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Frozen-thawed Spermatozoa (frozen-thawed + spermatozoa)
Selected AbstractsMitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilizationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2006Esther Velilla Abstract The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617,626, 2006 © 2006 Wiley-Liss, Inc. [source] Effect of Carbohydrates on the Ability of Bull Sperm to Bind to Bovine Oviduct Epithelial CellsREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2009Y Kon Contents In the present study, we investigated the effect of various carbohydrates on the ability of bovine spermatozoa to bind to the bovine oviduct epithelial cells (OECs). We also examined the fertilization competence and motility of spermatozoa that bind to OECs in the presence of carbohydrates. Frozen-thawed spermatozoa were incubated with OECs, with and without various carbohydrates. The sperms were then divided into two fractions: OEC-binding sperms (B-sperm) and non-OEC binding sperms (NB-sperm). The fertilization rate, ability to bind the zona pellucida, and membrane integrity of the spermatozoa as determined using a hypo-osmotic-swelling test (HOST) were lower in NB-sperm than in the unseparated spermatozoa (control). The motility of the B-sperm was maintained for a longer time than that of the control spermatozoa. The addition of N -acetyl- d -glucosamine (GlcNAc, 5 mm) to the sperm-OEC mixture increased the number of B-sperm. D -mannose (5 mm) and D -fucose (5 mm) had no effect on the number of B-sperm. The motility of B-sperm, which bound to OECs in the presence of GlcNAc, however, was not maintained. When either OECs or the spermatozoa were treated with GlcNAc prior to sperm-OEC co-incubation, only sperm-side treatment enhanced sperm-OEC binding, but B-sperm motility was not maintained. The motility of spermatozoa incubated with GlcNAc was lower than that of controls. These results indicate that GlcNAc enhances sperm binding to OECs, probably via sperm surface modification, but does not promote increased sperm survival. [source] Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junctionEQUINE VETERINARY JOURNAL, Issue 2 2003L. H. A. MORRIS Summary Reasons for performing study: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6,12 h of ovulation with a minimum of 300,500 × 106 frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5,20 × 106 spermatozoa are available for insemination. Objectives: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. Methods: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3,14 × 106) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. Results: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 × 106 motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 × 106 spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P<0.05). Conclusions: When inseminating mares with <10 × 106 frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. Potential clinical relevance: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon)1. Furthermore, these results were obtained when mares were inseminated with 14 × 106 progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility. [source] Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm,Egg Binding, Fertilization and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2008RF Gonçalves Contents Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 105 frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events. [source] Testing Usability of Butylated Hydroxytoluene in Conservation of Goat SemenREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2008TAA Khalifa Contents The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled-stored semen as well as motility and kidding rate of frozen-thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk-based and egg yolk-free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above-mentioned bucks, split and diluted with egg yolk-free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk-based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3-ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk-free extenders containing 0.6 mm BHT and egg yolk-based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120,140 × 106 progressively motile spermatozoa was used for a single cervical insemination of cloprostenol-synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk-deficient (2.5%) extenders significantly improved viability of chilled-stored semen together with motility (48.5%) and fertility (62.5%) of frozen-thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled-stored semen but also post-thaw motility (47.5%) and fertility (53.75%) of frozen-thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm. [source] Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric SortingREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2007P Klinc Contents The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37°C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37°C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 ± 6.9% vs 30.3 ± 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 ± 5.2%, 36.0 ± 12.5% and 35.1 ± 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 ± 6.3%, 7.8 ± 4.7% and 7.4 ± 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 ± 7.5%, 23.4 ± 5.4% and 28.8 ± 6.3% vs 25.9 ± 14.4%, 38.5 ± 16.7% and 79.8 ± 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 ± 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa. [source] Effect of freezing-thawing on the expression of mannose-ligand receptors on human spermatozoa: the impact on sperm capacitation and acrosome reactionANDROLOGIA, Issue 5 2001H. Yavetz Summary The aim of this study was to evaluate the change in the expression of mannose-ligand receptors following a freezing-thawing procedure, in order to assess its impact on sperm capacitation and acrosome reaction. Twenty semen samples were obtained from fertile donors. Sperm samples were divided into two equal volumes. One aliquot was cryopreserved and the other aliquot was incubated at 32°C. After 2 h the frozen sample was thawed and both samples were further incubated at 32°C to allow capacitation. Mannose receptors were examined following 4 and 22 h of incubation using a mannosylated-BSA-FITC probe. The expression of mannose-ligand receptors on the sperm plasma membrane was determined according to the fluorescence pattern: pattern I represents pre-capacitation, pattern II represents capacitated spermatozoa and pattern III represents acrosome-reacted spermatozoa. After 4 h incubation in capacitating medium, the percentages of patterns I, II and III were 90, 7 and 3% for fresh spermatozoa and 89, 8 and 3% for frozen-thawed spermatozoa, respectively (P>0.05). Following 22 h of incubation, the percentages of patterns I, II and III were 84, 11 and 5 for fresh spermatozoa and 83, 11 and 6% for frozen-thawed spermatozoa, respectively (not significant at P>0.05). The percentages of patterns II and III in fresh and frozen-thawed spermatozoa were increased by the same magnitude with longer incubation in the capacitating conditions. It was concluded that the freezing-thawing procedure for human spermatozoa does not affect the expression of mannose-ligand receptors and the dynamics of sperm pre-fertilization processes. [source] Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extenderANIMAL SCIENCE JOURNAL, Issue 2 2009Tetsuji OKAZAKI ABSTRACT Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa. [source] Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoaAQUACULTURE RESEARCH, Issue 9 2006Nabil Mansour Abstract The effects of extender composition and freezing rate on motility and fertility of frozen-thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L,1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N- dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose,methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post-thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen-thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose,DMA extender significantly improved the fertilization percentages of frozen-thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L,1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min,1, mean±SD from ,5 to ,55°C) is a promising protocol for cryopreservation of Arctic char semen. [source] | |||||||||||||