The Effect of Mycotoxins and Their Mixtures on Bovine Spermatozoa Characteristics.

There is growing concern about the effects of mycotoxins on mammalian reproduction. Although the effects of single mycotoxins have been well documented, the impact of their mixtures on spermatozoon quality is less known. Here, frozen-thawed semen (n = 6 bulls) was in-vitro-cultured (2 h) without (control) or with (i) a single mycotoxin [zearalenone (ZEN), ochratoxin A (OTA), toxin 2 (T2), and diacetoxyscirpenol (DAS)] in a dose-response manner; (ii) binary mixtures (OTA + T2, OTA + ZEN, OTA + DAS, ZEN + T2, DAS + T2 and ZEN + DAS); or (iii) ternary mixtures (OTA + DAS + T2, OTA + ZEN + T2, and ZEN + DAS + T2). Then, the spermatozoa quality was characterized according to its plasma- and acrosome-membrane integrity, mitochondrial membrane potential, and oxidation status by a flow cytometer. Exposure to single mycotoxins or binary mixtures did not affect the spermatozoa characteristics. However, exposure to the ternary mixtures, OTA + DAS + T2 and OTA + ZEN + T2, reduced (p < 0.05) the mitochondrial membrane potential relative to the control. In addition, OTA + ZEN + T2 increased (p < 0.05) the proportion of spermatozoa with reactive oxygen species relative to the control. The most suggested interaction effect between the mycotoxins was found to be an additive one. A synergistic interaction, mainly regarding the oxidation status of the spermatozoa, was also found between the mycotoxins. The current study sheds light on the potential risk of exposing spermatozoa to a mycotoxin mixture.

Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics.

Aflatoxins are considered as reproductive toxins for mammalian species. Here, we studied the effect of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) on the development and morphokinetics of bovine embryos. Cumulus oocyte complexes (COCs) were matured with AFB1 (0.032, 0.32, 3.2, 32 µM) or AFM1 (0.015, 0.15, 1.5, 15, 60 nM), then fertilized and the putative zygotes were cultured in an incubator equipped with a time-lapse system. Exposing COCs to 32 µM AFB1 or 60 nM AFM1 reduced the cleavage rate, whereas exposing them to 3.2 or 32 µM AFB1 further reduced the blastocyst formation. A delay was recorded for the first and second cleavages in a dose-dependent manner for both AFB1- and AFM1-treated oocytes. A delay was recorded in the third cleavage in the AFM1-treated group. To explore potential mechanisms, subgroups of COCs were examined for nuclear and cytoplasmic maturation (n = 225; DAPI and FITC-PNA, respectively), and mitochondrial function was examined in a stage-dependent manner. COCs were examined for their oxygen consumption rates (n = 875; Seahorse XFp analyzer) at the end of maturation, MII-stage oocytes were examined for their mitochondrial membrane potential (n = 407; JC1), and putative zygotes were examined using a fluorescent time-lapse system (n = 279; IncuCyte). Exposing COCs to AFB1 (3.2 or 32 µM) impaired oocyte nuclear and cytoplasmic maturation and increased mitochondrial membrane potential in the putative zygotes. These alterations were associated with changes in the expression of mt-ND2 (32 µM AFB1) and STAT3 (all AFM1 concentrations) genes in the blastocyst stage, suggesting a carryover effect from the oocyte to the developing embryos.

Seasonal variation in the morphokinetics of in-vitro-derived bovine embryos is associated with the blastocyst developmental competence and gene expression.

Summer heat stress is a major cause of reduced development of preimplantation embryos. Nevertheless, seasonal effects on embryo morphokinetics have been less studied. We used a non-invasive time-lapse system that allows continuous monitoring of embryos to study the seasonal impact on embryo morphokinetics. The experiments were performed during the cold and the hot seasons. Cumulus-oocyte complexes were aspirated from ovaries, in-vitro-matured, and fertilized. Putative zygotes were cultured in an incubator equipped with a time-lapse system. The cleavage and blastocyst formation rates were lower in the hot vs. the cold season (p < 0.01). The kinetics of the embryos differed between seasons, reflected by a delay in the second cleavage in the hot vs. the cold season (p < 0.03). The distribution of the embryos into different morphological grades (good, fair, and poor) throughout the first three cleavages differed between seasons, with a higher proportion of good-grade embryos in the hot season (p < 0.03). Cleaved embryos were categorized as either normal or abnormal, based on their first cleavage pattern. Normal cleavage was defined as when the first cleavage resulted in two equal blastomeres and further classified as either synchronous or asynchronous, according to their subsequent cleavages. Abnormal cleavage was defined as when the embryo directly cleaved into more than two blastomeres, it cleaved unequally into two unevenly sized blastomeres, or when the fusion of already divided blastomeres occurred. The proportion of abnormally cleaved embryos was higher in the hot season vs. the cold one (p < 0.01), reflected by a higher proportion of unequally cleaved embryos (p < 0.02). In the cold season, abnormally cleaved embryos had a lower potential to develop into blastocysts relative to their normally cleaved counterparts (p < 0.001). Blastocysts that developed in the cold and the hot seasons differed in the expression of genes that related to the cell cycle (STAT1; p < 0.01), stress (HSF1; p < 0.03), and embryo development (ZP3; p < 0.05). A higher expression level was recorded for the STAT1 and UHRF1 genes in blastocysts that developed from unequally vs. the synchronously cleaved embryos (p < 0.04). We provide the first evidence for a seasonal effect on embryo morphokinetics, which might explain the reduced embryo development during the hot season.

Association between the morphokinetics of in-vitro-derived bovine embryos and the transcriptomic profile of the derived blastocysts.

The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo's morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.

Progesterone supplementation to improve fertility of selected subgroups of lactating cows during the summer and fall.

One major cause of low fertility of cows in the summer is progesterone deficiency. We found that insertion of a controlled intravaginal drug-releasing (CIDR) device containing progesterone after artificial insemination (AI) increases pregnancy per AI (P/AI) in cows with uterine disease and low body condition score after calving. Here, we treated only these two subgroups, during the summer and autumn. Control (n = 191 AI) and treatment (n = 230 AI) cows were inseminated at estrus and the treated group received a CIDR device on day 5 post-AI, for 14 days. Overall analysis of data during the summer and autumn indicated no significant differences between treatment and control groups. Analysis of the summer data only indicated a significant effect of treatment: P/AI was higher in CIDR-treated vs. control groups (34.2% vs. 19.3%; p < .038). Results indicated a 15% increase in P/AI during the summer for CIDR-treated cows in subgroups that had responded positively to the progesterone treatment.

Carryover effects of feeding bulls with an omega-3-enriched-diet-From spermatozoa to developed embryos.

The impact of omega-3 nutritional manipulation on semen cryosurvival and quality post thawing is controversial. Our aim was to examine how feeding bulls with omega-3 supplementation from different sources affects the spermatozoa quality parameters. Fifteen Israeli Holstein bulls were fed for 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation: fish or flaxseed oil or saturated fatty acids (control). Ejaculates were collected before, during, and after the feeding trial. Frozen-thawed samples were evaluated by a flow cytometer for spermatozoa viability, mitochondrial membrane potential, the level of reactive oxygen species (ROS), acrosome membrane integrity, DNA fragmentation, phosphatidylserine translocation, and membrane fluidity. Both fish and flaxseed oil treatment resulted in lower ROS levels vs. control groups, during and after the feeding trial. Fewer spermatozoa with damaged acrosomes were observed in the fish oil group after the feeding trial. The spermatozoa membrane fluidity was altered in both the fish and flaxseed oil groups throughout the feeding trial, but only in the flaxseed oil group after the feeding trial. The proportion of spermatozoa with fragmented DNA was lower in the flaxseed oil group after the feeding trial. The spermatozoa fertilization competence did not differ between groups however, blastocyst formation rate was higher in the fish and flaxseed oil groups relative to the control. This was associated with differential gene expression in the blastocysts. Overall, the omega-3-enriched food improved the spermatozoa characteristics; this was further expressed in the developing blastocysts, suggesting a carryover effect from the spermatozoa to the embryos.

New evidence for deleterious effects of environmental contaminants on the male gamete.

The decreasing trend in human and domestic animal fertility in recent decades has resulted in the question of whether reduced sperm quality is associated with changes in global climate and the environment. Proposed causes for reduced sperm quality include environmental contaminants, which enter into the body of animals through the food chain and are transported to the reproductive tract, where contaminating agents can have effects on fertilization capacities of gametes. In this review, there is a focus on various environmental contaminants and potential effects on male fertility. Human-derived contaminants, particularly endocrine-disrupting phthalates and the pesticide atrazine, are discussed. Naturally occurring toxins are also addressed, in particular mycotoxins such as aflatoxin which can be components in food consumed by humans and animals. Mechanisms by which environmental contaminants reduce male fertility are not clearly defined; however, are apparently multifactorial (i.e., direct and indirect effects) with there being diverse modes of action. Results from studies with humans, rodents and domestic animals indicate there are deleterious effects of contaminants on male gametes at various stages of spermatogenesis (i.e., in the testis) during passage through the epididymis, and in mature spermatozoa, after ejaculation and during capacitation. Considering there is never detection of a single contaminant, this review addresses synergistic or additive effects of combinations of contaminants. There is new evidence highlighted for the long-lasting effects of environmental contaminants on spermatozoa and developing embryos. Understanding the risk associated with environmental contaminants for animal reproduction may lead to new management strategies, thereby improving reproductive processes.

Association between Fatty Acid Composition, Cryotolerance and Fertility Competence of Progressively Motile Bovine Spermatozoa.

An association between progressive motility (PM) and spermatozoa fertility competence has been suggested. However, the mechanism that underlies PM is not clear enough. We examined physiological characteristics and fatty acid composition of fresh spermatozoa with high and low PM. Additional analysis of fatty acid composition and structural characteristics was performed on spermatozoa samples with high and low progressively motile spermatozoa's survival (PMSS), i.e., the ratio between the proportion of progressively motile spermatozoa after and before cryopreservation. Finally, a fertility field trial was conducted to examine the association between the number of PM spermatozoa within the insemination straw post thawing and conception rate. Analysis of fresh spermatozoa revealed a higher omega-6 to omega-3 ratio in ejaculates with low PM relative to those with high PM (p < 0.01). The proportion of polyunsaturated fatty acids was higher in low-PMSS fresh samples (p < 0.05) relative to their high-PMSS counterparts. Fresh samples with high-PMSS expressed a higher mitochondrial membrane potential (p < 0.05) and a higher proportion of viable cells that expressed reactive oxygen species (ROS; p < 0.05). Post-thawing evaluation revealed a reduced proportion of progressively motile sperm, with a prominent effect in samples with high PM relative to low PM, defined before freezing (p < 0.01). No differences in spermatozoa mitochondrial membrane potential or ROS level were found post-thawing. A fertility study revealed a positive correlation between the number of progressively motile spermatozoa within a standard insemination straw and conception rate (p < 0.05). Considering these, the bull PMSS is suggested to be taken into account at the time of straw preparation.

Administration of GnRH at Onset of Estrus, Determined by Automatic Activity Monitoring, to Improve Dairy Cow Fertility during the Summer and Autumn.

We examined gonadotropin-releasing hormone (GnRH) administration at onset of estrus (OE), determined by automatic activity monitoring (AAM), to improve fertility of dairy cows during the summer and autumn. The study was performed on two dairy farms in Israel. The OE was determined by AAM recorded every 2 h, and a single im dose of GnRH analogue was administered shortly after OE. Pregnancy was determined by transrectal palpation, 40 to 45 d after artificial insemination (AI). Conception risk was analyzed by the GLIMMIX procedure of SAS. Brief visual observation of behavioral estrus indicated that about three-quarters of the events (n = 40) of visually detected OE occurred within 6 h of AAM-detected OE. Accordingly, the GnRH analogue was administered within 5 h of AAM-detected OE, to overlap with the expected endogenous preovulatory LH surge. Overall, pregnancy per AI (P/AI) was monitored over the entire experimental period (summer and autumn) in 233 first, second or third AI (116 and 117 AI for treated and control groups, respectively). Least square means of P/AI for treated (45.8%) and control (39.4%) groups did not differ, but group-by-season interaction tended to differ (p = 0.07), indicating no effect of treatment in the summer and a marked effect of GnRH treatment (n = 58 AI) compared to controls (n = 59 AI) on P/AI in the autumn (56.6% vs. 28.5%, p < 0.03). During the autumn, GnRH-treated mature cows (second or more lactations), and postpartum cows exhibiting metabolic and uterine diseases, tended to have much larger P/AI than their control counterparts (p = 0.07-0.08). No effect of treatment was recorded in the autumn in first parity cows or in uninfected, healthy cows. In conclusion, administration of GnRH within 5 h of AAM-determined OE improved conception risk in cows during the autumn, particularly in those exhibiting uterine or metabolic diseases postpartum and in mature cows. Incorporation of the proposed GnRH treatment shortly after AAM-detected OE into a synchronization program is suggested, to improve fertility of positively responding subpopulations of cows.

Melatonin slightly alleviates the effect of heat shock on bovine oocytes and resulting blastocysts.

Heat stress is associated with increased production of reactive oxygen species (ROS) and disruption of bovine oocyte function. Here, we examined whether the antioxidant melatonin can alleviate the deleterious effects of heat stress on oocyte developmental competence. Cumulus-oocyte complexes were matured for 22 h at 38.5 °C (control) or for 22 h at 41.5 °C (heat shock) with or without 1.0 × 10-7 M melatonin. At the end of maturation, a subgroup of oocytes was examined for nuclear and cytoplasmic maturation, ROS level and mitochondrial membrane potential. A second subgroup of oocytes underwent fertilization (18 h), and putative zygotes were cultured in an incubator equipped with a time-lapse system for ∼190 h. Cleavage rate and the proportion of blastocysts, as well as embryo kinetics were recorded. Expanded blastocysts were collected and their transcript abundance was evaluated. Heat shock increased ROS and reduced the proportion of oocytes that resumed meiosis and reached the metaphase-II stage. Exposing oocytes to heat shock with melatonin alleviated these effects to some extent, expressed by a marginal reduction in ROS level and increased proportion of metaphase-II stage oocytes. Neither the distribution of oocyte cortical granules nor polarization of the mitochondrial membrane differed between control and heat-shocked oocytes cultured with or without melatonin. Heat shock reduced the proportion of embryos that cleaved and developed to blastocysts, characterized by alterations in kinetics of the developed embryos expressed by a delay in the first cleavage, second cleavage and blastocyst formation for heat-shock vs. control groups. Melatonin did not restore the competence or kinetics of embryos developed from heat-shocked oocytes. However, expanded blastocysts developed from heat-shocked oocytes treated with melatonin expressed a higher transcript abundance of genes associated with mitochondrial function, relative to the control and heat-shock group. In summary, melatonin improved the oxidative status of heat-shocked oocytes to some extent and had a beneficial effect on maternal mitochondrial transcripts in the developed blastocysts.

Effect of environmental contamination on female and male gametes - A lesson from bovines.

Endocrine-disrupting compounds (EDCs) and foodborne contaminants are environmental pollutants that are considered reproductive toxicants due to their deleterious effects on female and male gametes. Among the EDCs, the phthalate plasticizers are of growing concern. In-vivo and in-vitro models indicate that the oocyte is highly sensitive to phthalates. This review summarizes the effects of di(2-ethylhexyl) phthalate and its major metabolite mono(2-ethyhexyl) phthalate (MEHP) on the oocyte. MEHP reduces the proportion of oocytes that fertilize, cleave and develop to the blastocyst stage. This is associated with negative effects on meiotic progression, and disruption of cortical granules, endoplasmic reticulum and mitochondrial reorganization. MEHP alters mitochondrial membrane polarity, increases reactive oxygen species levels and induces alterations in genes associated with oxidative phosphorylation. A carryover effect from the oocyte to the blastocyst is manifested by alterations in the transcriptomic profile of blastocysts developed from MEHP-treated oocytes. Among foodborne contaminants, the pesticide atrazine (ATZ) and the mycotoxin aflatoxin B1 (AFB1) are of high concern. The potential hazards associated with exposure of spermatozoa to these contaminants and their carryover effect to the blastocyst are described. AFB1 and ATZ reduce spermatozoa's viability, as reflected by a high proportion of cells with damaged plasma membrane; induce acrosome reaction, expressed as damage to the acrosomal membrane; and interfere with mitochondrial function, characterized by hyperpolarization of the membrane. ATZ and AFB1-treated spermatozoa show a high proportion of cells with fragmented DNA. Exposure of spermatozoa to AFB1 and ATZ reduces fertilization and cleavage rates, but not that of blastocyst formation. However, fertilization with AFB1- or ATZ-treated spermatozoa impairs transcript expression in the formed blastocysts, implying a carryover effect. Taken together, the review indicates the risk of exposing farm animals to environmental contaminants, and their deleterious effects on female and male gametes and the developing embryo.

Effect of aflatoxin B1 on bovine spermatozoa's proteome and embryo's transcriptome.

This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV-) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV- spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa's DNA integrity, additional damage mechanisms are involved.

Involuntary human exposure to carbamazepine: A cross-sectional study of correlates across the lifespan and dietary spectrum.

Treated wastewater (TWW) is increasingly used for agricultural irrigation, especially in arid and semi-arid regions. Carbamazepine is among the most frequently detected pharmaceuticals in TWW. Moreover, its uptake and accumulation have been demonstrated in crops irrigated with TWW. A previous controlled trial found that urine concentrations of carbamazepine were higher in healthy volunteers consuming TWW-irrigated produce as compared to freshwater-irrigated produce. The aim of the current study was to assess whether carbamazepine is quantifiable in urine of Israelis consuming their usual diets and whether concentrations vary according to age, personal characteristics and diet. In this cross-sectional study, we recruited 245 volunteers, including a reference group of omnivorous healthy adults aged 18-66; pregnant women; children aged 3-6 years; adults aged >75 years; and vegetarians/vegans. Participants provided spot urine samples and reported 24-hour and "usual" dietary consumption. Urinary carbamazepine levels were compared according to group, personal characteristics, health behaviors, and reported diet. Carbamazepine was detectable (≥1.66 ng/L) in urine of 84%, 76%, 75.5%, 66%, and 19.6% of the reference group, vegetarians, older adults, pregnant women, and children, respectively. Quantifiable concentrations (≥5.0 ng/L) of carbamazepine were found in 58%, 46%, 36.7%, 14%, and 0% of these groups, respectively (p = 0.001 for comparison of proportions across groups). In adults, higher carbamazepine concentrations were significantly associated (p < 0.05) with self-defined vegetarianism, usual consumption of dairy products and at least five vegetables/day, and no meat or fish consumption in the past 24-hours. This study demonstrates that people living in a water-scarce region with widespread TWW irrigation, are unknowingly exposed to carbamazepine. Individuals adhering to recommended guidelines for daily fresh produce consumption may be at higher risk of exposure to TWW-derived contaminants of emerging concern.

Oocyte maturation in plasma or follicular fluid obtained from lipopolysaccharide-treated cows disrupts its developmental competence.

Mastitis has deleterious effects on ovarian function and reproductive performance. We studied the association between plasma or follicular fluid (FF) obtained from endotoxin-induced mastitic cows, and oocyte developmental competence. Lactating Holstein cows were synchronized using the Ovsynch protocol. On Day 6 of the synchronized cycle, an additional PGF2α dose was administered, and either Escherichia coli endotoxin (LPS, 10 µg; n = 3 cows) or saline (n = 3 cows) was administered to one udder quarter per cow, 36 h later. Milk samples were collected and rectal temperatures recorded. Cows treated with LPS showed a typical transient increase in body temperature (40.3 °C ±â€¯0.4), whereas cows treated with saline maintained normal body temperature (38.9 °C ±â€¯0.04). A higher (P < 0.05) somatic cell count was recorded for cows treated with LPS. Plasma samples were collected and FF was aspirated from the preovulatory follicles by transvaginal ultrasound probe, 6 h after LPS administration. Radioimmunoassay was performed on plasma samples to determine estradiol and cortisol concentrations. Either FF or plasma was further used as maturation medium. In the first experiment, oocytes were matured in TCM-199 (Control) or in FF aspirated from cows treated with saline (FF-Saline) or LPS (FF-LPS). Cleavage rate to the 2- to 4-cell stage embryo did not differ among groups. However, the proportion of developed blastocysts on Day 7 postfertilization in the FF-LPS group tended to be lower for that in FF-Saline and was lower (P < 0.05) than that in the Control groups (10.6 vs. 22.4 and 24.4%, respectively). In the second experiment, oocytes were matured in TCM-199 (Control), or in plasma obtained from cows treated with saline (Plasma-Saline) or LPS (Plasma-LPS). Similar to the FF findings, cleavage rate did not differ among groups; however, the proportion of developing blastocysts tended to be lower in the Plasma-LPS group than in the Plasma-Saline group and was lower (P < 0.05) from that in the Control group (11.0 vs. 25.5 and 34.7%, respectively). The proportion of apoptotic cells per blastocyst, determined by TUNEL assay, did not differ among the experimental groups. The findings shed light on the mechanism by which mastitis induces a disruption in oocyte developmental competence. Further studies are required to clarify whether the negative effect on oocyte developmental competence is a result of LPS, by itself, or due to elevation of secondary inflammatory agents.

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos.

PURPOSE: This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos. METHODS: Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates. RESULTS: Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates. CONCLUSION: This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

Carryover Effects of Acute DEHP Exposure on Ovarian Function and Oocyte Developmental Competence in Lactating Cows.

We examined acute exposure of Holstein cows to di(2-ethylhexyl) phthalate (DEHP) and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P < 0.05) in the DEHP-treated group. Estradiol concentration in the follicular fluid was lower in the DEHP-treated group than in controls, and associated with a higher number of follicular pathologies (follicle diameter >25 mm). The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05). The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl) phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04) and 7-day blastocysts (P < 0.06). The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence.

Involvement of the sphingolipid ceramide in heat-shock-induced apoptosis of bovine oocytes.

Programmed cell death via the sphingomyelin pathway has been suggested to underlie heat-shock disturbance of oocyte developmental competence. A series of experiments were performed to characterise the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation can be regulated. Bovine cumulus-oocyte complexes (COCs) were aspirated from ovaries collected in the cold season (November-April), in vitro-matured, fertilised and cultured for 8 days. Exposure of COCs to heat shock (41°C) during maturation reduced cleavage rate and blastocyst formation relative to the control group (38.5°C). Annexin-V binding (V-FITC assay), which is associated with the early apoptotic event of membrane phosphatidylserine turnover, was higher in oocytes exposed to short-term versus long-term heat shock, suggesting that heat-shock-induced apoptosis involves membrane alterations. Similar to heat exposure, oocyte maturation with C(2)-ceramide had a dose-dependent deleterious effect on the first cleavages and subsequent embryonic development in association with increased annexin-V binding. Blocking endogenous ceramide generation with fumonisin B1, a specific inhibitor of dihydroceramide synthase (i.e. de novo formation), moderated, to some extent, the effects of heat shock on oocyte developmental competence, suggesting that ceramide plays an important role in heat-shock-induced apoptosis.