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 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

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 &lt; 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

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 themiceMII oocytes regained isotonic volumes and all (100%) the survivingwere 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 devicewill 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

<div><p>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 (<i>P</i> < 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 (<i>P</i> < 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 (<i>P</i> < 0.04) and 7-day blastocysts (<i>P</i> < 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.</p></div

DEHP-metabolite concentrations in the plasma before, during and after DEHP exposure.

<p>Cows were administered with water (control) or 100 mg/kg DEHP per day on days 1–3 of the experiment (DEHP-treated). Blood samples were taken before (day 0), during (days 2 and 4) and after (days 11, 19 and 24) DEHP exposure. Samples were analyzed for concentrations of DEHP metabolites mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl] phthalate (2cx-MMHP) by LC–MS/MS.</p

Developmental competence of bovine oocytes matured in follicular fluid (FF) aspirated from control (FF-control) and DEHP-treated (FF-DEHP) cows.

<p>(A) Proportion of oocytes cleaved to 2- to 4-cell-stage embryos 42–44 h postfertilization, and (B) proportion of embryos developed to the blastocyst stage on day 7 postfertilization, calculated from total oocytes or from cleaved embryos, respectively. Data are presented as means ± SEM; <i>P</i>-value indicates for treatment effect within embryonic stages between experimental groups. (C) Representative images of oocytes that were fertilized and cleaved into 2-cell-stage embryos, 4-cell-stage embryos, and further developed to 7-day blastocysts.</p

Estradiol and phthalate-metabolite concentrations in the follicular fluid (FF).

<p>Cows were tube-fed DEHP or water (control) on days 1–3 of the experiment. FFs of the preovulatory follicles were aspirated before (day 0) and after (day 29) DEHP treatment. Data presented as mean ± SEM; <i>P</i>-value indicates for treatment effect within experimental groups on each examined day. (B) Phthalate-metabolite concentrations in FF on day 0 and day 29 of the experiment. MMP, mono-methyl phthalate; MEP, mono-ethyl phthalate; MBP, mono-n-butyl phthalate; MEHP, mono(2-ethylhexyl) phthalate.</p

Schematic illustration of the experimental design.

<p>In the in-vivo part (A), cows were synchronized according to the ‘Ovsynch’ protocol. Cows were then divided into control (n = 5) and DEHP-treated (n = 4) groups. Samples of blood, urine and milk were collected on day 0 (before), days 2 and 4 (during) and days 11, 19 and 24 (after) DEHP administration. On day 7 of the experiment, cows were resynchronized and ovarian follicular dynamics was monitored by linear ultrasonographic scanner through the entire third synchronized cycle (days 9 to 29 of the experiment). For each experimental group, FF were pooled and analyzed for phthalate metabolite and estradiol concentrations (Asp. 1 and 2). In the second part of the study (B), FF aspirated from control and DEHP-treated cows were used as oocyte maturation medium. Cumulus oocyte complexes were aspirated and in-vitro matured in the aspirated FF (IVM in FF; 22h). Subgroups of mature oocytes were collected for examination of nuclear meiotic stages (DAPI staining), cortical granule distribution (FITC–PNA) and cumulus cell expansion. Another subgroup was in-vitro fertilized (IVF) for 18 h then in-vitro cultured (IVC) for 8 days. Oocyte developmental competence was evaluated as the proportion of oocytes that cleaved into 2- to 4-cell-stage embryos and developed to blastocysts 44 h and 7 days postfertilization, respectively.</p