The molecular mechanism by which sperm triggers Ca 2C oscillation, oocyte activation, and early embryonic development has not been clarified. Recently, oocyte activation has been shown to be induced by sperm-specific phospholipase Cz (PLCz). The ability of PLCz to induce oocyte activation is highly conserved across vertebrates. In the present study, porcine PLCz cDNA was identified and the nucleotide sequence was determined. The expression pattern of porcine PLCz mRNA during the period of postnatal testicular development was shown to be similar to that of mouse PLCz. PLCz mRNA expression in the pig and mouse was detected only in the testes when the elongated spermatids had differentiated, and was detected from day 96 after birth in the pig. Histological examination of porcine testis during the period of postnatal development revealed the presence of spermatozoa from day 110 after birth. These findings suggest that the synthesis of PLCz mRNA starts when spermiogenesis is initiated. Microinjection of porcine PLCz complementary RNA into porcine oocytes demonstrated that porcine PLCz has the ability to trigger repetitive Ca 2C transients in porcine oocytes similar to that observed during fertilization. It was also found that porcine PLCz cRNA has the potential to induce oocyte activation and initiate embryonic development up to the blastocyst stage.
Preimplantation genomic selection using genomic estimated breeding values (GEBVs) based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. To develop a preimplantation genomic selection system for carcass traits in Japanese Black cattle, we investigated the accuracy of genomic evaluation of carcass traits using biopsied embryonic cells (Experiment 1); we also performed an empirical evaluation for embryo transfer (ET) of vitrified GEBV-evaluated blastocysts to assess the efficiency of the preimplantation genomic selection system (Experiment 2). In Experiment 1, the mean call rate for SNP genotyping using approximately 15 biopsied cells was 98.1 ± 0.3%, whereas that for approximately 5 biopsied cells was 91.5 ± 2.4%. The mean concordance rate for called genotypes between ~15-cell biopsies and the corresponding biopsied embryos was 99.9 ± 0.02%. The GEBVs for carcass weight, ribeye area, and marbling score calculated from ~15-cell biopsies closely matched those from the corresponding calves produced by ET. In Experiment 2, a total of 208 in vivo blastocysts were biopsied (~15-cell) and the biopsied cells were processed for SNP genotyping, where 88.5% of the samples were found to be suitable for GEBV calculation. Large variations in GEBVs for carcass traits were observed among full-sib embryos and, among the embryos, some presented higher GEBVs for ribeye area and marbling score than their parents. The conception rate following ET of vitrified GEBV-evaluated blastocysts was 41.9% (13/31). These findings suggest the possible application of preimplantation genomic selection for carcass traits in Japanese Black cattle.
Background Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. Results A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900–3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1–0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29–0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000–11,000 animals, the accuracy of GEBV for carcass traits can range 0.73–0.79, which is comparable to estimated breeding value obtained in the progeny test. Conclusion Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000–11,000 animals can be a sufficient size of reference population for genomic prediction.
Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.
Genomic selection based on a high-throughput microarray for genotyping single nucleotide polymorphism (SNP) is expected to accelerate genetic improvement in cattle. Recently, a genomic evaluation system for carcass traits, such as carcass weight and marbling score, is being established in Japanese Black cattle. To further increase genetic improvement efficiency in this breed, establishing a genomic evaluation system for pre-implantation embryos before embryo transfer (ET) is required. Here, we examined the correlation between genomic estimated breeding value (GEBV) of carcass traits calculated from embryonic (blastocyst) biopsy cells and from a corresponding calf produced by ET (Experiment 1); we also evaluated the pregnancy rate following ET of GEBV-evaluated blastocysts (GEBV blastocysts) preserved by vitrification (Experiment 2). In total, 16 Japanese Black dams and cryopreserved semen from 6 Japanese Black sires were used for producing in vivo blastocysts (Day 7-8). In Experiment 1, four blastocysts (IETS code 1) were divided into biopsy cells (15-20 cells) and biopsied embryos using a micromanipulator equipped with a micro blade. Biopsy cells were processed for DNA extraction and whole-genome amplification. Freshly biopsied embryos were transferred to recipient cows, and DNA was extracted from the blood or ear cells of the resulting 4 calves. Then SNP genotyping was performed using Illumina bovine LD BeadChip (Illumina, San Diego, CA, USA). The GEBV of 6 carcass traits (carcass weight, ribeye area, rib thickness, subcutaneous fat thickness, estimated yield percent, and marbling score) were calculated using phenotypic and genotypic data from 4,311 Japanese Black steers, and these were compared between biopsy cells and the corresponding calf. In Experiment 2, 134 blastocysts (IETS code 1 and 2) in total were biopsied (10-20 cells), and the biopsied embryos were vitrified by the cryotop method. Biopsy cells were processed for SNP genotyping as in Experiment 1, and the samples in which the call rate was more than 85% were used for GEBV calculation. Based on GEBV records, 24 vitrified GEBV blastocysts were warmed, cultured for 3 to 5h, and 22 GEBV blastocysts that survived (re-expanded) post-culture were transferred to recipient cows. Pregnancy in these cows was diagnosed using ultrasonography during Day 55 to 60 of gestation. In Experiment 1, the SNP call rates of the biopsy cells and corresponding calf were 98.5 to 99.3% and 99.7 to 99.8%, respectively. The GEBV of 6 carcass traits from biopsy cells and from the corresponding calf had almost the same values. In Experiment 2, the SNP call rates of the biopsy cells were ranged from 26.1 to 99.3%. The GEBV of 6 carcass traits varied among full-sib embryos. The pregnancy rate following ET of vitrified GEBV blastocysts was 40.9% (9/22). These results suggest the possible application of a genomic evaluation system for carcass traits at the blastocyst stage in Japanese Black cattle. Further large-scale assessment of pregnancy rates following ET of cryopreserved GEBV blastocysts is required for practical application of the evaluation system.
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