Association of L-arginine with heparin on the sperm capacitation improves in vitro embryo production in bovine (original) (raw)
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TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES, 2016
The present study aimed at comparing the relative efficacy of different capacitating agents used for sperm pretreatment during in vitro fertilization (IVF) and subsequent embryo development of caprine oocytes. In experiment 1, the effect of different concentrations of heparin (0, 20, and 50 µg/mL) on sperm pretreatment for different periods of time (30 and 60 min) was studied. In experiment 2, the dose-dependent effect of calcium ionophore (0, 0.1, 0.2, and 0.5 µM) on sperm pretreatment was assessed. Experiment 3 validated and compared the effects of both capacitating agents on sperm pretreatment for capacitation based on the results of the first two experiments. The treated sperms from each group were used for in vitro fertilization and subsequent embryo development. The results indicated that in experiment 1, capacitation with heparin at 20 µg/mL for 60 min had significantly higher (P < 0.05) cleavage and blastocyst production. In experiment 2, capacitation with calcium ionophore at 0.1 µM had significantly more morulae (P < 0.05) and numerically more blastocyst production. In experiment 3, capacitation with heparin at 20 µg/mL had a significantly higher (P < 0.05) blastocyst production rate as compared to calcium ionophore treatment. Based on this study, heparin can be used to enhance capacitation of freshly collected sperms during in vitro fertilization and subsequent embryo development.
Capacitation of Bovine Sperm by Heparin1
Biology of Reproduction, 1988
Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 , g/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 pg/ml LC for 15 mm. When sperm were incubated for 4 h with beparin, exposure to 100 ,4g/ml LC for 15 mm had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 mm, and differed (p-<0.OO1) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 Mg/mI heparin. The correlation between the mean fertilization and LCinduced AR percentages was 0.997 (p<0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.
The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6 ± 3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58 ± 1.8 % of them intact after 18 h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87 ± 1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37 • C, and centrifuged for 30 minutes at 600g in a 45-90% Percoll gradient made with modified Tyrode's medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1 × 10 6 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12 µg mL −1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18 h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849.
Effect of heparin on in vitro capacitation of boar sperm
Biological Research, 2006
Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 μM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 μg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.
Theriogenology, 1998
The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk-or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 FL of c-SOF+NEA for 9 d. The percental:es of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%) respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility aher cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.
Animal Reproduction Science, 2013
Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P < 0.05) for COC that were IVF in the presence of heparin (54%) than without heparin (42%) or oocytes already denuded at collection that were IVF with or without heparin (41%; 38%; respectively). In Experiment 2, the developmental potential of oocytes denuded at collection was reduced (cleavage and blastocyst rates calculated from total oocytes: 34%; 11%, respectively) as compared to COC (77%; 59%, P < 0.05). However, when equal numbers of both were mixed at the start of IVM, the rates were not significantly different to COC alone (68%; 45%), but when both were mixed equally only for IVF, the rates were reduced (57%; 40%, P < 0.05). Denuded oocytes co-cultured with cumulus cells were not significantly different to intact COC (76%; 55%). The effect of adding COC during IVF to oocytes denuded after IVM was similar to adding cumulus cells to the same type of oocytes. In conclusion, both the use of heparin and the association of oocytes with cumulus cells, either detached or in intimate contact, during IVM and/or IVF significantly improve IVP of goat embryos. Furthermore, some oocytes that are already denuded at collection will develop satisfactorily to blastocysts when matured and fertilized with intact COC.
Bovine in vitro fertilization: In vitro oocyte maturation and sperm capacitation with heparin
Theriogenology, 2014
As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitro fertilization, the bovine is now one of the important models for development. Further, the current production of bovine embryos in vitro rivals that of in vivo embryo production for commercial applications. Researchers of today may be unaware of why decisions were made in the procedures. This review addresses the state of the art at the time of the work by Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986;25:591-600), and how later work would explain success or failure of competing procedures. Important was the use of frozen semen and heparin capacitation, because this allowed future researchers/practitioners to change sperm numbers and capacitation conditions to adjust for variations among bulls. The large numbers of citation of the original work stand the testament of time in the repeatability and success of the procedures. The work was done within the environment of the N.L. First laboratory and the unique interactions with a large number of talented graduate students, postdoctoral researchers, and technicians.
Use of Heparin to Accelerate Capacitation of Equine Spermatozoa In Vivo1
Biology of Reproduction
The ability of heparin to accelerate capacitation of stallion spermatozoa in vivo was studied using one stallion and 32 mares. Mares were inseminated either 0-12 h or 12-24 h after ovulation, with semen diluted in a nonfat milk-glucose extender, with or without added heparin (final concentration of 10 ng/ml). Timing of postovulation insemination tended to affect pregnancy rate (p = 0.07), with an advantage to earlier postovulation breeding. For mares bred within 12 h after ovulation, insemination with heparinized semen yielded a higher (p = 0.003) pregnancy rate (7 of 7; 100%) than did insemination with nonheparinized semen (3 of 7; 43%). For mares bred at 12-24 h after ovulation, a treatment effect was not detected (p-0.17). Over all data, insemination with heparinized semen increased (p = 0.01) the pregnancy rate in mares (12 of 16; 75%), as compared to pregnancy rate following insemination with nonheparinized semen (5 of 16; 31%). The effect of heparin on spermatozoa should be tested on additional stallions to determine whether individual stallion variation exists. Nonetheless, these data suggest that heparin accelerates capacitation of spermatozoa under in vivo conditions, thereby enhancing fertilization of oocytes prior to loss of oocyte viability, when insemination is performed after ovulation.
Theriogenology, 2015
Please cite this article as: García-Álvarez O, Maroto-Morales A, Jiménez-Rabadán P, Ramón M, del Olmo E, Iniesta-Cuerda M, Anel-López L, Fernández-Santos MR, Garde JJ, Soler AJ, Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum, Theriogenology (2015),
Heparin-induced in vitro capacitation changes of swamp buffalo spermatozoa
TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES, 2015
The aim of this study was to evaluate heparin-induced in vitro capacitation-associated changes in spermatozoa of swamp buffalo. Therefore, freshly ejaculated and washed spermatozoa of 8 swamp buffalo bulls were capacitated in vitro in TALP medium supplemented with BSA, heparin, and HEPES buffer at a concentration of 6 × 10 9 spermatozoa/mL at 37 °C for 6 h. Capacitation status of spermatozoa in terms of the hyperactivated motility, acrosome membrane integrity, total hypoosmotic swelling test (HOST), activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and sperm membrane protein (SMP) and cholesterol content were estimated for each ejaculate at 1-h intervals from 0 to 6 h of incubation. The results revealed that the highest hyperactivation of spermatozoa (74.50 ± 1.78%) and live acrosome reaction (56.92 ± 1.88%) was recorded at 4 h of incubation while the total HOSTreacted spermatozoa and SMP and cholesterol levels decreased significantly (P < 0.01) with increasing period of incubation. The AST and ALT activities increased significantly (P < 0.01) as incubation period increased. In conclusion, heparin induces in vitro capacitation changes in swamp buffalo spermatozoa as evidenced by the highest hyperactivation of spermatozoa and live acrosome reaction at 4 h of incubation.