Trace mineral mixture supplemented to in vitro maturation medium improves subsequent embryo development and embryo quality in cattle.

Trace minerals participate in reproductive processes and are crucial for oocyte maturation. The objective of the present study was to investigate the effect of combined supplementation with copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) during bovine in vitro maturation (IVM) on subsequent embryo development and quality. The IVM medium was supplemented as follows: a) Control (no mineral supplementation); b) MScz (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 400 ng/mL Zn); c) MScZ (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 1200 ng/mL Zn); d) MSCz (6 ng/mL Mn + 100 ng/mL Se + 600 ng/mL Cu + 400 ng/mL Zn). Supplementation with MScz and MSCz produced more blastocysts compared with the control. Total blastocyst cell number was higher when minerals were added at any combination. Day-8 blastocysts derived from oocytes treated with minerals had lower intracellular reactive oxygen species concentration and lipid content than the control. In conclusion, combined supplementation with Cu, Mn, Se and Zn during bovine oocyte IVM increased in vitro production performance, improving embryo developmental ability and quality.

Zinc supplementation within the reference ranges for zinc status in cattle improves sperm quality without modifying in vitro fertilization performance.

Zinc (Zn) has important functions in mammalian reproductive processes. In cattle, Zn status can be classified as deficient, marginal, and adequate, depending on the plasma Zn concentration. In addition, Zn deficiency can lead to reproductive failure. The aim of this study was to investigate the effect of maternal Zn status at the beginning of a fixed-time artificial insemination (FTAI) treatment regimen on pregnancy rate in cattle, and evaluate the effect of supplementing in vitro fertilization (IVF) medium with Zn concentrations within the reference range for Zn status on sperm quality and IVF performance. Pregnancy rates of animals with marginal and adequate Zn status did not differ, and there were no Zn-deficient animals detected. Supplementation of 0.8 µg/mL Zn to IVF medium enhanced progressive motility, sperm viability, functional sperm membrane integrity (HOST), acrosomal integrity and sperm-zona binding, without modifying pronuclear formation, or development of embryos to the cleavage or blastocyst stage after IVF. In conclusion, the present results indicate pregnancy rates are not associated with maternal Zn status at the beginning of the FTAI treatment regimen if Zn status is marginal or adequate. Furthermore, supplementation of IVF medium with Zn at concentrations which is considered adequate for Zn status in cattle led to improved sperm quality, without having effects on embryo development in cattle.

Parenteral Zinc Supplementation Increases Pregnancy Rates in Beef Cows.

Zinc (Zn) is required for normal reproductive performance in cattle. The aim of this study was to evaluate the effect of subcutaneous injection of 400 mg Zn at the beginning of fixed-time artificial insemination (FTAI) on preovulatory follicle and corpus luteum (CL) size, plasma estradiol (E2) and progesterone (P4) concentrations, and pregnancy rates in beef cows. Copper (Cu) concentration and alkaline phosphatase (ALP) activity in plasma were also evaluated. Zinc supplementation at the beginning of the FTAI protocol (day 0) increased the area of preovulatory follicle (APF, day 9; P = 0.042) and plasma P4 concentration (day 16; P = 0.01), whereas plasma E2 concentration (day 9) and area of CL (ACL; day 16) were not modified by Zn supplementation in cows with adequate plasma Zn concentration. Zinc supplementation in Zn-deficient cows increased ACL with respect to controls (P = 0.048) but did not modify plasma E2 concentration. Pregnancy rate on day 41 after FTAI was higher in cows supplemented with Zn compared with controls (80.95% and 51.61%, respectively; P = 0.042). Plasma Zn and Cu concentrations on days 7, 9, and 16 were not affected by Zn supplementation. In conclusion, the results obtained in the present study determined that parenteral Zn supplementation at the beginning of the FTAI protocol increased preovulatory follicle size, plasma P4 concentration, and pregnancy rates in beef cows.

The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality.

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 µg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 µg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm-zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process.

Analyses of apoptosis and DNA damage in bovine cumulus cells after in vitro maturation with different copper concentrations: consequences on early embryo development.

The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus-oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.

The importance of having zinc during in vitro maturation of cattle cumulus-oocyte complex: role of cumulus cells.

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus-oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 µg/ml Zn compared with 0.7, 1.1 and 1.5 µg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 µg/ml Zn than with 0 µg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 µg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 µg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.

Effect of different manganese concentrations during in vitro maturation of bovine oocytes on DNA integrity of cumulus cells and subsequent embryo development.

Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH-GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte-complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures 'normal' intracellular GSH content in COCs and protects DNA integrity of cumulus cells.

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes.

THE OBJECTIVES WERE TO EVALUATE: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 µg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 µg/dL Cu) and small (82 ± 22.1 µg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 µg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 µg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 µg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10(6) cumulus cells) and 6 µg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10(6) cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 µg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 µg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 µg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 µg/mL Cu relative to 0 Cu (IVM alone) and 2 µg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 µg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 µg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 µg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 µg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 µg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 µg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 µg/mL Zn relative to 0 Zn (IVM alone) and 0.7 µg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.