Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Evaluating the performance of temporal and spatial early warning statistics of algal blooms.

Regime shifts have large consequences for ecosystems and the services they provide. However, understanding the potential for, causes of, proximity to, and thresholds for regime shifts in nearly all settings is difficult. Generic statistical indicators of resilience have been proposed and studied in a wide range of ecosystems as a method to detect when regime shifts are becoming more likely without direct knowledge of underlying system dynamics or thresholds. These early warning statistics (EWS) have been studied separately but there have been few examples that directly compare temporal and spatial EWS in ecosystem-scale empirical data. To test these methods, we collected high-frequency time series and high-resolution spatial data during a whole-lake fertilization experiment while also monitoring an adjacent reference lake. We calculated two common EWS, standard deviation and autocorrelation, in both time series and spatial data to evaluate their performance prior to the resulting algal bloom. We also applied the quickest detection method to generate binary alarms of resilience change from temporal EWS. One temporal EWS, rolling window standard deviation, provided advanced warning in most variables prior to the bloom, showing trends and between-lake patterns consistent with theory. In contrast, temporal autocorrelation and both measures of spatial EWS (spatial SD, Moran's I) provided little or no warning. By compiling time series data from this and past experiments with and without nutrient additions, we were able to evaluate temporal EWS performance for both constant and changing resilience conditions. True positive alarm rates were 2.5-8.3 times higher for rolling window standard deviation when a lake was being pushed towards a bloom than the rate of false positives when it was not. For rolling window autocorrelation, alarm rates were much lower and no variable had a higher true positive than false positive alarm rate. Our findings suggest temporal EWS provide advanced warning of algal blooms and that this approach could help managers prepare for and/or minimize negative bloom impacts.

Pratylenchus Araucensis (Rhabditida: Pratylenchidae) a Widely Distributed Nematode in Musa Spp. from Colombia.

Pratylenchus is one of the most limiting nematodes of Musaceae production in the world. Knowledge of the nematode species is one of the requirements for its management in the field. This study aimed to identify up to the species level Pratylenchus populations associated with plantain and banana crops in the states of Caldas, Quindío, and Valle del Cauca in Colombia. In these regions, Pratylenchus has been reported to affect these crops in the past, but with records of the nematode only up to the genus level. For this purpose, five populations of Pratylenchus extracted from samples composed of roots and rhizospheric soil, four from plantain and one from banana, were identified through morphological, morphometric, and molecular analysis (sequencing of the D2-D3 of rRNA and cytochrome oxidase subunit I of mtDNA). All populations were identified as P. araucensis, a species reported previously in eastern Colombia, and one that the present study found in the center and southwest of the country, indicating that this species of nematode is widely distributed in the Musaceae-producing areas of Colombia. The present study reports the first COI mtDNA sequences for this species of nematode.

Dynamic reflective color pixels based on molybdenum oxide.

Active materials which show phase transitions, usually known as Phase Change Materials (PCM), have paved the way to a new generation of reconfigurable plasmonic platforms. Tunable color devices have experienced a great development in the recent years. In particular, reflective color filters can take advantage from sunlight to select and reflect a specific resonant wavelength in the visible spectrum range. Reflective displays are usually structural color filters based on asymmetric Fabry-Perot cavities (AFPCs). For a fixed geometry, most of AFPCs filters generate static color, limiting their potential as tunable color devices. Dynamic color is achieved by introducing an active layer whose optical properties can be modulated by an external stimuli. In this paper, we propose AFPCs based on molybdenum oxide (MoOx, 2

Lipid peroxidation in bull semen influences sperm traits and oxidative potential of Percoll®-selected sperm.

Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

An improved acetic acid-urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Influence of seminal plasma during different stages of bovine sperm cryopreservation.

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 106 sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (-65.2%), acrosomal membrane (-34.0%) and mitochondrial potential (-48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.

Endoplasmic reticulum distribution during bovine oocyte activation is regulated by protein kinase C via actin filaments.

Fertilization-induced [Ca2+ ]i oscillations generally depend on the release of calcium ions from the endoplasmic reticulum (ER). Since ER is the main store of calcium ions, it plays an important role in oocyte fertilization. However, the mechanism of ER organization at oocyte activation is unknown. Here, we show that protein kinase C (PKC) is involved in ER distribution during bovine oocyte activation, but not involved in cell cycle resumption and spindle organization. Actin filaments were affected by PKC pharmacological inhibition. In addition, similar to PKC results, the actin-depolymerizing drug cytochalasin B affected the ER distribution during oocyte activation. Specifically, we have demonstrated that ER organization during bovine oocyte activation is regulated by PKC possibly through its action on actin filaments regulation. Taken together, the results presented here provide further information on the pathway involved in the regulation of ER organization during oocyte activation and new insight into the functional role of PKC and actin filaments during this process.

Sperm DNA fragmentation: causes and identification.

Sperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to fragmentation. Routine semen analysis is unable to estimate sperm chromatin damage. Sperm DNA integrity influences sperm functional capability, therefore tests that measure sperm DNA fragmentation are important to assess fertility disorders. Actually, there is a considerable number of methods for assessing sperm DNA fragmentation and chromatin integrity, sperm chromatin stability assay (SCSA modified), sperm chromatin dispersion (SCD), comet assay, transferase dUTP nick end labelling (TUNEL); and protamine evaluation in sperm chromatin assay, such as toluidine blue, CMA3, protamine expression and evaluation of cysteine radicals. This review aims to describe the main causes of sperm DNA fragmentation and the tests commonly used to evaluate sperm DNA fragmentation.

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status.

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.

Distribution and Pathogenicity of Colletotrichum Species Associated With Mango Anthracnose in Mexico.

Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango (Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides. The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2/MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum, C. asianum, C. fructicola, C. siamense, and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum, C. fructicola, C. siamense, and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

Physically meaningful Monte Carlo approach to the four-flux solution of a dense multilayered system.

Due to the complexity of the radiative transfer equation, light transport problems are commonly solved using either models under restrictive assumptions, e.g., N-flux models where infinite lateral extension is assumed, or numerical methods. While the latter can be applied to more general cases, it is difficult to relate their parameters to the physical properties of the systems under study. Hence in this contribution we present, first, a review of a four-flux formalism to study the light transport problem in a plane-parallel system together with a derivation of equations to evaluate the different contributions to the total absorptance and, second, as a complementary tool, a Monte Carlo algorithm with a direct correspondence between its inputs and the properties of the system. The combination of the four-flux model and the Monte Carlo approach provides (i) all convergence warranties since the formalism has been established as a limit and (ii) new added capabilities, i.e., both temporal (transient states) and spatial (arbitrarily inhomogeneous media) resolution. The support between the theoretical model and the numerical tool is reciprocal since the model is utilized to set a Monte Carlo discretization criterion, while the Monte Carlo approach is used to validate the aforementioned model. This reinforces the parallel approach used in this work. Furthermore, we provide some examples to show its capabilities and potential, e.g., the study of the temporal distribution of a delta-like pulse of light.

Deterioration of nutritional status of patients with locally advanced cervical cancer during treatment with concomitant chemoradiotherapy.

BACKGROUND: In Mexico, 80% women with cervical cancer are diagnosed at locally advanced stages and are treated with concomitant chemoradiotherapy. The treatment modality and catabolic state confer a nutritional risk. The present study aimed to thoroughly evaluate the nutritional status and change in body composition of locally advanced cervical cancer (LACC) patients throughout treatment. METHODS: An observational prospective study, carried out at the Mexican National Cancer Institute, included 55 LACC patients. Nutritional status was evaluated before, during and after treatment, using anthropometric, dietary and biochemical measurements. Body composition was analysed using computed tomography images obtained at the time of diagnosis and approximately 4 months after treatment completion. Clinical outcomes were associated with changes in body composition. RESULTS: At the time of diagnosis, no patients were clinically malnourished, although 33.3% presented sarcopenia and most were overweight; by the end of treatment, 69% became clinically malnourished and 58% were sarcopenic. Average weight loss was 7.4 kg (P = 0.001). Adequacy of energy intake was reduced to 54%, obtained predominantly from carbohydrates. By the week 9, 62.8% patients became anemic and 34.5% had low albumin levels. Body composition analysis revealed that patients lost both, muscle and adipose tissues, although 27% patients were muscle depleted by the end of treatment. Patients who lost ≥10% skeletal muscle presented a higher tumour recurrence (hazard ratio = 2.957, P = 0.006) and a tendency towards diminished overall survival (hazard ratio = 2.572, not significant). CONCLUSIONS: The nutritional status of cervical cancer patients deteriorates during treatment with concomitant chemoradiotherapy and, most importantly, muscle loss impacts the clinical outcome of patients.

Immunolocalization of antioxidant enzymes in testis of rams submitted to long-term heat stress.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.

Emergent behaviors in RBCs flows in micro-channels using digital particle image velocimetry.

The key points in the design of microfluidic Lab-On-a-Chips for blood tests are the simplicity of the microfluidic chip geometry, the portability of the monitoring system and the ease on-chip integration of the data analysis procedure. The majority of those, recently designed, have been used for blood separation, however their introduction, also, for pathological conditions diagnosis would be important in different biomedical contexts. To overcome this lack is necessary to establish the relation between the RBCs flow and blood viscosity changes in micro-vessels. For that, the development of methods to analyze the dynamics of the RBCs flows in networks of micro-channels becomes essential in the study of RBCs flows in micro-vascular networks. A simplification in the experimental set-up and in the approach for the data collection and analysis could contribute significantly to understand the relation between the blood non-Newtonian properties and the emergent behaviors in collective RBCs flows. In this paper, we have investigated the collective behaviors of RBCs in a micro-channel in unsteady conditions, using a simplified monitoring set-up and implementing a 2D image processing procedure based on the digital particle image velocimetry. Our experimental study consisted in the analysis of RBCs motions freely in the micro-channel and driven by an external pressure. Despite the equipment minimal complexity, the advanced signal processing method implemented has allowed a significant qualitative and quantitative classification of the RBCs behaviors and the dynamical characterization of the particles velocities along both the horizontal and vertical directions. The concurrent causes for the particles displacement as the base solution-particles interaction, particle-particle interaction, and the external force due to pressure gradient were accounted in the results interpretation. The method implemented and the results obtained represent a proof of concept toward the realization of a general-purpose microfluidic LOC device for in-vitro flow analysis of RBCs collective behaviors.

Goldspotted spinefoot Siganus punctatus (Siganidae) age-based reproductive life history and fisheries vulnerability.

Between February 2015 and 2016, samples of the Indo-Pacific goldspotted spinefoot Siganus punctatus were taken from local fish markets, feeding sites and nursery grounds on the main island of Pohnpei, Micronesia, to ascertain sexual pattern, reproductive seasonality, age, growth and mortality. Microscopic examinations of gonads identified two seasonal peaks in reproduction: February to May and September to December, with evidence of some spawning activity in most months. Ripe females were observed 4 days on either side of the new moon. Females first matured at c. 180 mm fork length (LF ) and 1 year of age, which coincides with their entry into the fishery. Ninety five per cent of individuals were less than 3 years and the oldest fish were 8 years. To examine the species vulnerability to fishing, a tag-and-recapture study was conducted over 4 months in 2015 in a locally managed marine area and at an unprotected site. Findings suggest high residency and high vulnerability to fishing at shallow-water feeding sites and restricted migration overall. The placement of marine protected areas within critical habitat appears to be an effective conservation strategy for this species, particularly when combined with gear and seasonal market restrictions during vulnerable life-history phases.

Extension of the Kubelka-Munk theory to an arbitrary substrate: a Monte Carlo approach.

In this work we review and-to some extent-upgrade one of the main theories of light flux through homogeneous isotropic media, namely, the Kubelka-Munk (K-M) theory, and in particular the later expansion made by Kubelka to obtain the reflectance of a specimen when a substrate lies underneath. We have completed this solution by calculating the transverse energy density in the specimen and the transmission of the whole. We show that this last result-compatible with Kubelka's upgrade for layered media-also allows for the calculation of the specimen/substrate absorption split. In order to validate these expressions, the results were reproduced by means of a Monte Carlo simulation working on a layered medium under the same assumptions as the K-M theory. Interestingly, the numerical procedure introduces new capabilities in the model regarding the history of any absorbed or outgoing elemental light beam, such as the recording of its time-of-flight through a given system.

Growth and reproduction of the highfin grouper Epinephelus maculatus.

Highfin grouper Epinephelus maculatus sampled in Chuuk, Micronesia, exhibited a moderate growth rate and a relatively short life span compared to other epinephelids of a similar size. Combined gonad and otolith analysis provide preliminary evidence that the species conforms to a protogynous sexual pattern. Mean total length at maturity for females was 308 mm with first age at maturity 2·8 years for females and 4 years for males, which differs from other regional studies. Based on the gonado-somatic index and microscopic analysis of gonads, E. maculatus in Chuuk have a 4 month spawning season (January to April) that corresponds with seasonal lows in sea surface water temperature and overlaps with that of other aggregating epinephelids. The estimated von Bertalanffy growth factor (K) was 0·51 year(-1) , while total mortality was 0·34 year(-1) . Current management for E. maculatus in Chuuk includes a January to May catch, sale and export ban, which overlaps with its reproductive season. The effectiveness of these arrangements will require on-going monitoring to determine whether alternative management strategies are required to ensure population persistence.

System for evaluation of oxidative stress on in-vitro-produced bovine embryos.

This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.