<i>In vitro</i> capacitation of Siberian tiger spermatozoa

Byers, A. P.; Hunter, A.G.; Hensleigh, Hugh C.; Kreeger, Terry J.; Binczik, G. A.; Reindl, N. J.; Seal, Ulysses S.; Tilson, Ronald L.

doi:10.1002/zoo.1430060404

Cited by 8 publications

(6 citation statements)

References 14 publications

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“…The capacitation results of these experiments using fresh spermatozoa from 5 Siberian tigers confirm the preliminary results reported previously on one tiger (Byers et al, 1987), namely in-vitro capacitation of Siberian tiger spermatozoa requires a 2-h preincubation period at 37°C in capaci¬ tation medium or in seminal plasma. The decapacitation factor in the seminal plasma of several other species (rabbit: Chang, 1957; (1988) reported that removal of seminal plasma was not required for in-vitro capacitation of domestic cat spermatozoa before penetration of zona-free hamster ova.…”

Section: Discussionsupporting

confidence: 90%

“…hCG (Sigma) 55 h later. At 17 h after hCG the animals were killed, oocytes harvested and zona-free oocytes prepared as described by Byers et al (1987). The zona-free eggs were then washed 3 times and 20 eggs were added to each 50 µ droplet of spermatozoa (prepared as described below for each experiment) under mineral oil.…”

Section: Methodsmentioning

confidence: 99%

“…Siberian tiger spermatozoa are capable of penetrating zona-free hamster eggs (Byers et al, 1987). The SPA is presently the best in-vitro test available to provide information about the functional capacity of spermatozoa (Yanagimachi, 1984).…”

Section: Introductionmentioning

confidence: 99%

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In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

Byers¹,

Hunter²,

Seal³

et al. 1989

Reproduction

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Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg-sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37 degrees C results in significantly higher penetration rates than does a 22 degrees C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen-thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze-thaw procedure results in a shortening of the required capacitation time.

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Section: Discussionsupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

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In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

Byers¹,

Hunter²,

Seal³

et al. 1989

Reproduction

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“…Additionally, the conditions for in vitro capacitation of domestic cat sperm are less complex than for other species that require exposure to a hypertonic medium [281, to heparin [261, or to a high calcium concentration [25]. On the contrary, removing seminal plasma is unnecessary for cat oocyte penetration; simple semen dilution is sufficient for gamete interaction in the domestic cat [1] and tiger (Panthera tigris) [29]. The discovery that domestic cat sperm exhibit a relatively low frequency of spontaneous acrosomal loss in vitro also differs from that observed in sheep [30] and dog [31] sperm.…”

Section: Discussionmentioning

confidence: 99%

Sperm Capacitation and the Acrosome Reaction are Compromised in Teratospermic Domestic Cats1

Long

Wildt

Wolfe

et al. 1996

Biology of Reproduction

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The efficiency of sperm capacitation and of the acrosome reaction was studied in the teratospermic domestic cat to evaluate further the etiology of compromised zona pellucida penetration and oocyte fertilization. Specific objectives were to compare normospermic and teratospermic cat ejaculates for 1) the kinetics and timing of sperm capacitation in vitro as determined by an ionophore-induced acrosome reaction; 2) the incidence of spontaneous acrosomal loss; 3) the ability of capacitated, swim-up processed sperm to acrosome-react in response to chemical (calcium ionophore) or physiological (solubilized zonae pellucidae) inducers; and 4) differences in acrosomal ultrastructure by use of transmission electron microscopy (TEM). Acrosomal status was determined with the fluorescent probe Arachis hypogaea (peanut) agglutinin. The timing of in vitro capacitation differed (p < 0.05) between cat populations. Normospermic samples were capacitated at 2.0 h postcentrifugation, whereas teratospermic samples required 2.5 h to become capacitated. At 2.5 h, sperm from teratospermic males were less capable (p < 0.05) of completing the acrosome reaction after ionophore exposure (49.3 +/- 8.0%) than sperm from normospermic males (73.3 +/- 3.8%). Levels of spontaneous acrosomal loss/reaction over time were similar (p > 0.05) between cat groups (range, 7.6-17.8%). In swim-up separated sperm from normospermic cats, ionophore A23187 was a more potent inducer (p < 0.05) of the acrosome reaction (70.1 +/- 6.5%) than solubilized zonae pellucidae (31.1 +/- 1.2%). Swim-up separated sperm from teratospermic cats, however, were compromised in the ability to acrosome react, regardless of inducer (ionophore, 23.9 +/- 3.3%; solubilized zonae pellucidae, 23.9 +/- 4.7%; p > 0.05). Sperm motility patterns over time indicated that differences in acrosomal status were not influenced by cell death. The frequency of abnormal acrosomes detected by TEM was higher (p < 0.05) in teratospermic (30.0 +/- 3.9%) than in normospermic (3.1 +/- 1.3%) samples. Swim-up separation failed to reduce (p > 0.05) the proportion of sperm cells with malformed acrosomes (swim-up, 33.5 +/- 3.5%; washed, 26.6 +/- 4.6%). These results indicate that sperm from teratospermic cats exhibit a high incidence of malformed acrosomes detectable only at the ultrastructural level. Nevertheless, acrosomal dysfunction is not related exclusively to structural defects because > 40.0% of swim-up separated sperm with structurally normal acrosomes still are incapable of completing the acrosome reaction. This suggests that compromised capacitation and acrosomal dysfunction may be responsible for low fertilization success in the teratospermic domestic cat.

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“…Siberian tigers are the largest of the tiger subspecies and 366-406 Siberian tigers exist in the wild (WWF). There are many researches in favor of saving tigers (Tilson and Seal, 1986;Byers et al, 1987;Donoghue et al, 1990Donoghue et al, , 1992Miller et al, 1990). However, there population has gradually been reduced (International Union for Conservation of Nature and Natural Resources, IUCN).…”

Section: Introductionmentioning

confidence: 99%

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

Hashem

Bhandari

Kang

et al. 2006

Molecular Reproduction Devel

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The present study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors (PI), and different levels of dimethylsulfoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts. We also compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Cell cycle synchronization between nuclear donor and recipient cells is considered to be one of the most crucial factors for successful cloning. Five experiments were performed each with a one-way completely randomized design involving three replicates of all treatments. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants like b-Mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 4 hr. In Experiment III, three PI, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ ml) were used in the sane manner as in Experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0%, and 2.5% were tested on different cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In Experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the G 0 /G 1 stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%; P < 0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of G 0 þ G 1 phases than serum starved and fully confluent. After nuclear transfer we did not see any significant differences on the development of tigerporcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter G 0 /G 1 and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs in Siberian tiger, because there are negative effects, such as apoptosis associated with serum starvation. Mol. Reprod. Dev. 74: 403-411, 2007. ß

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In vitro capacitation of Siberian tiger spermatozoa

Cited by 8 publications

References 14 publications

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

Sperm Capacitation and the Acrosome Reaction are Compromised in Teratospermic Domestic Cats1

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

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