Effect of hyaluronic acid on development of in vitro produced bovine embryos

The present study was carried out to evaluate the effect of hyaluronic acid (HA) added to the culture medium on bovine embryo development to the blastocyst stage as well as embryo quality and viability after freezing and thawing. In vitro matured and fertilized (IVM/IVF) bovine oocytes from slaughterhouse ovaries were cultured for 8 d in SOFm supplemented with 4 mg/mL fatty acid-free BSA, either in the absence or presence of 1 or 0.5 mg/mL HA. There was a significant increase in blastocyst yield in the presence of 1 mg/mL HA (P < 0.01), whereas 0.5 mg/mL HA was ineffective. Cleavage rate and mean number of days to blastocyst formation were unaffected by HA at any concentration. At 1 mg/mL, HA did not affect either post-freeze survival of Grade 1 and 2 blastocysts or the number of nuclei per blastocyst. Supplementation with HA at 1 mg/mL also significantly enhanced embryo development up to the blastocyst stage (P < 0.05) in a chemically-defined culture medium without a protein source. It is concluded that supplementation of both semi-defined and defined culture media with 1 mg/mL HA improves the development of IVM/IVF bovine embryos to the blastocyst stage, without affecting embryo quality and post-freeze survival. These results open the possibility of including HA in culture media in order to increase the efficiency of in vitro blastocyst production from in vitro-matured bovine oocytes.     

Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system

In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization.     

几种动物肝脏糖胺聚糖的醋酸纤维素薄膜电泳分析

采用酶解和离子交换色谱的方法,从兔、鸡、猪和羊肝组织中提取和纯化得到了糖胺聚糖（GAGs）.通过比较透明质酸（HA）、硫酸软骨素A（CS-A）、硫酸软骨素C（CS-C）、硫酸皮肤素（DS）、肝素（HP）、硫酸乙酰肝素（HS）等标准品在醋酸钡、醋酸锌、吡啶-甲酸等几种不同缓冲体系下的醋酸纤维素薄膜电泳行为,结合灰度积分建立了适合于微量GAGs定性和定量分析的电泳方法.将从不同动物肝脏组织中提取的GAGs运用该方法进行分析,发现 不同动物肝脏组织中,GAG含量和组成均有较大差异：羊肝中GAGs含量最高（0.52 mg/g 组织干粉）,种类也最丰富,含有HA、HS、DS和CS,其中HA所占比例最高;鸡肝中GAGs含量最少（0.18 mg/g组织干粉）,主要含有HA和DS;兔肝GAGs种类与猪肝相似,均含有HA、HS和DS,但HS是猪肝GAGs的主要成分,DS是兔肝GAGs的主要成分.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.     

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.     

In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media

This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertilization and development of porcine oocytes fertilized in vitro (IVF). The effect of porcine oviduct epithelial cell aggregates (POECA) on in vitro development of IVF embryos was also examined. Oocytes matured in vitro for 48 to 50 h were inseminated with epididymal spermatozoa preincubated at 2 sperm concentrations (1 - 2 x 10(8)/ ml vs 4 - 5 x 10(8)/ ml) for 3 h in either Dulbecco's phosphate buffered saline (PBS) or Brackett and Oliphant medium (BO). For capacitation, spermatozoa were treated with heparin (100microg / ml) for 15 min at 38.5 degrees C under 5% CO(2) in air. Cleavage and development to the blastocyst stage were evaluated on Day 3 and Day 8 after culture with or without POECA. The effect of sperm concentration on preincubation did not affect the fertilization rate, but preincubation in PBS medium did result in a higher fertilization rate (P < 0.05) than did the BO medium. The proportion of embryos undergoing cleavage and development to the blastocyst stage was significantly higher (P < 0.05) in the POECA co-culture group than in the group without POECA co-culture. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro culture improved the development of IVF oocytes to the blastocyst stage.     

Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.     

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.     

Ultra-structural changes and developmental potential of porcine oocytes following vitrification

This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P<0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P<0.05) in activated group than the group without activation. Significant differences (P<0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development.     

Exogenous hyaluronic acid enhances porcine parthenogenetic embryo development in vitro possibly mediated by CD44

Exogenous hyaluronic acid (HA) has been reported to improve early embryo development in vitro in pigs and cows. Although early embryo development in vitro is improved by exogenous HA, the mechanism mediating the action of HA is not clearly defined. In the present study, two possible HA actions on early embryo development were proposed to understand interactions between HA and the embryos using porcine parthenotes. We hypothesized that improvement of early embryo development mediated by HA would be caused by embryo-derived growth factors due to the high molecular weight of HA or cellular response through its receptor (CD44). We examined the effects of HA molecular weight on parthenogenetic embryo development, permeability of HA into the zona pellucida, expression of CD44 in porcine parthenotes at various stages, and blocking interactions between HA and CD44 by monoclonal anti-CD44 antibody (mCD44Ab). As a result, although development of porcine parthenotes to the blastocyst stage was significantly enhanced by exogenous HA with various molecular weights, there was no difference in blastocyst formation among the various molecular weights (P < 0.05). Immunofluorescence revealed that exogenous HA was accessible to CD44 through the zona pellucida, irrespective of the oocyte activation and that CD44 was also expressed in both oocytes and parthenotes at all developmental stages. In addition, development of parthenotes was partially blocked by mCD44Ab. In conclusion, we demonstrated that exogenous HA enhanced development of porcine parthenotes in vitro. This improvement mediated by exogenous HA on parthenogenetic embryo development was possibly caused by cellular response via CD44.     

Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats

Investigations on in vitro embryo production in goats in comparison with other domestic species, especially cattle, have been the subject of few reports despite their usefulness for both basic research and commercial application. The objectives of this study were to compare the efficiency of IVP in goats using immature follicular oocytes recovered from FSH-primed and control goats. After IVM, oocytes were fertilized with fresh or frozen-thawed semen capacitated with or without heparin. Mature oocytes were fertilized in vitro with fresh and frozen-thawed sperm of a single buck. Sperm preparation included swim-up separation and heparin treatment (50 micrograms/ml of sperm suspension for 45 min) before spermatozoa were added to oocytes in TALP-IVF. After IVF, the zygotes were cultured for 24h and cleaved embryos were further cultured with goat oviduct epithelial cells or transferred to synchronized recipients. Mean number of oocytes recovered from FSH-primed goats (24.5 +/- 8.6) was significantly higher (P < 0.01; t test) in comparison to control does (14.7 +/- 4.7). Irrespective of fresh or frozen semen, no differences were observed in blastocyst yield when sperm was treated with heparin. However, the highest cleavage rate (99/126; 79.4%) as well as blastocyst yield (47/126; 37.3%) was obtained after IVF with fresh sperm capacitated without heparin. Contrary to fresh sperm, heparin treatment of frozen-thawed sperm significantly improved (P < 0.01) embryo cleavage. No differences between in vivo developmental competence of embryos related to sperm origin were found after transferring into recipients. Overall, more than 60% of the recipients became pregnant and 20% of all transferred embryos survived delivering 13 healthy kids. Our caprine IVP system allows obtaining embryos with developmental competence comparable to bovine IVP.     

Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture

The viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs-Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose-dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1-x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE-2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.     

The effect of hyaluronan concentrations in hST-supplemented TCM 199 on in vitro nuclear maturation of bitch cumulus-oocyte complexes

In this study, the effect of hyaluronan (HA) on in vitro nuclear maturation of bitch oocytes was evaluated. Cumulus-oocyte complexes (COCs) were cultured for 48 h at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes with one or more layers of intact cumulus cells and dark cytoplasm were allocated to the following treatments: (A) TCM 199 supplemented with 25 mM Hepes/L (v/v), with 10% heat inactivated estrous cow serum (ECS), 50 microg/mL gentamycin, 2.2 mg/mL sodium bicarbonate, 22 microg/mL pyruvic acid, 20 microg/mL estradiol, 0.5 microg/mL FSH, 0.03 IU/mL hCG and 1 microg/mL human somatotropin (hST) (control medium), (B) Treatment A + 0.5 mg/mL HA and (C) Treatment A + 1.0 mg/mL HA. Supplementation with HA did not increase the number of oocytes that resumed meiosis. Additionally, there were no differences among treatments in the cumulus cell expansion of oocytes. In conclusion, the addition of HA to hST-supplemented TCM 199 did not improve the in vitro nuclear maturation of bitch oocytes, and therefore appeared to be unsatisfactory as supplement for in vitro maturation (IVM) of canine oocytes.