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ABSTRACT: Long-term preservation of mammalian spermatozoa is regarded as being essential for the conservation of endangered species and for assisted reproduction. Although the method generally applied for semen preservation is storage at -196°C, liquid nitrogen and special equipment might not be available in every situation. Additionally, logistical difficulties make cryopreservation difficult under extreme conditions, such as in the wilderness. Fortunately, with the development of intracytoplasmic sperm injection (ICSI), preserved spermatozoa do not need to maintain their motility. The aim of the study was to investigate alternative preservation methods for spermatozoa without freezing in liquid nitrogen and to examine the effects of the preserved sperm on fertilization after injection into in vitro-matured pig oocytes. The pig was chosen as the model for investigations, and the alternative preservation methods included heat-drying, flame-drying, or freezing (-20°C, household refrigerator) of spermatozoa. After semen collection and swim up, sperm concentrations were adjusted to 0.5 to 1×10(5)cellsmL(-1). For heat-drying, aliquots of sperm suspension (50µL) were dehumidified at 50, 56, or 90°C each for 45min, or at 120°C for 20min. Flame-drying was performed by flaming 5µL of semen suspension or sperm-rich fraction for up to 2s on glass slides. Dried semen samples were stored at 4°C. Further, sperm samples were frozen (100µL/tube) without cryoprotectant and kept at -20°C. The specimens were stored up to 5 days. Before ICSI, heat-dried and flame-dried samples were rehydrated with 50 and 10µL of ultrapure water (Millipore(®), Millipore Corp., Billerica, MA, USA), respectively. Frozen sperm were thawed for 10min at room temperature. Afterward, spermatozoa were injected into in vitro-matured pig oocytes at metaphase II, and sperm-injected oocytes were activated artificially by 10% ethanol. Subsequently, in vitro culture in mTCM-199(®) [20µgmL(-1) of insulin, 0.08mgmL(-1) of L-glutamine, 50µgmL(-1) of gentamicin, and 20% FCS (vol/vol)] with 10µgmL(-1) of cycloheximide was performed for 24h. Afterward, oocytes were fixed, stained (aceto-orcein staining), and investigated for signs of fertilization. Positive fertilization criteria included extrusion of the second polar body, formation of two pronuclei, and the presence of a visible sperm tail. The fertilization ability of heat-dried samples achieved 9.0% (6/67), 8.8% (6/68), 2.7% (2/75), and 0% (0/60) at 50, 56, and 90°C for 45min and 120°C for 20min, respectively. Flame-drying reached a fertilization rate of 14.0% (6/43) v. 4.4% (3/69) for the sperm-rich fraction v. swim-up spermatozoa, respectively. With frozen semen (-20°C), 9.4% (13/138) fertilized oocytes were obtained. These results imply that ICSI with spermatozoa, which were alternatively preserved at various temperatures and circumstances in combination with artificial oocyte activation, may fertilize the oocytes. The best results were accomplished with the flame-dried sperm-rich fraction.
Reproduction Fertility and Development 12/2012; 25(1):286-7. · 2.11 Impact Factor
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ABSTRACT: Several factors have been shown to alter the sex ratio of bovine embryos generated in vitro, i.e. the maturity of the oocyte at the time of insemination, the duration of sperm-oocyte co-incubation and the culture conditions after in vitro fertilization. It has been shown that the presence of glucose during in vitro culture reduced the development of female embryos to the blastocyst stage compared with controls cultured in the absence of glucose. The sex ratio of bovine embryos has also been linked with changes in the composition of the follicular fluid in which the oocyte undergoes growth and maturation, i.e. the intrafollicular testosterone concentration. However, no information is available regarding the effect of intrafollicular glucose concentration on the sex ratio of embryos after in vitro production (IVP). The purpose of this study was to determine whether different glucose concentrations in the follicular fluid at the time of cumulus-oocyte complex (COC) collection have an effect on the sex ratio of the resulting blastocysts after IVP. Ovaries from a local abattoir were transported to the laboratory within 2h of slaughter. Follicles (3-8mm) were individually dissected and the glucose concentration of each follicle was measured using a blood glucose monitoring system (Freestyle Freedom Lite, Abbott, Germany). Based on a glucose concentration, COC [low glucose:<1.1mM (group 1) and high glucose:>1.1mM (group 2)] were pooled in groups and used for blastocyst production employing standard protocols for IVP. Developmental rates were recorded at Day 3 (cleavage) and Day 7/8 (blastocyst stage). Total cell number of blastocysts was determined after Hoechst staining. Sex of the embryos was analysed via PCR using bovine X- and Y-chromosome specific primers. Developmental rates for COC stemming from follicles with different glucose concentrations did not show significant differences (P>0.05) compared to each other [Cleavage rate: group 1: 81.8±4.7% (93/117); group 2: 79.3±4.9% (94/123); blastocyst rate: group 1: 35.6±5.2% (38/117); group 2: 31.6±5.2% (38/123)]. Total cell numbers were similar in embryos of both groups [Group 1: 117.7±8.1 (n=18); group 2: 117.2±6.4 (n=18)]. The overall sex ratio significantly differed (P<0.05) from 1:1 in favour of females in both groups [Group 1: 85 v. 15% (n=20); group 2: 63.6 v. 36.4% (n=22)]. No significant difference (P>0.05) in the overall sex ratio was detected in blastocysts produced under standard IVP conditions employed in the laboratory [without measurement of follicular glucose concentration, 55.0 v. 45.0%, (n=20)]. In conclusion, under the conditions used in the present study, the intrafollicular glucose concentration from which the immature COC was collected affects the sex of the resulting embryo after IVP, favouring females. Further studies are needed to confirm these findings in living cows using the ovum pickup technique.
Reproduction Fertility and Development 01/2011; 23(1):160. · 2.11 Impact Factor
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Reproduction in Domestic Animals 10/2007; 19(4):193 - 204. · 1.36 Impact Factor
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ABSTRACT: In order to study in more detail the functional significance of the granulosa cell reaction for the in vitro oocyte maturation in the pig, 412 oocytes taken from Graafian follicles and 510 taken from tertiary follicles were grown on different culture media. 147 oocytes, which during in vitro maturation showed a granulosa cell reaction, were transplanted and were utilized to study their embryonic developmental potentialities.It was possible to induce the granulosa cell reaction in about two-thirds of oocytes by the addition of the fluid taken from Graafian follicles. Denudation of oocytes led to a severe inhibition of maturation.Only one-third of the transplanted oocytes showed normal embryonic developmental potentialities.The findings suggest that maturation of the oocyte nucleus is related directly to the granulosa cell reaction, while the maturation of the cytoplasm is independent of it.
Anantomia Histologia Embryologia 06/2007; 7(1):58 - 69. · 0.90 Impact Factor
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ABSTRACT: The diploid/triploid (60,XX/90,XXY) condition in Bos taurus is very rare and only three cases have been published previously. The present animal exhibited an aplastic vulva, penis and clitoris agenesis, a male-like urethra located in a pseudoprepuce opening between the mammary complexes and a well developed M. rectipeninus. A normal (60,XX) female karyotype was detected in lymphocyte cultures whereas uterus and tendon cells revealed a 60,XX/90,XXY mixoploidy. Quantification of X and Y chromosome-specific sequences using RT-PCR revealed extraordinary high Y chromosome equivalents in the sample recovered from the male-like transformed vestibulum vaginae suggesting a causative relationship. The pathogenesis of the missing clitoris and penis, which is contrasted by the concomitant presence of a well developed M. rectipeninus, remains difficult to explain. A chimeric origin is suggested despite the fact that microsatellite analysis of the animal's blood cells displayed no un- usual allele accumulation.
Sexual Development 02/2007; 1(1):59-65. · 2.27 Impact Factor
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Animal Reproduction Science 11/2005; 89(1-4):206-8. · 1.75 Impact Factor
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ABSTRACT: Molar transformations of the bovine placenta are extremly rare phenomenona and the aetiology of this genuine placental disease is still unknown. In the present study, an uncommon case of a German Holstein Friesian foetus co-twinned with a hydatidiform mole is described. Cytogenetic and fluorescence in situ hybridization analysis of cell cultures as well as prove of the presence of the SRY gene sequence revealed a heterosexual twin pregnancy. A chimeric condition of the mole was also established. In addition, an XO cell population was detected in the co-twin as well as in the mole. Upon examination of microsatellites of the parents, the mole and the co-twin an androgenetic origin of the mole is suggested, supporting the hypothesis that molar transformation of the bovine placenta may be caused by an androgenetic origin. Furthermore, the present observation demonstrates that the freemartin condition in cattle can be induced even in cases where severe placental transformations had subsequently occurred and no foetus proper could be detected at delivery.
Placenta 02/2003; 24(1):107-12. · 3.69 Impact Factor
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ABSTRACT: In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 microg/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4-5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1-3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4-7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2-5 h earlier in comparison with untreated oocytes.
Reproduction in Domestic Animals 09/2001; 36(3-4):183-8. · 1.36 Impact Factor
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ABSTRACT: During the breeding season, 42 adult German Improved Fawn nanny goats were superovulated with a single administration of pFSH, hMG or eCG at the end or a single dose of pFSH 36h before the end of estrus synchronization. Development of follicles and corpora lutea were observed by ultrasonic scanning of the ovaries every 8h from gonadotrophin treatment, until the end of estrus and once daily for the following 6 days. Differences in follicular dynamics could be realized in the four superovulation groups, and the duration of the stimulatory action following the single gonadotrophin challenge was estimated to last for 40-72h in the case of pFSH and to exceed 72h in the case of hMG and eCG treatment groups. Corpora lutea could first be detected 3 days after estrus, whereas an exact count was not possible until day 6. The ovulation rates were satisfactory, suggesting that a single injection of pFSH or hMG provides an adequate stimulus to induce a superovulatory reaction. Premature regression of corpora lutea could not be identified ultrasonographically at these early stages of the luteal phase. However, ultrasonography is a suitable method to follow follicular dynamics after superovulation and to estimate the superovulatory response in goats.
Small Ruminant Research 05/2001; 40(1):83-93. · 1.29 Impact Factor
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ABSTRACT: During the breeding season, 42 adult German Improved Fawn nanny goats were superovulated with a single dose of pFSH, hMG or eCG at the end or a single application of pFSH 36h before the end of synchronization treatment using flugestone acetate (FGA). Plasma sampling was performed immediately before and 1h after gonadotrophin treatment, twice daily during pre-estrus and estrus and once daily during post-estrus in order to determine peripheral estradiol-17beta and progesterone levels. During that period, ovarian dynamics was followed by serial ultrasound scans (8h interval during pre-estrus and estrus and once daily during post-estrus). Estradiol-17beta profiles differed between the treatment groups exhibiting significantly positive correlations between the mean estradiol-17beta concentrations and the numbers of developing large and medium-sized follicles during estrus. The early bolus application of FSH 36h before the end of synchronization treatment induced an additional advanced estradiol-17beta peak during gestagen dominance. A sharp decrease of estradiol-17beta at the end of estrus seems to play a major role for normal luteal development. Progesterone profiles during the early luteal phase revealed high premature luteal regression. An early progesterone increase was accompanied by low premature regression rates.Although simple B-mode ultrasonography is suitable to follow follicular growth patterns and to determine the ovulation rate following different superovulation regimen endocrinological supervision is required in order to detect a premature corpus luteum insufficiency.
Small Ruminant Research 05/2001; 40(1):73-82. · 1.29 Impact Factor
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ABSTRACT: Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be convenient and less expensive to use a single dose of FSH, but results of various trials have been controversial. We wished to investigate ovarian dynamics using ultrasonography after superovulation with a single dose of pFSH and hMG as compared with a single dose of eCG. Estrus was synchronized during the breeding season with fluorogestone acetate-containing intravaginal sponges in adult German Merino ewes (n = 38). They were superovulated with single doses of pFSH (17 mg; n = 10), hMG (600 IU FSH and 600 IU LH; n = 9) or eCG (1250 IU; n = 10) given at the time of sponge removal, or pFSH (17 mg; n = 9) given 36h before sponge removal. Follicular and luteal development were observed by ultrasonic scanning every 8 h from the gonadotrophin injection until the end of estrus, and then once daily until Day 6 after estrus. Jugular venous blood was collected starting immediately before and 1 h after superovulation treatment, then twice daily until the end of estrus and once daily for the following 7 days. Concentrations of estradiol-17beta (E2) and progesterone (P4) were measured in plasma. Differences in the follicular dynamics of the 4 superovulation groups were obvious. The functional duration of the pFSH action was estimated to last approximately 48 h, whereas eCG and hMG were active for up to 72 h. The diameter of the ovulatory follicles proved to be smaller than it was described for unstimulated ewes. Single applications of pFSH or hMG can induce a superovulatory response, although the post-estrus progesterone profile revealed a high premature luteal regression rate in the different superovulation groups. Premature corpus luteum regression could not be seen by ultrasonography at this early stage of the luteal phase, indicating that the technique may fail to detect these corpora lutea in an embryo transfer program. However, ultrasonography represents a suitable method to observe follicular dynamics following different superovulation regimens in sheep.
Theriogenology 04/2001; 55(4):847-65. · 1.96 Impact Factor
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ABSTRACT: Intact ovine and caprine d7/d8 blastocysts and inner cell masses, or embryonic discs (d10/d11) isolated mechanically were cultured under different conditions in order to establish which of the feeder cell types, medium supplements or developmental stages used are most suitable for the isolation of ES-like cells in both species. Subcultures were most successful with isolated embryonic discs on inactivated bovine fetal liver fibroblasts, but ES-like cells did not develop beyond the 44th passage. Morphological ES-like cells were analyzed fluorescence microscopically after treatment with monoclonal antibodies against cytoskeleton markers or after marking with DBA- and PNA-agglutinin. Cells were Cytokeratin 18- and DBA-negative, but vimentin- and PNA-positive. In suspension culture these cells developed embryoid body-like structures, and following induced differentiation tubular structures and epithelial-, fibroblast-, and neuron-like cells. ES-like cells (> 10 passages) were analysed in the nuclear transfer assay. Initial development could be initiated, but development beyond the 12cell stage did not occur.ZusammenfassungIsolierung ES-hnlicher Zellinien von ovinen und caprinen Embryonen der PrimplantationsphaseIntakte ovine und caprine d7/d8 Blastozysten und mechanisch isolierte Embryonalknoten oder Keimscheiben (d10/d11) wurden unter verschiedenen Bedingungen in vitro kultiviert, um festzustellen welche Nhrzelltypen und Mediumzustze und welche Entwicklungsstadien zur Isolierung ES-hnlicher Zellen beider Spezies besonders geeignet sind. Erfolgreiche Passagen stammzellhnlicher Zellen konnten besonders bei Kokultivierung mit inaktivierten fetalen bovinen Leberfibroblasten durchgeführt werden, wobei ES-hnliche Zellinien etabliert wurden, die sich jedoch nicht über die 44. Passage hinaus in vitro entwickelten. Es erwies sich als vorteilhaft, isolierte Keimscheiben als Ausgangsstadium einzusetzen. Die vitalzytologisch als ES-hnlich eingestuften Zellen wurden mit monoklonalen AK gegen Zytoskelettmarker oder mit DBA- und PNA-Agglutinin markiert und fluoreszenzmikroskopisch analysiert. Sie erwiesen sich als Zytokeratin 18- und DBA-negativ, jedoch Vimentin- und PNA-positiv. In Suspensionskultur entwickelten sich von diesen Zellen ‘embryoid body'-hnliche Strukturen und als Folge einer induzierten Differenzierung tubulre Strukturen und epithel-, fibroblasten- und neuronenhnliche Zellen. Bei einer Überprüsfung der in vitro etablierten stammzellhnlichen Zellen (< 10 Passagen) nach Kerntransfer, kam es nur gelegentlich zu einer Initiierung von Furchungen, doch wurde das 12Zellstadium nicht überschritten.
Journal of Animal Breeding and Genetics 01/1996; 113(1‐6):413 - 426. · 1.46 Impact Factor
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ABSTRACT: The effects of alpha-amanitin on cumulus enclosed and denuded porcine oocytes exposed to this heterogeneous nuclear RNA (hnRNA) synthesis inhibitor at different times (0, 0.5, 1, 2, 4, 6, 8, 12 and 20 h) after the start of culture was investigated. A further objective was to determine the sequence of nuclear progression after removing the inhibitor. The addition of 10 micrograms alpha-amanitin ml-1 to a gonadotrophin containing medium (10 iu PMSG ml-1 in TCM 199) at 0, 0.5 or 1 h after the onset of culture prevented cumulus expansion, and only 4.4, 6.4 and 3.5% of oocytes underwent germinal vesicle breakdown. This inhibitory effect was considerably reduced by delaying the addition of the drug to the culture medium for 2-8 h (2 h: 34.9%, 4 h: 53.5%, 6 h: 46.9%, 8 h: 59.2% germinal vesicle breakdown), and no inhibition of nuclear maturation was observed when alpha-amanitin was added after 12 or 20 h following explantation of the oocytes. When cumulus-oocyte complexes were cultured for > or = 2 h in inhibitor-free medium and then transferred to medium supplemented with alpha-amanitin, full cumulus expansion was observed in all cases, at the end of the 44 h culture. Denudation of the oocytes before culture in either medium supplemented with alpha-amanitin or microinjection of alpha-amanitin into the ooplasm at concentrations of 1.0 and 10.0 mg ml-1 remained without any effect on nuclear progression.(ABSTRACT TRUNCATED AT 250 WORDS)
J Reprod Fertil 05/1993; 98(1):195-201.
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DTW. Deutsche tierärztliche Wochenschrift 05/1988; 95(4):167-74. · 0.41 Impact Factor
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ABSTRACT: A comparative experiment in oestrous synchronized and superovulated ewes and does is described on which fertilization was studied following natural mating or transmural intrauterine insemination by laparoscopy. Thirty-two ewes were inseminated into each uterine horn with 50-100 microliter of fresh undiluted semen, whereas 26 ewes were mated by fertile rams. Fertilization rates of eggs recovered 5-7 days later were 93.7% (163/174) and 60.5% (78/129) respectively. Eight does were inseminated by laparoscopy while 12 does were mated by fertile bucks. Numbers of eggs fertilized of those recovered 6-7 days later were 42/47 (89.4%) in the intrauterine inseminated group and 51/60 (85%) in the naturally mated group. These results indicate that high levels of fertilization can be obtained in ewes following intrauterine insemination with fresh semen with the aid of a laparoscope and that it is possible to overcome the reduced fertilization rates after natural mating. In contrast to the ewe the intrauterine semen deposition in does did not improve the high fertilization rates obtained after natural mating.
Tierärztliche Praxis 02/1986; 14(1):35-41. · 0.28 Impact Factor
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Zentralblatt für Veterinärmedizin. Reihe A 07/1984; 31(5):370-85.
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Zentralblatt für Veterinärmedizin. Reihe A 03/1983; 30(2):146-53.
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Berliner und Münchener tierärztliche Wochenschrift 04/1982; 95(6):107-11. · 0.82 Impact Factor
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Zentralblatt für Veterinärmedizin. Reihe A 03/1980; 27(1):65-9.
