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.

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.

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.

Atrazine-induced toxicity in goat spermatozoa is alleviated to some extent by polyphenol-enriched feed.

Atrazine (ATZ) is one of the most extensively used herbicides to control growth of broadleaf and grassy weeds in crops. ATZ and its metabolites have deleterious effect on sperm quality. ATZ is also known for its ability to induce oxidative stress. Pistacia lentiscus (PL) is an evergreen shrub, with a high content of polyphenols in leaf extracts, with a known anti-inflammatory and antioxidant properties. The protective effect of PL or its extracts against ATZ-induced damage have not been yet evaluated. We examined the harmful effects of atrazine (ATZ) exposure on male reproductive system, using goat (Capra hircus) model spermatozoa and the protective effects of PL and PL ethanolic extract (PLE). In in-vivo experiments, male goats were fed a standard ration or one supplemented with 15 mg ATZ/kg body weight daily, for 6 months. Exposure to ATZ impaired the spermatozoa's morphology, viability, mitochondrial membrane potential and cell lipid composition. These alterations may in turn lead to reduced fertilization competence of the exposed spermatozoa. In an ex-vivo experiment, spermatozoa from male goats fed a standard ration or one supplemented with PL or PLE for 90 days and then were exposed to 1 µM ATZ or 10 µM of its major metabolite diaminochlorotriazine (DACT) through in-vitro capacitation. Prefeeding with PL or PLE partially attenuated the harmful effects of ATZ and DACT. Dietary supplementation with polyphenol-enriched feed can protect, to a certain extent, spermatozoa in males exposed to environmental toxicants.

Effect of the herbicide atrazine and its major metabolite, DACT, on bovine sperm cryotolerance.

During freezing and thawing procedures, sperm are exposed to chemical and/or physical stressors that may cause adverse and harmful changes to sperm membranes. Accurate evaluation of the structural and functional integrity of fresh as well as cryopreserved sperm is highly important in predicting sperm fertilization capacity and success of artificial insemination (AI). The herbicide atrazine (ATZ) and its major metabolite, diaminochlorotriazine (DACT) are considered a ubiquitous environmental contaminants and endocrine disruptors, which deleteriously effect sperm function. Taking into consideration possible damage caused by environmental contaminants to sperm membranes, additive effects during cryopreservation cannot be ruled out. The aim of the current study was to evaluate the effect of ATZ (0.1 or 1 µM) and DACT (1 or 10 µM) exposure during or after cryopreservation on bovine sperm cryotolerance. Sperm membrane integrity and functionality were evaluated using fluorimetric probes: (1) double-stranded DNA was examined by 4',6-diamidino-2-phenylindole; (2) plasma membrane integrity was examined by propidium iodide; (3) acrosome reaction (AR) was examined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; mitochondrial membrane potential (ΔΨm) was examined by 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe. The findings demonstrate, that exposure of sperm to ATZ (0.1 or 1 µM) or DACT (1 or 10 µM) during cryopreservation increased the proportion of dead sperm relative to the control (P < 0.09); exposure to DACT (1 or 10 µM) increased ΔΨm (P < 0.03). Neither ATZ nor DACT affected spontaneous AR. In contrast, the proportion of sperm with Ca++ ionophore-induced AR was lower after exposure to 1 µM DACT (P < 0.05). Following freezing and thawing procedures, exposing sperm to 1 µM ATZ increased the proportion of dead sperm relative to the control (P < 0.05), but had no significant effect on sperm ΔΨm or AR. In conclusion, exposing sperm to endocrine-disrupting chemicals such as ATZ or DACT during cryopreservation reduces sperm cryotolerance and resistance post-thawing.

Fluorimetric Techniques for the Assessment of Sperm Membranes.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

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.

Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality.

Atrazine (ATZ), one of the most extensively used herbicides, is considered a ubiquitous environmental contaminant. ATZ is a known endocrine disruptor, and deleterious effects on reproductive function have been shown, even at low, ecologically relevant doses (0.1-3µg/L). Once it enters the body, ATZ is metabolized to various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). The current study focuses on direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). Sperm were incubated under capacitation conditions with or without 0.1-10µM ATZ or 1-100µM DACT. The integrity and functionality of sperm membranes (plasma, acrosomal and mitochondrial) were examined simultaneously by fluorescence staining at 0, 2 and 4h of incubation. Acrosome reaction (AR) was induced by Ca++ ionophore, after capacitation. The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low (0.1 or 1µM) concentrations; pseudo-AR and that induced by Ca++ ionophore were both affected by exposure to 0.1µM ATZ (P<0.05 and P<0.00004, respectively). A similar pattern was documented for sperm isolated from ejaculates (P<0.002 and P<0.001, respectively). ΔYm was affected by ATZ in sperm isolated from the epididymis tail (1µM, P<0.0009), but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1µM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P<0.005). DACT at 100µM did not induce pseudo-AR in sperm isolated from the ejaculate, but did in sperm isolated from the epididymis tail (P<0.05). Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100µM DACT (P<0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100µM DACT (P<0.0004). These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.

Ploidy of spermatogenic cells of men with non-mosaic Klinefelter's syndrome as measured by a computerized cell scanning system.

PURPOSE: This study aims to characterize the origin of testicular post-meiotic cells in non-mosaic Klinefelter's syndrome (KS). METHODS: The study included testicular tissue specimens from 11 non-mosaic KS patients, with (6 positive) and without (5 negative) spermatozoa presence. The obtained testicular cells were affixed and stained for morphology followed by fluorescence in situ hybridization (FISH) for centromeric probes X, Y, and 18. We used a computerized automated cell scanning system that enables simultaneous viewing of morphology and FISH in the same cell. RESULTS: A total of 12,387 cells from the positive cases, 11,991 cells from the negative cases, and 1,711 cells from the controls were analyzed. The majority of spermatogonia were 47, XXY in both the positive and negative KS cases (88.9 ± 4.76 % and 90.6 ± 4.58 %) as were primary spermatocytes (76.8 ± 8.14 % and 79.6 ± 7.30 %). The respective rates of secondary spermatocytes and post-meiotic cells (round, elongating spermatids and sperm cells) were 1.1 ± 1.39 % in the positive cases, 2.9 ± 3.33 % in the negative cases, compared to 67.6 ± 6.22 % in the controls (P < 0.02). Pairing of both 18 and XY homologous chromosomes in 46,XY primary spermatocytes was 2.5 ± 2.31 % and 3.4 ± 2.39 %, respectively, compared to 19.8 ± 8.95 % in the control group (P < 0.02) and in 47,XXY primary spermatocytes in 2.4 ± 3.8 % in the positive group and 3.2 ± 2.26 % in the negative group. CONCLUSIONS: This study presents data to indicate that the majority of primary spermatocytes in the testes of non-mosaic KS patients are 47,XXY and could possibly develop into post-meiotic cells.

Conventional IVF versus ICSI in sibling oocytes from couples with endometriosis and normozoospermic semen.

PURPOSE: This study compares the fertilization rate and embryonic development of oocytes randomly inseminated by conventional IVF or ICSI in patients with endometriosis and normozoospermic semen during IVF cycles. METHODS: Sibling oocytes were randomized to be inseminated either by ICSI or IVF. Rates of fertilization, cleavage, blastulation and embryonic morphology were assessed. RESULTS: A total of 786 sibling cumulus-oocyte complexes (COC) were randomized between insemination by conventional IVF (387 COC) or ICSI (399 COC). A significantly higher fertilization rate was found in the ICSI group (ICSI versus IVF, 73.3±23 % versus 54.7±31.9 % respectively; P=0.003), yielding a higher mean number of day 2 embryos (5.2±3.4 versus 3.6±2.9 respectively; P=0.002). Triploid fertilization rate (3PN/COC) was significantly higher in the IVF group compared to the ICSI group (3.9±8.7 % versus 0.9±3.1 % respectively; P=0.02). The morphology score and rate of development of day 2 and 3 embryos were not different between the two groups. Comparison of embryo transfer cycles in which either IVF or ICSI only embryos were transferred did not reveal any statistically significant differences in pregnancy or implantation rates. CONCLUSION: ICSI appears to be a better treatment option than conventional IVF in endometriosis-associated infertility, since it offers the advantages of higher fertilization rate and mean number of embryos and lower rate of total fertilization failure and triploid fertilization.

Computerized cell-scanning system for evaluating human spermatogenesis in non-obstructive azoospermic patients.

There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction (TESE) outcome. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE and autopsied fertile controls was developed. Testicular cells stained and scanned automatically for morphology underwent fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 after destaining. Cells were automatically classified according to ploidy, and ratios of haploid cells and autosomal (18) and sex-chromosome bivalent rates were calculated. Identification of testicular cells in suspension enabled prediction of spermatogenesis in seven of eight Sertoli-cell-only syndrome patients. Haploid/diploid cell ratios were 67.6:32.2 for controls and 9.6:90.4 for patients. Both autosomal (18) and sex-chromosome bivalents were present in patients (4.1 ± 5.82%) and controls (19.7 ± 8.95%). Few tetraploid pachytene spermatocytes were observed. More secondary spermatocytes with NOA showed two distinct signals for chromosome 18 (27.9 ± 32.69%) compared with controls (0.4 ± 0.35%). The computerized cell-scanning system enables simultaneous application of morphology and chromosome analysis of testicular cells, which enhance assessing different pathological disturbances and estimating the likelihood of a successful second TESE procedure.