Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.

This study aimed to investigate the function of the constitutively activating mutation D540G on eel FSHR activity by in vitro functional studies. Site-directed mutagenesis was carried out to generate the D-to-G mutation at position 540 of the pcDNA3-eel FSHR construct. Vectors expressing either wild type or mutant receptor were transfected into Chinese hamster ovary (CHO-K1) cells. The functional characteristics of both the wild type and mutant receptors were analyzed by a cAMP assay. cAMP accumulation was highly increased in cells transfected with the D540G mutant receptor in a dose-dependent manner. Of note, basal cAMP levels were remarkably increased (~13.1-fold) with expression of this mutant when compared to wild type receptor. These findings suggest that the D540G mutation in the eel FSHR may contribute to ovulation during eel sex maturation as well as play a pivotal role in inducing FSHR activity.

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and 'EOMES transcription factor mRNAs' were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

This study was conducted to examine the oocyte recovery efficiency through having an OPU session once and twice a week. Also, the oocyte recovery efficiency was examined by using OPU after two and three months of rest period. Six cows were used for oocytes collection and were randomly divided into two groups. In experiment 1, OPU sessions were conducted once and twice a week to collect oocytes. The collected oocytes between once and twice OPU groups were classified into four groups (grade 1, 2, 3 and 4) according to the quality of cumulus cells and ooplasm. Based on the result, the percentage of collected oocytes per aspirated follicle number was similar between once and twice OPU session groups (65.5 ± 1.9 and 68.7 ± 1.4 vs.). However, the percentage of grade 1 oocytes from the twice OPU session group was significantly high compared with that of the once a week OPU session group (25.3 ± 0.9 and 32.5 ± 1.2% vs. once and twice session group, respectively, p < 0.05). In experiment 2, the group with three months of rest period tended to have a high percentage of collected oocyte compared with the group with two months of rest period (64.6 and 70.9% vs. 2 and 3 months rest group, respectively, p = 0.62). The percentage of grade 4 in the group with three months of rest period was significantly low compared with the group with two months of rest period group (27.3 and 36.5% vs. two and three months rest group, respectively, p = 0.05). In conclusion, twice a week OPU session is suitable for collection of high quality oocytes by using OPU, and three months of rest period is needed for the recovery of oocyte quality of a donor cow.

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.

Reproductive potential decreases with age. A decrease in male fertility is due to a combination of morphological and molecular alterations in the testes. Transient receptor potential vanilloid receptor-1 (TRPV1) is associated with aging and lifespan, and its activation causes apoptotic cell death in various cell types. However, the effect of TRPV1 on testicular apoptosis in aged mice has not yet been reported. TRPV1 knockout (KO) mice had a longer lifespan than that of wild-type (WT) mice. Lifespan was increased by 11.8% in male TRPV1 KO mice compared to that in WT mice. TRPV1 KO males lived approximately 100 days longer than WT males on average, and the maximum lifespan was markedly extended in TRPV1 KO mice compared with that in WT mice. The TRPV1 expression levels were highly increased in the testes of older mice. TRPV1 was expressed in the entire testes region of the old mice. In addition, old TRPV1 KO mice had lower testicular apoptosis than that of WT mice. Our results show that TRPV1 induces testicular apoptosis and suggest that TRPV1 may be associated with testicular aging.

Recent studies showed that tight junctions (TJs) integrity and assembly are required for blastocyst development in mouse and pig models. However, the biological functions of TJs associated with embryo implantation and maintenance of pregnancy were not investigated yet. To examine whether disrupted TJs affect further embryo development, we employed RNAi approach and inhibitor treatment. The embryos were injected with Cxadr (Coxsackievirus and adenovirus receptor) siRNA for knock down (KD) and treated with Adam10 (A Disintegrin and Metalloproteinase specific inhibitor 10; GI254023X; SI). We compared blastocyst development and paracellular sealing assay using FITC dextran uptake between control and KD or SI embryos. Finally, we transferred control and Cxadr KD or Adam 10 SI treated blastocyst to uteri of recipients. Cxadr KD and Adam 10 SI showed lower blastocyst development and more permeable to FITC-dextran. Moreover, we observed that half of KD and inhibited embryos failed to maintain pregnancies after the second trimester. Our findings suggested that TJs integrity is required for the maintenance of pregnancy and can be used as a selective marker for the successful application of assisted reproduction technologies.