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.

scholarly journals In Vitro Hepatic Assessment of Cineole and Its Derivatives in Common Brushtail Possums (Trichosurus vulpecula) and Rodents

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1326
Author(s):  
Ravneel R. Chand ◽  
Mhairi Nimick ◽  
Belinda Cridge ◽  
Rhonda J. Rosengren
Keyword(s):  
Catalytic Activity ◽  
Management Strategies ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Cytochrome P450 3A ◽  
Phascolarctos Cinereus ◽  
Catalytic Activities ◽  
Rats And Mice ◽  
Drug Metabolising Enzymes

Folivore marsupials, such as brushtail possum (Trichosurus Vulpecula) and koala (Phascolarctos cinereus), can metabolise higher levels of dietary terpenes, such as cineole, that are toxic to eutherian mammals. While the highly efficient drug metabolising enzymes, cytochrome P450 3A (CYP3A) and phase II conjugating enzymes (UDP-glucuronosyltransferase, UGT), are involved in the metabolism of high levels of dietary terpenes, evidence for inhibitory actions on these enzymes by these terpenes is scant. Thus, this study investigated the effect of cineole and its derivatives on catalytic activities of hepatic CYP3A and UGT in mice, rats, and possums. Results showed that cineole (up to 50 µM) and its derivatives (up to 25 µM) did not significantly inhibit CYP3A and UGT activities in mice, rats, and possums (both in silico and in vitro). Interestingly, basal hepatic CYP3A catalytic activity in the possums was ~20% lower than that in rats and mice. In contrast, possums had ~2-fold higher UGT catalytic activity when compared to mice and rats. Thus, these basal enzymatic differences may be further exploited in future pest management strategies.

Exogenous glycosaminoglycans (GAG) are able to modulate avian skin differentiation (epithelial keratinization and feather formation

Development ◽  
1984 ◽  
Vol 82 (1) ◽  
pp. 25-40
Author(s):  
E. Becchetti ◽  
G. Stabellini ◽  
A. Caruso ◽  
P. Carinci
Keyword(s):  
Hyaluronic Acid ◽  
Chick Embryo ◽  
Synthetic Medium ◽  
Calf Serum ◽  
Epithelial Differentiation ◽  
Dermal Fibroblasts ◽  
Conditioned Media ◽  
Lung Fibroblasts ◽  
Skin Explants

Several reports have suggested that mesenchymal glycosaminoglycans (GAG) may be involved in the regulatory role of epithelial differentiation. Some researchers have pointed out that exogenousGAG affects extracellular GAG accumulation. We have therefore examined the effect of added GAG on two typical processes of avian skin differentiation: keratinization and feather formation. Glycosaminoglycans, either obtained from fibroblasts cultures (conditioned media) or purified commercially available GAG were administered to 5/6-day chick embryo back skin explants. Control cultures were supported with 199 synthetic medium, chick embryo extract or calf serum. Explants have been examined by histological and histochemical procedures. Skin explants maintained in vitro for 7 days exhibited an epithelial differentiation and a dermal histochemical reactivity which were related to the composition of the culture medium. In conditioned media from dermal fibroblasts, but not from heart or lung fibroblasts, explants always exhibited keratinization. In purified-GAG-containing media, keratinization was observed with condroitinsulphates and not with hyaluronic acid. Keratinization was always related toprevalent accumulation of hyaluronic acid in the underlying mesenchyme whereas feather formation was in relation to deposits of condroitinsulphates in dermis pulp. The above findings demonstrate that exogenous GAG is able to modulate avian skin differentiation and that this regulation is linked to an influence on the mesenchymal GAG pattern.

scholarly journals 266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

2004 ◽  
Vol 16 (2) ◽  
pp. 253 ◽  
Author(s):  
L. Ferré ◽  
C. Ohlrichs ◽  
D. Faber
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Calf Serum ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Sexed Semen ◽  
Chi Square Analysis

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 202 ◽  
Author(s):  
O. Dochi ◽  
M. Tanisawa ◽  
S. Goda ◽  
H. Koyama
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Holstein Cattle ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Repeat Breeder

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

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.

272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE

2006 ◽  
Vol 18 (2) ◽  
pp. 243
Author(s):  
L. S. A. Camargo ◽  
J. H. M. Viana ◽  
W. F. Sa ◽  
A. M. Ferreira ◽  
A. A. Ramos ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Developmental Competence ◽  
Tropical Region ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Developmental Capacity

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

164 EFFECTS OF KINETICS AND MORPHOLOGY ON EARLY EMBRYONIC DEVELOPMENT IN BOVINE OPU-IVF EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 212
Author(s):  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Embryo Transfer ◽  
Calf Serum ◽  
Conception Rate ◽  
Culture Dish ◽  
Fresh Embryo Transfer ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Observation Method

In recent years, the use of ovum pick up (OPU) and IVF for embryo production has increased worldwide; however, the conception rate of embryo transfer is lower for OPU-IVF embryos than for in vivo-derived embryos. This study aimed to determine the efficacy of embryo selection by a 3-step observation method by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). In this study, 9 Holstein and 15 Japanese Black cows were used, and the OPU-IVF was conducted from October 2014 to May 2015. The collected cumulus-oocyte complexes (COC) were cultured for 22 h in 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH. Sperm (at a final concentration of 5 × 106 spermatozoa mL–1) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS by using a micro-well culture dish. Kinetics and morphology were observed at 27, 31, and 55 h post-insemination (hpi). The presumptive zygotes were divided to 3 groups (more than 2 cells, 2 cells, and no cleavage) at 27 and 31 hpi. Then, embryos at the 2-cell stage at 31 hpi were divided into 2 groups: 2-cell with normal cleavage and 2-cell embryos with abnormal cleavage (unequal cleavage, 2-cell with fragments, and 2-cell with protrusion). Subsequently, embryos were classified as 8-cell and more than 8 cell, or less than 8 cell at 55 hpi. The blastocyst rate (BL%) was analysed at 7, 8, and 9 days post IVF. Embryos selected by the 3-step observation method were used for fresh embryo transfer. The data were analysed by chi-squared test. In total, 856 oocytes were collected by OPU and 633 oocytes were cultured, of which 39.7% (263/663) developed to the blastocyst stage. The BL% of 2-cell embryos (72.5%, 116/160) was significantly higher (P < 0.01) than that of no cleavage (47.0%, 117/249) at 27 hpi. The BL% of 2-cell (65.4%, 206/315) and more than 2-cell (53.0%, 35/66) was significantly higher (P < 0.01 and P < 0.05) than that of embryos divided as no cleavage (25.9%, 22/85) at 31 hpi. The BL% was not significantly different between 2-cell with normal cleavage (68.5%, 172/251) and abnormal cleavage (53.1%, 34/64). The BL% of 8-cell and more than 8-cell stage (72.8%, 182/250) was significantly higher (P < 0.01) than that of embryos with less than 8 cells (38.8%, 81/209) at 55 hpi. Overall, 2-cell embryos at 27 hpi, 2-cell embryos with normal cleavage at 31 hpi, and 8-cell and more than 8 cell at 55 hpi showed the highest BL% (82.1%, 78/91). The conception rate was higher for following the selected fresh embryo transfer that was 70.6% (12/17) than average of in vitro fertilization embryos transfer that was 40.0%. These results demonstrate that the 3-step observation method used in this study can be effectively applied for the selection of IVF embryos that have a strong ability to develop into blastocysts and high competence for conception.

Prefertilization gamete maturation events in marsupials

1994 ◽  
Vol 6 (4) ◽  
pp. 473 ◽  
Author(s):  
JC Rodger
Keyword(s):  
Acrosome Reaction ◽  
Cortical Granules ◽  
Vitro Fertilization ◽  
Australian Species ◽  
Egg Membrane ◽  
Cytoplasmic Maturation ◽  
Ovulatory Period ◽  
And Function ◽  
Australian Marsupial

Despite many fundamental similarities between the gametes of marsupials and placental mammals, the regulation and timing of prefertilization gamete maturation are quite different. The marsupial acrosome is remarkably stable and an acrosome reaction (AR) is not induced by reagent effective for the sperm of placental mammals. The ultrastructure of the marsupial sperm AR is essentially similar to that of placental mammals, however, whether an equatorial segment (ES) persists to serve as the site of sperm-egg membrane fusion is unclear. Diacylglycerol induction of the AR suggests that the sperm of Australian species lack an ES, yet an ES-like region appears to be involved in fertilization in the opossum Monodelphis. The marsupial oocyte, unlike those of placentals, continues to grow throughout follicular life and major cytoplasmic maturation events occur late in oocyte development. Cortical granules only become evident shortly before ovulation and mature dark granules may only appear after ovulation. Further, the zona pellucida (ZP) changes in character and function during the peri-ovulatory period. In vitro fertilization has been achieved for an opossum but not for any Australian marsupial, owing to failure of sperm-ZP binding. Requirement for a sperm maturation process is likely, but capacitation treatments used for placental sperm in vitro have been ineffective. Since it is now feasible to experimentally manipulate marsupial gametes in vitro major advances in our understanding of their function can be expected.

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