Biomedical subjects

D E Wildt

Publications and source records attributed to D E Wildt.

At least 37 records · Page 2Linked to original sources

PubMed2000

Age-dependent changes in sperm production, semen quality, and testicular volume in the black-footed ferret (Mustela nigripes).

The black-footed ferret (Mustela nigripes), which was extirpated from its native North American prairie habitat during the 1980s, is being reintroduced to the wild because of a successful captive-breeding program. To enhance propagation, the reproductive biology of this endangered species is being studied intensively. The typical life span of the black-footed ferret is approximately 7 yr. Female fecundity declines after 3 yr of age, but the influence of age on male reproduction is unknown. In this study, testis volume, seminal traits, sperm morphology, and serum testosterone were compared in 116 males from 1 to 7 yr of age living in captivity. Results demonstrated that testes volume during the peak breeding season was similar (P > 0.05) among males 1 to 5 yr of age, reduced (P < 0.05) among males 6 yr of age, and further reduced (P < 0.05) among males 7 yr of age. Motile sperm/ejaculate was similar in males 1 to 6 yr of age but diminished (P < 0.05) in those 7 yr of age. Males at 6 and 7 yr of age produced fewer (P < 0.05) structurally normal sperm than younger counterparts; however, serum testosterone concentrations were not reduced (P > 0.05) in older males. Histological comparison of testicular/epididymal tissue from 5- and 7-yr-old black-footed ferrets confirmed that the interval between these two ages may represent a transitional period to reproductive senescence. In summary, functional reproductive capacity of male black-footed ferrets exceeds that of females by at least 2 yr. Testes and seminal quality are indistinguishable among males 1 to 5 yr of age, with progressive reproductive aging occurring thereafter.

Aging↗

PubMed2000

Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and peregrine falcon spermatozoa.

Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonelli's eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21 degrees C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR-14/propidium iodine). Sperm viability at osmolalities >/=800 mOsm was higher (P: < 0.05) in raptor than poultry semen. Return to isotonicity after exposure to hypertonicity (3000 mOsm) decreased (P: < 0.05) number of viable spermatozoa in chicken, turkey, and golden and Bonelli's eagle spermatozoa but not in imperial eagle or peregrine falcon spermatozoa. Differences were found in spermatozoa resistance to hypotonic conditions, with eagle species demonstrating the most tolerance. Semen, equilibrated for 1 h (4 degrees C) in diluent containing DMA (> or =2.06 M), experienced decreased (P: < 0. 05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21 degrees C) markedly reduced (P: < 0.05) spermatozoa survival in all species except the Bonelli's eagle and turkey. Rapid cooling was detrimental (P: < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.

Animals↗

PubMed1999

Scimitar-horned oryx (Oryx dammah) spermatozoa are functionally competent in a heterologous bovine in vitro fertilization system after cryopreservation on dry ice, in a dry shipper, or over liquid nitrogen vapor.

A heterologous bovine in vitro fertilization (IVF) system was used to study the functional competence of scimitar-horned oryx spermatozoa after cryopreservation. Four sperm-freezing methods were compared after dilution of ejaculates from six oryx with an equine semen extender: 1) dry ice, 2) dry shipper one-step, 3) dry shipper two-step, and 4) liquid nitrogen vapor. Post-thaw sperm motility, longevity, and acrosomal status were assessed and zona pellucida penetration, fertilization, and embryo cleavage were evaluated after coincubation of thawed oryx spermatozoa with in vitro-matured domestic cow oocytes. Sperm motility index (SMI) decreased (p < 0.05) over a 6-h period, but a high percentage (>/= 65%) of spermatozoa contained intact acrosomes in all treatments. Despite differences in sperm motility among methods, oocyte penetration, fertilization, and embryo cleavage did not differ (p >/= 0.05). However, cleavage success was < 50% across all treatments. There were positive correlations (p < 0.05; r = 0.81-0.97) between sample SMI at 3 and 6 h and fertilization, penetration, and cleavage, but no correlations (p >/= 0.05) between SMI at 0 or 1 h and IVF success. This study demonstrates that compatible heterologous gamete interaction allows thorough assessment of post-thaw sperm function in an endangered antelope. Scimitar-horned oryx spermatozoa appear relatively tolerant of varied cryopreservation methods, and preserved samples exhibit adequate post-thaw function to warrant use for assisted reproduction.

Acrosome↗

PubMed1999

Prolonged mating in prairie voles (Microtus ochrogaster) increases likelihood of ovulation and embryo number.

Prairie voles are induced ovulators that mate frequently in brief bouts over a period of approximately 24 h. We examined 1) impact of mating duration on ovulation and embryo number, 2) incidence of fertilization, 3) temporal pattern of embryo development, 4) embryo progression through the reproductive tract over time, and 5) embryo development in culture. Mating was videotaped to determine first copulation, and the ovaries were examined and the reproductive tracts flushed at 6, 8, 10, 12, 16, 20, and 24 h and 2, 3, and 4 days after first copulation. The number of mature follicles and fresh corpora lutea and the number and developmental stage of embryos were quantified. One, two-, and four-cell embryos were cultured in Whitten's medium. Mature follicles were present at the earliest time examined (6 h). Thirty-eight percent of females that had been paired for < 12 h after the first copulation ovulated, whereas all females paired >/= 12 h after the first copulation ovulated. Virtually all (> 99%) oocytes recovered from females paired for >/= 12 h after first copulation were fertilized. Pairing time after first copulation and mean copulation-bout duration were significant (p < 0.05) determinants of embryo number. Embryos entered the uterine horns and implanted on Days 3 and 4, respectively, after first copulation (Day 0). Embryos cultured in vitro underwent approximately one cell division per day, a rate similar to that in vivo. We conclude that prairie voles ovulate reliably after pairing for >/= 12 h, although some females showed exceptional sensitivity not predicted by the variables quantified. Prolonged mating for longer than 12 h increased the total embryos produced. This mechanism likely has adaptive significance for increasing offspring number.

Animals↗

PubMed1999

Circannual variations in intraovarian oocyte but not epididymal sperm quality in the domestic Cat.

Ovaries and testes were collected throughout the year from domestic cats spayed and neutered at local veterinary clinics. Fresh oocytes recovered from minced ovaries were subjected to in vitro maturation and then stained to determine stage of maturation or were inseminated with conspecific sperm. The cauda and corpus regions of each epididymis were dissected into pieces and placed in medium; 30 min later, the epididymal tissue was removed, the medium centrifuged, and the sperm pellet resuspended. Samples were assessed for total sperm count and sperm motility traits, morphology, acrosomal integrity, and ability to penetrate cat oocytes in vitro. Fewer excellent (grade I) oocytes were recovered per ovarian pair during September-November (mean +/- SEM, 19.2 +/- 2.1%) than during January-July (36.8 +/- 3.6%, p < 0.05), while the remaining months had intermediate percentages of grade I oocytes (p > 0.05). A high percentage of oocytes recovered from November-April completed nuclear maturation (64.3 +/- 6.8%), which was different (p < 0.05) from the values for May-July (32.2 +/- 3.8%) and August-October (10.4 +/- 2.9%). Percentage of oocytes with bound sperm was lowest (p < 0.01) in September and October (32.0 +/- 3.1%) compared to February and March (91.4 +/- 1.7%). Percentage of oocytes with sperm within the perivitelline space was highest (p < 0.05) in May-August (33.8 +/- 4.6%) compared to all other months. In contrast, the period of highest (p < 0.01) fertilization (i.e., >/= 4-cell embryo formation) was March-April (51.7 +/- 3.1%) as compared to May-July (17.2 +/- 1.8%) or November-January (12.4 +/- 2.6%). Negligible numbers of oocytes recovered during August-October developed beyond the 2-cell stage (1.1 +/- 0.3%). Blastocyst development from cleaved embryos was highest during February-April (44.3 +/- 2.3%) and lowest during August-October (0.6 +/- 0.1%; p < 0.01). Sperm recovered from the epididymides throughout the year did not differ (p > 0.05) in concentration or in any of the motility, structural, or functional variables evaluated. In summary, cat oocyte nuclear maturation in vitro is depressed during August-October, and the ability to form cleaved embryos remains low even when the capacity to achieve nuclear maturation is relatively high (November-January and May-July). In contrast, male cats are capable of consistently producing viable, progressively motile sperm throughout the year.

Animals↗

PubMed1998

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors.

Spermatozoa from teratospermic domestic cats (> 60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (< 40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)-induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase-dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males.

Acrosome↗

PubMed1998

Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah).

Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 microM heparin, 3) TALP + 20 microM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p < 0.05) sperm incubated in TALP + 10 microM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p < 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to > or = 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting that the two bovid species have similar fertilization mechanisms.

Acrosome↗

PubMed1998

Viability of small preantral ovarian follicles from domestic cats after cryoprotectant exposure and cryopreservation.

About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40-90 micron) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0 degrees C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at O h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P > 0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P < 0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2'-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% +/- 2.9%, 18.8% +/- 1.9% and 16.2% +/- 1.6% were intact after O h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2'-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.

Animals↗

PubMed1997

Water distribution and permeability of zebrafish embryos, Brachydanio rerio.

Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the distribution to permeability to water. These embryos are a complex multi-compartmental system composed of two membrane-limited compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the complexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequally in each compartment. At the 6-somite stage, the percent water (V/V) was distributed as follows: total in embryo = 74%, total in yolk = 42%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenological water permeability parameter, Lp, assuming intracellular isosmotic compartments of either 40 or 300 mosm. This analysis revealed that the membrane permeability changed (P < 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability remained constant (Lp = 0.022 +/- 0.002 micron x min-1atm-1 [mean +/- S.E.M.] assuming isosmotic is 40 mosm or Lp = 0.049 +/- 0.008 micron x min-1atm-1 assuming isosmotic is 300 mosm). However, at the 6-somite stage, Lp increased twofold (Lp = 0.040 +/- 0.004 micron x min-1atm-1 assuming isosmotic is 40 mosm or Lp = 0.100 +/- 0.017 micron x min-1atm-1 assuming isosmotic is 300 mosm). Therefore, the low permeability of the zebrafish embryo coupled with its large size (and consequent low area to volume ratio) led to a very slow osmotic response that should be considered before formulating cryopreservation protocols.

Animals↗

PubMed1997

Follicle-oocyte atresia and temporal taphonomy in cold-stored domestic cat ovaries.

In vitro oocyte maturation followed by in vitro fertilization (IVM/IVF) success in the domestic cat remains inferior to commonly studied livestock or laboratory species. The objectives here were (1) to histologically assess atresia status of freshly excised follicle/oocyte complexes, and (2) to evaluate taphonomic change (deterioration after excision) of these complexes after ovarian cold storage for up to 48 h. After excision of 50 ovarian pairs, one ovary was preserved immediately and the other stored in phosphate buffered saline (4 degrees C) for 4, 8, 12, 24, or 48 h before fixation and examination. Ovaries were classified as luteal if prominent corpora lutea (CL) were present or as follicular if antral follicles and no CL were present. Two classes of follicle-oocyte complexes (preantral and antral) were microscopically evaluated. Of the 2,280 complexes examined, 64.3% demonstrated clear evidence of slight to severe degeneration, with various stages being described and photographed for the first time. There was no histological evidence indicating distinctive morphological differences between oocytes recovered from follicular versus luteal donors. Storage of whole ovaries in cold saline inhibited taphonomic changes for 48 h after excision. In summary, there is marked variability in the number and quality of follicle populations in cat ovaries. A high percentage of full-sized follicular oocytes are undergoing atresia at any given time. However, additional gross degeneration as a result of cold-storage appears modest for up to 48 h. Nonetheless, this high level of natural atresia in the cat likely contributes to comparatively lower IVM/IVF success than in other species.

Animals↗

PubMed1997

Transition from maternal to embryonic control of development in IVM/IVF domestic cat embryos.

The timing of the transition from maternal to zygotic control of development (MZT) and the initiation of transcription was studied in domestic cat embryos to determine if there is a temporal association between these phenomena and the development block observed in cat embryos fertilized in vitro. Embryos were derived from in vitro-matured, in vitro-fertilized (IVM/IVF) oocytes. In Experiment 1, embryos (n = 52) were cultured continuously in the presence of 10 micrograms/ml alpha-amanitin (a transcriptional inhibitor) from 12-hr postinsemination (hpi), and cleavage stage was evaluated every 24 hr. The proportion of embryos cleaving to at least the 5-8 cell stage in the presence of alpha-amanitin (32/52) was similar (P > 0.05) to that of controls cultured without alpha-amanitin (25/50). In contrast, only 7.7% of alpha-amanitin-treated embryos cleaved to the 9-16-cell stage, compared with 38.0% of the controls (P < 0.05), indicating that products of embryonic transcription were required for cleavage beyond the 5-8-cell stage. In Experiment 2, embryos were cultured in the presence of 20 microM 3H-uridine for 12 hr beginning at 24, 36, 48, or 60 hpi and subjected to autoradiography. Embryos of 5-8-cell and 9-16-cell stages (14 of 27 and 8 of 12, respectively) clearly demonstrated nuclear labeling, a finding also confirmed by computer-aided densitometry. It is concluded that embryonic transcription and the MZT occur by the 5-8-cell stage of IVM/IVF domestic cat embryo development and the MZT is not directly related to the partial morula-to-blastocyst developmental block observed in cultured IVF cat embryos.

Amanitins↗

PubMed1997

DNA degradation in mural granulosa cells of non- and slightly atretic follicles of fresh and cold-stored domestic cat ovaries.

Apoptosis of granulosa cells is associated with follicular atresia and may occur before atresia becomes morphologically evident. Detection of DNA fragmentation by in situ end-labeling (ISEL) with terminal transferase allows the histological assessment of apoptotic cells on conventional histological sections. Degradation of DNA also may occur after prolonged cold storage of ovaries caused by the release of lysosomal enzymes. The objectives of this study were to assess follicle atresia and the impact of cold storage for 8, 12, 24, and 48 hr after ovarian excision by assessing DNA degradation in mural granulosa cells of cat ovaries. Follicles were distinguished by morphological criteria as nonatretic (NA), slightly atretic (SA), or atretic, and the mean number (+/-SEM) of granulosa cells labeled by ISEL was determined. About 50% of follicles showed some sign of atresia independent from the stage of the reproductive cycle of the ovarian donor. Number of ISEL-stained granulosa cells for NA and SA, freshly collected follicles was 7.5 +/- 0.6 and 9.3 +/- 0.8 cells/field, respectively, compared to 16.2 +/- 0.8 cells/field in the wall of atretic follicles (P < 0.001). Fresh NA follicles from luteal phase ovaries had more (P < 0.05) labeled granulosa cells (9.2 +/- 0.7 cells/field) than measured in follicles of cats in a follicular phase (5.7 +/- 0.7). During cold storage, DNA degradation began within 12 hr (NA, 12.2 +/- 0.7 cells/field; SA, 13.3 +/- 0.5), both values being different (P < 0.05) from fresh controls. By 24 hr, DNA degradation was at the level of a positive control subjected to DNAse treatment. In summary, results reveal that granulosa cell DNA degeneration precedes the loss of developmental capacity of cat oocytes during atresia and postexcision storage. Finding irreversible changes in granulosa cell DNA after storage of cat ovaries for > 12 hr may be important for developing oocyte rescue protocols for rare felids in cases where prolonged storage and transport may be required.

Animals↗

PubMed1997

Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio.

The zebrafish (Brachydanio rerio) was used as a model for basic studies of the chilling sensitivity, permeability and toxicity of cryoprotectants. In both intact and dechorionated embryos, early-stage embryos (1.25, 1.5, 1.75, and 2 h) were more susceptible (P < 0.05) to chilling injury at 0 degrees C than late-stage embryos (50, 75, and 100% epiboly and three-somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eight-hour zebrafish embryos tolerated short-term exposures to temperatures ranging from 4 to 23 degrees C for 3.5 h with no detrimental developmental effects. The permeability of dechorionated embryos to cryoprotectants was examined by measuring the kinetics of volumetric change at various developmental stages (16 cells to six somites or ca. 1.25 to 14 h postfertilization) at 28.5 degrees C. The dechorionated zebrafish embryo is composed of two complex cellular compartments (i.e., a large yolk and the developing blastoderm). From 40 to 100% epiboly, the volumes of yolk and blastoderm remained constant, ca. 82 and 18%, respectively. However, these volumes changed rapidly after epiboly. For example, at the six-somite stage, the yolk composed 61% of the total volume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurement of volume changes indicated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solution, but not for 3-somite embryos. For up to 30 min at room temperature, these cryoprotectant solutions were toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclude that the complex nature of the zebrafish embryo reduces the effectiveness and predictive value of light microscopical measurements for cryoprotectant permeability studies.

Animals↗

PubMed1997

New approaches for studying the permeability of fish embryos: toward successful cryopreservation.

This paper describes some new approaches for understanding the permeability of teleost embryos. The dechorionated zebrafish (Brachydanio rerio) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells. Cellular water was distributed unequally in each compartment. Measurements indicated that the total water in the embryo was 74%, while the total water in the yolk was 42%, and total water in the blastoderm was 82%. The internal isosmotic value for the zebrafish embryo is unknown. However, for one-compartment modeling studies of membrane permeability, the mean Lp (+/- SEM) values were 0.022 +/- 0.002 to 0.049 +/- 0.008 microns x min-1 atm-1 at 40 mOsm (assuming this was one possible internal isosmotic value for the entire embryo) and 0.040 +/- 0.004 to 0.1 +/- 0.017 microns x min-1 atm-1 at 300 mOsm (assuming this was another possible internal isosmotic value for the entire embryo). When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume decrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0 M solution. Magnetic resonance (MR) images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permeation into the yolk over 2.5 h. The results from MR spectroscopy and cryoprotectant microinjections into the yolk suggested that the yolk syncytial layer plays the critical limiting role for cryoprotectant permeation throughout the embryo.

Animals↗

PubMed1997

Seasonal effects on ovarian responsiveness to exogenous gonadotrophins and successful artificial insemination in the snow leopard (Uncia uncia).

Ovaries of the seasonally-breeding snow leopard (Uncia uncia) were examined to determine whether they were responsive to exogenous gonadotrophins throughout the year. The potential of laparoscopic artificial insemination (AI) also was assessed for producing offspring. During the non-breeding, pre-breeding, breeding and post-breeding seasons, females (n = 20) were treated with a standardized, dual-hormone regimen given intramuscularly (600 I.U. of equine chorionic gonadotrophin followed 80-84 h later with 300 I.U. of human chorionic gonadotrophin (hCG)). Laparoscopy was performed 45-50 h after administration of hCG, and all ovarian structures were described. Females with fresh corpora lutea (CL) were inseminated, and anovulatory females were subjected to follicular aspiration to examine oocyte quality. Snow leopards responded to exogenous gonadotrophins throughout the year. Mean number of total ovarian structures (distinct follicles mature in appearance plus CL) did not differ (P > or = 0.05) with season, but the proportion of CL: total ovarian structures was greater (P < 0.01) for the breeding season compared with all other seasons. The proportion of females ovulating was greater (P < 0.05) during the breeding and post-breeding seasons than during the pre-breeding and non-breeding seasons respectively. No Grade-1 quality oocytes were recovered from follicles of anovulatory females. Serum concentrations of oestradiol-17 beta appeared elevated in all females, and neither oestradiol-17 beta concentrations nor progesterone concentrations differed (P > or = 0.05) among seasons. Of 15 females artificially inseminated, the only one that was inseminated in the non-breeding season became pregnant and delivered a single cub. This is the first successful pregnancy resulting from AI in this endangered species.

Animals↗

PubMed1997

Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard.

Ovarian sensitivity to exogenous gonadotropins was assessed in the cheetah (Acinonyx jubatus) and clouded leopard (Neofelis nebulosa) to help optimize artificial insemination (AI). Eighteen female cheetahs were used on 29 occasions and were given i.m. injections of 100, 200, or 400 IU eCG and 100 or 250 IU hCG 80 h later. Twenty-three female clouded leopards were treated i.m. on 27 occasions with 25, 50, 75, 100, 200, or 400 IU eCG followed 80 h later with 75, 140, or 280 IU hCG. Ovaries were examined laparoscopically at 43-48 h after hCG in cheetahs and 39-50 h in clouded leopards. All gonadotropin dosages stimulated ovarian activity in both species, but ovulation success and corpus luteum (CL) morphology varied (p < 0.05) with treatment. For both species, the highest and intermediate eCG dosages resulted in ovulation in a high proportion (72-100%) of females. The lowest eCG dosage, although capable of stimulating follicular development, compromised ovulation and resulted in few (< 26%) postovulatory females. For each species, small CL (2-4-mm diameter) were observed with the highest and lowest eCG dosage, and large CL (5-8-mm diameter) were associated with intermediate eCG dosages. Aged CL (10-12 mm diameter) were observed in 4 of 23 (17.4%) clouded leopards with no prior male exposure, indicating occasional spontaneous ovulation. Nineteen laparoscopic intrauterine AI procedures were performed in eCG/hCG-treated postovulatory cheetahs. Eighteen AI procedures were conducted in eCG/hCG-treated postovulatory clouded leopards. Six of the 13 cheetahs (46%), all in the 200-IU eCG/100-IU hCG group, became pregnant, in contrast to none of the clouded leopards. This study has revealed differences in ovarian activity in two wild felid species as a result of changes in exogenous gonadotropin dosage. Because of this dose-effect response, this comparative approach is necessary to identify a gonadotropin regimen that can mimic "normalcy." Even then, the relatively high AI success in the cheetah compared to the clouded leopard suggests that factors other than ovarian response can dictate the efficiency of assisted reproduction in this taxon.

Acinonyx↗

PubMed1997

Effects of equine chorionic gonadotropin, human chorionic gonadotropin, and laparoscopic artificial insemination on embryo, endocrine, and luteal characteristics in the domestic cat.

The effects of gonadotropin treatment and laparoscopic artificial insemination (AI) on embryo quality, serum progesterone and estradiol concentrations, and luteal progesterone content were examined in the domestic cat. These data were compared to similar historical data reported for naturally estrual, mated queens. All queens in this study (n = 32) were treated with eCG followed by 1) natural breeding (eCG-NB), 2) NB and hCG (eCG-NB-hCG), 3) NB and a sham AI procedure (eCG-NB-sham AI), or 4) hCG and actual AI (eCG-hCG-AI). Queens ovulating in response to treatment were ovariohysterectomized, and oviducts and uteri were flushed to collect embryos. Ovarian structures were recorded, corpora lutea (CL) were excised and evaluated for progesterone content, and serum was analyzed for estradiol-17beta and progesterone. Follicle and CL numbers ranged from 0 to 28 and 2 to 42 per cat, respectively, and treatment means did not differ (p > or = 0.05) among groups. Embryos were recovered from oviducts and uterine horns in all treatment groups, and recovery ranged from 60-96%. Mean embryo number per queen ranged from 8.2 +/- 2.6 to 23.2 +/- 3.8 and did not differ (p > or = 0.05) among groups. However, the proportions of unfertilized oocytes were greater (p < 0.05) for groups treated with hCG and/or artificially inseminated, and the proportion of blastocysts produced (31 of 107, 29.0%) was lower (p < 0.05) in the eCG-hCG-AI group than for any other treatment (range, 59 of 116 [50.9%] to 67 of 116 [57.8%]). Not all queens in each group produced good-quality embryos (eCG-NB, 5 of 5; eCG-NB-hCG, 5 of 8; eCG-NB-sham AI, 2 of 5; and eCG-hCG-AI, 3 of 6). Serum progesterone and estradiol-17beta, and total luteal progesterone per ovary did not differ (p > or = 0.05) among treatments. Compared to historical controls (naturally estrual, mated queens), eCG-NB queens produced > 4 times as many good-quality embryos and blastocysts. Similarly, eCG-hCG-AI-treated queens produced > 4 times the number of oocytes and embryos, although a high proportion of these were poor quality and did not develop to blastocysts. Together, these results indicate that queens treated with eCG are capable of consistently producing many good-quality embryos, at least half of which develop to blastocysts in culture. These data support the use of eCG in felids and suggest that other factors are responsible for reduced pregnancy success and small litter sizes following assisted reproduction.

Animals↗

PubMed1997

Pharmacokinetics and ovarian-stimulatory effects of equine and human chorionic gonadotropins administered singly and in combination in the domestic cat.

Pregnancy success and embryo survival are low with the use of assisted reproduction in felids treated with exogenous gonadotropins. In this study, the pharmacokinetics and ovarian-stimulatory effects of eCG and hCG were evaluated in the domestic cat. Catheterized anestrual queens (n = 4 per treatment [Trt] group) were given 100 IU eCG i.v. (Trt 1), 100 IU eCG i.m. (Trt 2), 75 IU hCG i.v. (Trt 3), 75 IU hCG i.m. (Trt 4), or 100 IU eCG i.m. followed 80 h later by 75 IU hCG i.m. (Trt 5). Blood samples were collected at 0, 5, 30, and 60 min and 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h postinjection, and serum samples were analyzed for estradiol-17beta, progesterone, eCG, and hCG. Pharmacokinetic traits (volume of distribution, Vd; elimination half-life, t1/2beta; clearance rate, Clr) were calculated for eCG and hCG. When i.v. and i.m. administration were compared, no differences (p > 0.05) were observed in follicle or corpus luteum (CL) number or hormone concentrations for queens receiving eCG or hCG alone. Number of mature ovarian follicles (> or = 2 mm diameter) observed at 168 h postinjection did not differ (p > 0.05) for eCG (mean +/- SEM, 10.5 +/- 2.0) vs. hCG (11.1 +/- 3.0), indicating that these were equally effective in inducing follicular growth. In most queens (> 90%) given single gonadotropins (i.m. or i.v.), eCG and hCG persisted in circulation for at least 120 h and 96 h after injection, respectively, reflecting similar (p > 0.05) pharmacokinetic (i.v.) values for Vd (eCG, 91.4 +/- 24.8 ml/kg; hCG, 59.1 +/- 7.9 ml/kg), t1/2beta (eCG, 23.0 +/- 2.4 h; hCG, 22.9 +/- 4.1 h), and Clr (eCG, 2.7 +/- 0.5 ml/h per kg; hCG, 1.8 +/- 0.1 ml/h per kg). Sequential treatment with eCG+hCG did not affect (p > 0.05) the t1/2beta of individual gonadotropins. In summary, eCG and hCG have comparable pharmacokinetics and ovarian-stimulatory activity when administered alone to the domestic cat. These findings suggest that hCG promotes the ancillary follicle formation that is frequently observed after ovulation in cats treated with eCG+hCG regimens, possibly disrupting the maternal environment and decreasing fecundity following assisted reproductive procedures.

Animals↗