Pellet-freezing spermatozoa of two marsupials: the tammar wallaby, Macropus eugenii, and the brushtail possum, Trichosurus vulpecula

1996 ◽  
Vol 8 (4) ◽  
pp. 681 ◽  
Author(s):  
FC Molinia ◽  
JC Rodger
Keyword(s):  
Room Temperature ◽  
Tammar Wallaby ◽  
Brushtail Possum ◽  
Vitro Fertilization ◽  
Macropus Eugenii ◽  
Progressive Motility ◽  
Viable Population ◽  
Phosphate Buffered Saline ◽  
Breeding Techniques

A protocol was developed for pellet-freezing spermatozoa of the tammar wallaby and the brushtail possum. Seren was collected by electro-ejaculation and wallaby spermatozoa were washed by 'swim-up' into phosphate-buffered saline (PBS), whereas possum spermatozoa were not washed. Wallaby spermatozoa were screened for toxicity in diluents containing a range of cryoprotectants (0-10%): dimethyl sulfoxide (DMSO), ethylene glycol and propanediol. Possum spermatozoa were tolerant of diluents containing 17.5% glycerol. Wallaby and possum spermatozoa were diluted 1:1 with the most promising cryoprotective diluents (final concentrations in PBS: possum, 17.5% glycerol; wallaby, 7.5% glycerol + 10% DMSO) and, after 5 min equilibration at room temperature, were pellet-frozen. Pellets were thawed (35 degrees C) and wallaby spermatozoa were washed by centrifugation (200 g for 5 min) and resuspended in PBS to minimize cryoprotectant toxicity. A high proportion of possum spermatozoa was recovered after freezing (67.5%), having good progressive motility (3.6 on a 0-5 scale). The progressive motility of frozen-thawed wallaby spermatozoa was also high (3.0), but only 10% of motile spermatozoa were recovered. The pellet-freezing method in conjunction with the post-thaw washing procedure (wallaby) may produce a viable population of cryopreserved marsupial spermatozoa suitable for use in assisted-breeding techniques such as in vitro fertilization and artificial insemination.

Hormones of oestrus and ovulation and their manipulation in marsupials

1996 ◽  
Vol 8 (4) ◽  
pp. 661 ◽  
Author(s):  
LA Hinds ◽  
TP Fletcher ◽  
JC Rodger
Keyword(s):  
In Vitro Fertilization ◽  
Tammar Wallaby ◽  
Monodelphis Domestica ◽  
Ovarian Activity ◽  
Vitro Fertilization ◽  
Macropus Eugenii ◽  
Australian Species ◽  
Maturation In Vitro ◽  
American Laboratory

Oestrus and ovulation occur spontaneously in the majority of marsupials, with behavioural oestrus usually occurring 1-2 days before ovulation. The hormone changes that occur at this time have been described in the most detail for the monovular tammar wallaby Macropus eugenii. The respective roles of the Graafian follicle, corpus luteum and the pituitary in the events leading up to oestrus and ovulation in this species are also reviewed. Recently, various protocols have been developed for superovulation of marsupials, including Australian species, such as the brush-tailed possum, fat-tailed dunnart, brush-tailed bettong and tammar wallaby, and the American laboratory opossum, Monodelphis domestica. These protocols provide an opportunity for studying the regulation of ovarian activity and for the collection of larger quantities of material for the study of gamete maturation, in vitro fertilization and embryonic development.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

scholarly journals Epididimis: van hoofspeler tot bysaak?

1997 ◽  
Vol 16 (1) ◽  
pp. 10-15
Author(s):  
M. H. Fourie ◽  
M. S. Bornman ◽  
J. M. C. Oosthuizen
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Injection Techniques ◽  
Infertile Couples

During epididymal transit, sperm acquire the capacity for fertilization and progressive motility. The epididymis is functionally and mechanically indispensable for in vivo fertilization, but technology has progressed rapidly in the field of in vitro fertilization. Modern sperm-injection techniques have dispensed with the epididymis as a necessity in the treatment of individual infertile couples.

50 Effects of Cathepsin B Inhibitor E64 on the Survival Rate of Cryopreserved Semen from Korean Brindled Bulls

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
S. W. Kim ◽  
M.-S. Kim ◽  
C.-L. Kim ◽  
I. S. Jeon
Keyword(s):  
Cathepsin B ◽  
Room Temperature ◽  
Egg Yolk ◽  
Coat Colour ◽  
Control Group ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
The Difference ◽  
The Individual

Korean brindled cattle have distinctive coat colour and are regarded as rare cattle in the Korean peninsula. To preserve the line as a genetic resource for diversity in cattle, semen cryopreservation has been used for selection of individuals for breeding between local AI centers. Nevertheless, the survival and viability of frozen semen from Korean brindled bulls is not uniform due to the difference between the individual bulls or experimental techniques. In this study, E64, a cathepsin B inhibitor, was used at final concentrations of 0.05, 0.5, and 1 µM to test the viability of frozen semen. To prepare frozen semen, a triladyl-egg yolk diluent with 6.4% glycerol was used in a two-step freezing method with 4 different bulls with 3 repeats. A total of 12 ejaculates was diluted to a concentration of 50 to 100 × 106 mL−1 at room temperature, and slowly cooled from room temperature to 5°C in 2 to 3 h. The cooled semen was diluted 1:1 with a secondary diluent containing E64 to prepare the experimental group. After loading, 0.5-mL straws were immersed into liquid nitrogen after 10 min exposure at 5 cm above the nitrogen using a styrofoam box. The viability of spermatozoa after thawing at 37°C for 40 s was analysed by Student’s t-test. The rate of surviving sperm in the 1 µM E64 group (82 ± 4.3%) was significantly higher than that of the control group (71.2 ± 2.0%; P < 0.05). However, the 0.05 and 0.5 µM E64 treatment groups lead to similar rates (77.5 ± 2.0% and 75.0 ± 5.0%, respectively; P > 0.05). Based on these results, it is expected that E64 could be used for the improvement of productivity of frozen semen; further results on in vitro fertilization and development are ongoing.

scholarly journals Induction of sperm maturation in vitro in epididymal cell cultures of the tammar wallaby (Macropus eugenii): disruption of motility initiation and sperm morphogenesis by inhibition of actin polymerization

Reproduction ◽  
2002 ◽  
pp. 107-117 ◽  
Author(s):  
M Lin ◽  
R Hess ◽  
RJ Aitken
Keyword(s):  
Culture System ◽  
Actin Polymerization ◽  
Tammar Wallaby ◽  
Sperm Maturation ◽  
Actin Organization ◽  
Cell Monolayers ◽  
Macropus Eugenii ◽  
Maturation In Vitro ◽  
Marsupial Species

A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days. The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa. These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials. These observations also indicate that the marsupial co-culture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species.

Limb development in pouch young of the brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii)

2006 ◽  
Vol 0 (0) ◽  
pp. 060606025751023-???
Author(s):  
R. G. Lentle ◽  
M. C. Kruger ◽  
D. J. Mellor ◽  
M. Birtles ◽  
P. J. Moughan
Keyword(s):  
Limb Development ◽  
Tammar Wallaby ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Macropus Eugenii

scholarly journals In-vitro secretion of progesterone by the corpus luteum of the tammar wallaby, Macropus eugenii

Reproduction ◽  
1983 ◽  
Vol 67 (1) ◽  
pp. 57-63 ◽  
Author(s):  
L. A. Hinds ◽  
S. M. Evans ◽  
C. H. Tyndale-Biscoe
Keyword(s):  
Corpus Luteum ◽  
Tammar Wallaby ◽  
Macropus Eugenii

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 285-291 ◽  
Author(s):  
K.S. Sidhu ◽  
K.E. Mate ◽  
F.C. Molinia ◽  
D.K. Berg ◽  
J.C. Rodger
Keyword(s):  
Calf Serum ◽  
Explant Culture ◽  
Conditioned Media ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Large Variability ◽  
Vitro Fertilization ◽  
Ionic Calcium ◽  
Australian Marsupial

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

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