Interferon-Stimulated Gene Expression in Peripheral Blood Leucocytes as a Convenient Prediction Marker for Embryo Status in Embryo-Transferred Japanese Black Cows during the Peri-Implantation Period.

Pregnancy diagnosis during early gestation is important for cattle reproduction. The expression of interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) was studied in embryo-transferred (ET) Japanese Black cattle. ISGs in PBLs-ISG15, MX1, MX2, and OAS1-were detected in multiple ovulation ET cattle using a real-time quantitative polymerase chain reaction, and receiver operating characteristic (ROC) curve analysis was performed. Gestational status was predicted using the average ISG levels during the normal estrous cycle (AVE) and the Youden index from the ROC curve analysis as cutoff values. The ISG15, MX1, and MX2 levels were significantly higher in pregnant cattle (n = 10) than in non-pregnant cattle (n = 23) on gestation day 21, whereas the levels of all ISGs were similar between non-pregnant and non-pregnant cattle with late embryonic death (n = 7). ISG15, MX1, and MX2 appropriately predicted the gestational status of ET cows. The statistical evaluation of the diagnostic accuracy in ET cows on day 21 of gestation presented higher values of sensitivity, specificity, accuracy, and positive predictive values of ISG15, MX1, and MX2 using the Youden index than using the AVE. Therefore, ISG15, MX1, and MX2 are excellent biomarkers of gestational status during the peri-implantation period in ET cattle.

In vitro survival kinetics of microfluidic-sorted bovine spermatozoa.

BACKGROUND: The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples. OBJECTIVES: This study primarily aims to obtain baseline information on sorted spermatozoa according to its characteristics and in vitro life span. MATERIALS AND METHODS: Frozen-thawed bull semen was subjected to microfluidic sperm sorting using diffuser-type microfluidic sperm sorter (DMSS). After sorting, samples were collected as the sorted spermatozoa and unsorted residual spermatozoa and incubated at 37°C for subsequent evaluation. The samples were assessed at different time points (0 or 1, 6, and 24 h) in terms of motility, which was measured by computer-assisted sperm analysis (CASA), membrane integrity, mitochondrial function, and adenosine triphosphate (ATP) production after sorting (0 h). To determine the characteristics and efficiency of DMSS sorting, the sorted spermatozoa were compared with samples collected using the swim-up method, a conventional method in motile sperm selection. RESULTS: A comparison between the sorted and residual spermatozoa demonstrated significantly higher motility parameters, membrane integrity, and mitochondrial function of the sorted spermatozoa until 6 h after incubation. The time course decrement of membrane and mitochondrial status were subjected to curve fitting and theoretically supported. Sperm ATP production measured immediately after sorting showed higher ATP generation of the sorted spermatozoa compared with the unsorted, frozen-thawed spermatozoa. The motility parameters and mitochondrial activity of DMSS-sorted spermatozoa were higher than the swim-up-collected spermatozoa (p < 0.05). DISCUSSION AND CONCLUSION: These results indicate that DMSS sorting can strictly select highly motile spermatozoa with the ability to maintain its membrane integrity and mitochondrial function related to ATP production. We speculate that the device that is able to sort high-quality spermatozoa can have great potential in assisted reproduction.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle.

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination.

Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility.

BACKGROUND: The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process. RESULTS: Our strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization. CONCLUSIONS: These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility.

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.

Effect of pre-in vitro maturation with cAMP modulators on the acquisition of oocyte developmental competence in cattle.

The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2-4 mm) of FSH-unprimed animals. Pre-IVM with N6,2'-O-dibutyryladenosine 3',5'-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.

A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes.

Interferon tau plays an important role in establishing bovine pregnancy. Interferon-stimulated genes (ISGs) have been examined to identify a suitable indicator for the diagnosis of early gestation in cows. Although ISGs can be specifically detected in peripheral white blood cells during early gestation, its reliability remains to be validated. In the current study, a predictive threshold level of ISGs to determine pregnancy in cows during Days 20-22 of gestation was verified by analyzing the expression of ISGs in granulocytes and peripheral blood leucocytes (a total of 57 cows were used, 28 of which were pregnant and 29 were non-pregnant). Four genes, interferon-stimulated gene 15 ubiquitin-like modifier (ISG15), MX dynamin like GTPase (MX) 1, MX2, and 2'-5'-oligoadenylate synthetase 1 (OAS1), were analyzed via quantitative RT-PCR and a receiver operating characteristic (ROC) curve was produced to visualize diagnostic accuracy measures. The expression values of the four ISGs during the estrous cycle (100 collection points from 65 cattle) were used to determine a pregnancy prediction cutoff value. Pregnancy status was determined using these cutoff values and then confirmed by ultrasonography. ROC analysis was then applied to confirm the accuracy of the pregnancy statuses (positive and negative) statistically. The statistical evaluation of the diagnostic accuracy measurements suggested that the average values of ISG15 and MX2 in granulocytes were reliable indicators of pregnancy within the three weeks after insemination with 80% accuracy. Average ISG15 and MX2 levels during the estrous cycle were more reliable biomarkers for the prediction of gestation. They predicted negative and positive pregnancies efficiently within three weeks after artificial insemination.

Transcriptomic signature of the follicular somatic compartment surrounding an oocyte with high developmental competence.

During antral folliculogenesis, developmental competence of prospective oocytes is regulated in large part by the follicular somatic component to prepare the oocyte for the final stage of maturation and subsequent embryo development. The underlying molecular mechanisms are poorly understood. Oocytes reaching the advanced stage of follicular growth by administration of exogenous follicle-stimulating hormone (FSH) possess higher developmental competence than oocytes in FSH-untreated smaller follicles. In this study, the transcriptomic profile of the cumulus cells from cows receiving FSH administration (FSH-priming) was compared, as a model of high oocyte competence, with that from untreated donor cows (control). Ingenuity Pathway Analysis showed that cumulus cells receiving FSH-priming were rich in down-regulated transcripts associated with cell movement and migration, including the extracellular matrix-related transcripts, probably preventing the disruption of cell-to-cell contacts. Interestingly, the transcriptomic profile of up-regulated genes in the control group was similar to that of granulosa cells from atretic follicles. Interferon regulatory factor 7 was activated as the key upstream regulator of FSH-priming. Thus, acquisition of developmental competence by oocytes can be ensured by the integrity of cumulus cells involved in cell-to-cell communication and cell survival, which may help achieve enhanced oocyte-somatic cell coupling.

Promising system for selecting healthy in vitro-fertilized embryos in cattle.

Conventionally, in vitro-fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors--(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage--pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.

Follicular growth-stimulated cows provide favorable oocytes for producing cloned embryos.

We examined the influence of recipient oocytes on in vitro development, oxygen consumption, and gene expression in the resulting cloned bovine embryos. Oocytes derived from slaughterhouse ovaries and ovum pickup (OPU)-derived oocytes were used as recipient cytoplasts for the production of cloned embryos. A series of OPU sessions was conducted on Holstein cows without follicular growth treatment (FGT). In the same cows, we then performed dominant follicle ablation and subsequently administered follicle-stimulating hormone and prostaglandin F(2α) with controlled internal drug release device before a second series of OPU. Cumulus cells collected from single Holstein cows were used as donor cells. After measurement of oxygen consumption at the blastocyst stage with modified scanning electrochemical microscopy, analysis of 10 genes (CDX2, IFN-tau, PLAC8, OCT4, SOX2, NANOG, ATP5A1, GLUT1, AKR1B1, and IGF2R) was performed with real-time RT-PCR. Rates of fusion, cleavage, and blastocyst formation were not different among the treatment groups. Levels of oxygen consumption in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT were significantly lower than in blastocysts derived from artificial insemination (AI). However, oxygen consumption was increased in cloned blastocysts derived from OPU with FGT, depending on the individual oocyte donor. Furthermore, gene expression of IFN-tau and OCT4 in cloned blastocysts derived from OPU with FGT was similar to that in AI-derived blastocysts, whereas expression of those genes in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT was significantly different from that in AI-derived blastocysts. Thus, recipient oocytes collected by OPU in combination with manipulation of follicular growth in donor cows are suitable for producing cloned embryos.

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop.

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.