 for TNE and from -0.14 to 0.04 for NGE, and their standard errors ranged from 0.10 to 0.14. These results imply that the genetic relationship between the superovulatory response traits in Japanese Black donor cows and the carcass traits in their fattened progenies was weak to negligible. Therefore, we concluded that selecting donors with superior genetic ability for superovulatory responses would not have antagonistic effects on carcass performance in their fattened progenies.

Estimation of genetic parameters for superovulatory response traits in Japanese Black cows.

The aim of this study was to estimate genetic parameters for superovulatory response traits in order to explore the possibility of genetic improvement in Japanese Black cows. We analyzed 19 155 records of the total number of embryos and oocytes (TNE) and the number of good embryos (NGE) collected from 1532 donor cows between 2008 and 2018. A two-trait repeatability animal model analysis was performed for both. Because records of TNE and NGE did not follow a normal distribution, the records were analyzed following no, logarithmic, or Anscombe transformation. Without transformation, the heritability estimates were 0.26 for TNE and 0.17 for NGE. With logarithmic transformation, they were 0.22 for TNE and 0.18 for NGE. With Anscombe transformation, they were 0.26 for TNE and 0.18 for NGE. All analyses gave similar genetic correlations between TNE and NGE, ranging from 0.60 to 0.71. Spearman's rank correlation coefficient between breeding values of cows with more than 10 records was ≥0.95 with both transformations. Thus, the genetic improvement of TNE and NGE of donor cows could be possible in Japanese Black cattle.

Fertility risk factors in transferring Japanese Black embryos into dairy heifers: An epidemiological study.

The aims of this study were 1) to summarize the current status of Japanese Black (JB) embryo transfer into Holstein heifers, which is carried out on a commercial basis in Japan, and 2) to reveal fertility risk factors, including those from the environment (year and season of transfer), recipient (age, number of transfers, clinical status of the ovaries) and embryo (quality, stage, state, genetic background). We used data from 4467 JB fresh or frozen embryo transfers into Holstein heifers conducted by Zen-noh Embryo Transfer Center during 2016-2018, and the differences in fertility risk due to factors related to the environment, recipient, and embryo were statistically evaluated. Differences in fertility risk due to each variable were observed, leading to significant differences in fertility with respect to year of transfer, embryo quality, embryo state, and embryo breed. These results suggest that the fertility of JB embryos might depend on differences in genetic background. There have been no previous reports of differences in embryo fertility due to the differences among JB's bloodline combinations. In the future, overall reproductive efficiency must be monitored, including the effects of different bloodline combinations on the success of embryo recovery and transfer.

Intranasal midazolam administration enhances amnesic effect in rats.

Intranasal (i.n.) administration of midazolam has been shown to be effective and safe for its sedative, anxiolytic, and anticonvulsant effects. However, there has been no investigation on the influence of i.n. administration on midazolam-induced anterograde amnesia. In addition, although the potential of direct drug delivery from the nose to the central nervous system (CNS) has recently become a topic of great interest, it remains unclear whether this pathway is also involved after i.n. midazolam. In this study, we examined the efficacy and the underlying mechanism of i.n. administration compared with intramuscular (i.m.) administration on midazolam-induced amnesia in rats. Equivalent doses of 0.6 mg/kg midazolam were administered via either the i.m or the i.n. route. Anterograde amnesia was assessed by a contextual/cued fear conditioning test. Each animal was conditioned 20 min after drug administration and then tested for a freezing response 24 h later. Midazolam administration by either route produced a similar level of light sedation (minimum spontaneous activity). However, i.n. administration of midazolam induced significantly less freezing behavior compared with i.m. midazolam. Furthermore, in rats with disrupted electrical input from the olfactory epithelium after an olfactotoxicant 3-methylindole administration, the i.n.-mediated enhanced amnesic effect of midazolam was not observed. Our findings indicate that i.n midazolam could probably generate olfactory signals to the brain via benzodiazepine receptors and, compared with i.m. administration, can produce a more significant amnesic effect without alteration in sedative levels. Further clinical studies are warranted.

Anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, suppresses human to bovine cellular responses.

UNLABELLED: The effect of an anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, against the human to bovine cellular response was investigated. METHODS: Human peripheral mononuclear cells (PBMCs) were cocultured with inactivated self-PBMCs (Self), bovine PBMCs with control antibody (Xeno), or bovine PBMCs with IMMU-114 (IMMU-114). Cellular responses were investigated by thymidine incorporation assay, CFSE (carboxyfluorescein diacetate succinimidyl ester)-mixed lymphocyte reaction, and cytokine production in culture medium. RESULTS: Thymidine incorporation rates at a 1:1 responder to stimulator ratio for Xeno + control antibody, Xeno + IMMU-114, Self + control antibody, and Self + IMMU-114 were 14201.3 ± 1968.4, 513.0 ± 49.5, 952.7 ± 128.7, and 423.3 ± 138.8 cpm, respectively (P = 0.032). Those at a 1:2 ratio were 6518.0 ± 690.1, 896.6 ± 92.9, 1051.0 ± 123.6, and 736.0 ± 35.6 cpm, respectively (P = 0.036). CFSE-mixed lymphocyte reaction demonstrated that the frequencies of CFSE-low, CD4(+), and CD25(+) activating T cells in Self, Xeno, and IMMU-114 were 0.27 ± 0.04%, 3.65 ± 0.53%, and 1.23 ± 0.15%, respectively (P = 0.027). Cytokine production in culture medium indicated that IMMU-114 decreased Th1-type cytokines, including interleukin-2, interferon-γ, and tumor necrosis factor-α. CONCLUSION: IMMU-114 effectively suppresses human to bovine cellular responses. The mechanism involves direct inhibition of the interaction between class II human leukocyte antigen-DR-positive cells and CD4(+) T cells, and indirect suppression of Th1 cytokine production.

Donor cells at the G1 phase enhance homogeneous gene expression among blastomeres in bovine somatic cell nuclear transfer embryos.

The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the ß-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.

Comparison of early development in utero of cloned fetuses derived from bovine fetal fibroblasts at the G1 and G0/G1 phases.

In bovine somatic cell nuclear transfer (NT), embryos are more likely to develop to full term when they are derived from fibroblasts at the G1 phase instead of cells at the G0/G1 phase. To better understand the reason for this difference, we examined morphological development in the early pregnancy of NT embryos using G1 phase cells (G1-NT embryos) and G0/G1 phase cells (G0/G1-NT embryos). Blastocysts derived from G1 and G0/G1-NT embryos were transferred to recipient heifers, and the conceptuses at day 50 of gestation were retrieved non-surgically using prostaglandin F(2alpha) and oxytocin. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The percentages of embryos that developed to the blastocyst stage did not differ between G1 and G0/G1-NT embryos. Pregnancy rates at day 30 of recipient heifers carrying G1-NT, G0/G1-NT, IVF, parthenogenetic and AI embryos were similar (57-100%). Two recipient heifers carrying parthenogenetic embryos returned to estrus between days 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at day 50 of gestation of all experimental groups (83%) were recovered non-surgically by several PGF(2alpha) and oxytocin treatments. Recovery rates of normal fetuses derived from G1-NT embryos (83%), IVF embryos (80%) and AI embryos (88%) were greater than those of G0/G1-NT embryos (33%) and parthenogenetic embryos (0%). Our results suggest that NT embryos reconstructed with cells at the G1 phase have a high developmental competence from the time of embryo transfer to day 50 of gestation.

Administration of peripheral blood mononuclear cells into the uterine horn to improve pregnancy rate following bovine embryo transfer.

Embryo transfer (ET) has been used to improve reproductive efficiency and genetic make-up in bovine species. However, the success rate of ET has not been improved since its inception. Here we examined whether administration of autologous peripheral blood mononuclear cells (PBMCs) into the uterine horn can improve pregnancy rates following bovine ET. First we determined that the abundance of interleukin (IL)-1alpha, IL-1beta and IL-8 transcripts in PBMCs was greatest after 24h of culture. PBMCs that had been cultured for 24h were gently administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle. On day 7, the ET was carried out and the pregnancy rate in the PBMC-treated group was compared with that in the non-treated group. The pregnancy rate on day 60 in the PBMC-treated group (76.7%, 56/73) was significantly higher than that in the non-treated group (59.7%, 43/72, p<0.05). These results indicate that administration of autologous PBMCs into the uterine horn improves pregnancy rates following bovine ET.

Subjecting holstein heifers to stress during the follicular phase following superovulatory treatment may increase the female sex ratio of embryos.

The sex ratio of mammals has previously been shown to be affected by maternal stress. In our previous study, the proportion of female embryos collected from superovulated and artificially inseminated Holstein heifers that were frequently placed in stanchions and subjected to transrectal examinations of the ovaries during the follicular phase tended to be higher than the expected 50%. The goal of the present study was to test the validity of this observation using a greater number of heifers. Superovulated heifers were artificially inseminated at 56 and 72 h after PGF(2alpha) treatment using a single batch of frozen semen. Frequent capture (FC), transrectal examination and/or blood sampling were performed at 4-h intervals from 36 to 76 h after PGF(2alpha) treatment (n=13). Nine heifers were used as the Control (non-treatment). Seven-day-embryos were recovered by uterine flushing. Male and female embryos were separated using the loop-mediated isothermal amplification procedure. The proportion of female transferable embryos in the FC group (67.8%, 78/115) was significantly higher than that in the Control group (51.2%, 43/84, P<0.05). The peak concentration of plasma cortisol during the follicular phase following superovulatory treatment was 20.6 ng/ml in the FC group. These results suggest that subjecting heifers to stress during the follicular phase following superovulatory treatment may increase the female sex ratio of embryos.

Early development in utero of bovine nuclear transfer embryos using early G1 and G0 phase cells.

Bovine somatic cell nuclear transfer (NT) embryos can develop to normal calves, but the success rates are still quite low. Recently, enhanced development of bovine NT embryos to full term has been achieved using fibroblasts at the early G1 phase instead of cells at the quiescent (G0) phase. In the present study, we examined the morphological development in utero of NT embryos using early G1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). We produced eG1- and G0-NT blastocysts, and then they were transferred to recipient heifers for transient development in utero up to day 14 of gestation. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The rate of formation of embryonic disks of the recovered embryos was the same among the groups of eG1-NT, IVF, and AI embryos (p>0.05). The formation rate in eG1-NT embryos was significantly higher than that in G0-NT embryos (p<0.05). The lengths of eG1-NT embryos were the same as those of IVF, parthenogenetic, and AI embryos (p>0.05), but significantly shorter than those of G0-NT embryos (p<0.01). We conclude that the morphological development of day 14 embryos derived from eG1-NT embryos was mostly similar to that of AI embryos, but that the morphological development of G0-NT embryos was abnormally large and different from that of AI and eG1-NT embryos.

Relationships among estrous behavior, superovulatory response and grade 1 embryo sex ratio in superovulated Holstein heifers.

In this study, we first attempted to determine whether the timing of artificial insemination affects the sex ratio of seven-day-old embryos in superovulated Holstein heifers. The superovulatory treatment consisted of eight decreasing doses of FSH for 4 days and 2 doses of PGF(2alpha) given with the last two doses of FSH. The superovulated heifers were given a GnRH analogue 48 h after the first PGF(2alpha) treatment and were artificially inseminated 48 h (n=10) or 56 h (n=8) after the first PGF(2alpha) treatment. There were no significant differences in the percentages of unfertilized ova and transferable embryos (grades 1 to 3) between the two groups. The proportions of female grade 1 embryos did not significantly differ from the expected ratio of 50:50 (49.3% at 48 h and 52.5% at 56 h). We then compared the estrous behavior and superovulatory responses of the heifers with a proportion of female embryos of 50% or less (n=7, Low group) to those of the heifers with a proportion of female embryos of more than 50% (n=9, High group). The Low group had a longer duration of estrus and a higher superovulatory response than the High group. These findings offer little encouragement for prediction of the population of female embryos collected from superovulated heifers. Further studies are necessary to evaluate to what degree maternal hormone levels are related to estrus duration and sex ratio.

alpha1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and cre-expressing adenovirus.

BACKGROUND: Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle. METHODS: alpha1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using alpha1,3GT fetal fibroblasts, and one alpha1,3GT knockout calf was generated but died shortly after birth (day 287). RESULTS: Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. CONCLUSION: The alpha1,3GT knockout- and antibiotic-resistance gene free (alpha1,3GT(-/-)neo-) cells could be cloned normally. Thus, cloned cattle from alpha1,3GT(-/-) neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

Examination of a modified cell cycle synchronization method and bovine nuclear transfer using synchronized early G1 phase fibroblast cells.

Somatic cell nuclear transfer has a low success rate, due to a high incidence of fetal loss and increased perinatal morbidity/mortality. One factor that may affect the successful development of nuclear transfer embryos is the cell cycle stage of the donor cell. In order to establish a cell cycle synchronization method that can consistently produce cloned embryos and offspring, we examined the effects of different combinations of three cell treatments on the recovery rate of mitotic phase cells using bovine fetal fibroblasts. In the first experiment, we examined the recovery rate of mitotic phase cells by a combination of treatment with a metaphase arrestant (1 microM 2-methoxyestradiol), shaking the plate and selecting cells with a diameter of 20 microns. As a result, 99% of mitotic phase cells were recovered by repeating the combined treatment of metaphase arrestant and shaking, and collection of cells with a specific diameter. In the second experiment, nuclear transfer was carried out using early G1 phase cells by choosing pairs of bridged cells derived from mitotic phase cells recovered by the combined treatment of 1 microM 2-methoxyestradiol and shaking, and collection of cells with a diameter of 20 microns. The reconstructed embryos were transferred to recipient heifers to determine post-implantation development. Development of embryos reconstructed from early G1 phase cells from the >/=6 cells stage on Day 3 to the morula-blastocyst stage on Day 6 was 100%. Ten blastocysts constructed from two cell lines were transferred into 10 recipient heifers. Nine of the 10 recipients delivered single live calves. In conclusion, mitotic phase bovine fibroblast cells were easily recovered by the combined treatments of 1 microM 2-methoxyestradiol, shaking, and selecting cells of the appropriate diameter. Furthermore, nuclear transfer using cells in the early G1 phase as donor cells gave a high rate of offspring production.

Heterozygous disruption of the alpha1,3-galactosyltransferase gene in cattle.

BACKGROUND: Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the alpha1,3-galactosyltransferase (alpha1,3-GT) gene in cattle. METHODS: The alpha1,3-GT gene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine alpha1,3-GT gene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the alpha1,3-GT locus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer. RESULTS: To establish fetal fibroblasts that were heterozygously disrupted at the alpha1,3-GT locus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the alpha1,3-GT gene. CONCLUSIONS: Heterozygous knockout of the alpha1,3-GT gene was performed in JBC, and production of a homozygous alpha1,3-GT knockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.