Effect of Vitrification of Feline Ovarian Cortex on Follicular and Oocyte Quality and Competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.52 mm3) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

Effects of Hormonal Supplementation on Nuclear Maturation and Cortical Granules Distribution of Canine Oocytes During Various Reproductive Stages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus-Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulusoocyte complexes (COCs) vitrified with DAP 213 (2 M DMSO, 1 M acetamide, 3 M propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm3) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

Chemical Composition of Lipids Present in Cat and Dog Oocyte by Matrix-Assisted Desorption Ionization Mass Spectrometry (MALDI- MS)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolamento e caracterização ultraestrutural de folículos pré-antrais de vacas da raça Nelore (Bos taurus indicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Meiotic inhibition of bovine oocytes in medium supplemented with a serum replacer and hormones: effects on meiosis progression and developmental capacity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção in vitro de embriões bovinos: utilização de diferentes fontes de gonadotrofinas na maturação dos oócitos

O presente trabalho foi conduzido com o objetivo de avaliar o efeito da utilização de diferentes fontes de gonadotrofinas para maturação in vitro dos oócitos bovinos fecundados e desenvolvidos in vitro sobre as taxas de clivagem (TC) e de blastocistos (TBL). Oócitos imaturos provenientes de ovários de vacas de abatedouro foram submetidos a maturação in vitro sob diferentes condições: meio TCM 199, acrescido de 10% de soro de vaca em estro (SVE), aditivos, hepes, NaHCO3, piruvato de sódio, antibióticos (meio B-199), 20 UI/mL de PMSG e 10 UI/mL de hCG (PMSG/hCG) ou meio B-199, acrescido de 5 mig/mL de FSH e 5 mig/mL de LH (FSH/LH). Seguidos 24 h de cultura a 38,5ºC em atmosfera com 5% de CO2, os oócitos maturos foram incubados com sêmen descongelado durante 18 a 21 horas. Após esse período, os oócitos foram transferidos para placas contendo microgotas de meio Ménezo suplementado com 10% de SVE e células epiteliais do oviduto bovino em suspensão, cobertas com óleo de silicone, os quais permaneceram em cultura por mais 9 dias. Os dados foram analisados pelo teste do Qui-quadrado. A TC e a TBL, para PMSG/hCG e FSH/LH, foram 60 e 13,9% e 61,2 e 10,6%, respectivamente. Não houve diferença entre os tratamentos com relação a TC ou a TBL. Esses resultados sugerem que ambas as fontes de gonadotrofinas podem ser utilizadas para maturação in vitro dos oócitos fecundados e desenvolvidos in vitro.

Dinâmica reprodutiva e parasitária de quatro espécies de peixes das águas costeiras do Sudoeste do Oceano Atlântico, Brasil

The present study investigated the reproductive dynamics and parasitism of four species of marine fishes: serra Spanish mackerel, Scomberomorus brasiliensis, Atlantic leatherjacks, Oligoplites saurus and O. palometa,, and Atlantic bumper, C. chrysurus, during the period of August, 2005 to July, 2007, in the coastal waters of Southwest Atlantic Ocean, Brazil. The collected fish samples were measured, weighed, dissected, the gonads were weighed and examined to separate the sex. The gonadosomatic index (GSI), fecundity, type of spawning, the breeding season, the macro and microscopic characterization of the gonads were determined. The ectoparasites from the branchial chambers and bucal cavity of the fish were collected, measured, weighed and identified. The sex ratio of the study fish species were approximately 1M:1F, however, there was a predominance of males of O. palometa (3M:2F). The GSI of fishes varied according to their reproductive cycle and the stage of gonadal maturation. The highest values of GSI and the spawning period coincided with the rainy period of the region. The females presented total spawning and the fecundity was positively correlated with the weight of the ovary and the body. Four stages of development of the gonads immature, maturing, mature and spent were identified macroscopically and histological analyses of ovaries revealed the different phases of oocyte development. Three species of isopod parasites were identified in the study fishes: Livoneca redmmanni, Rocinela signata and Cimothoa spinipalpa. The first two species occurred in the branchial cavities of C. chrysurus and S. brasiliensis. The isopod C.spinipalpa (a new species) was registered for the first time in the bucal cavity of O. saurus and O. palometa. The parasitic isopods preferred the branchial chambers and the bucal cavity of the host fishes as these were protected microhabitats. The isopods parasitized the immature, maturing and mature fishes. The prevalence of infection of isopods in the hosts varied from 16 to 21%, though in O. palometa it was 60%. In the rainy period the highest isopod parasitic occurrence was registered, however, this did not prejudice the normal reproductive cycle of the host fish.

Impact of skeletal maturation on bone metabolism biomarkers and bone mineral density in healthy Brazilian male adolescents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Artificial oocyte activation using calcium ionophore in ICSI cycles with spermatozoa from different sources

The present study evaluated the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 oil intracytoplasmic sperm injection (ICSI) cycles using spermatozoa from different sources. The 314 cycles evaluated were divided into three groups according to sperm origin, the ejaculated group (n = 92), the epididymal group (n = 82). and the testicular roup (n = 140). Each group was further split into experimental subgroups, depending oil whether or no AOA was performed. In additions the cycles of women younger than 36 years were evaluated separately. For each experimental group, ICSI outcomes were compared between subgroups. No significant difference was observed between subgroups for all sperm origin groups. When evaluating only the cycles of women younger than 36 years of age, AOA increased the percentage of high-quality embryos (74.5 versus 53.0%. P = 0.011) and the implantation rate (19.3 versus 10.5%, P = 0.0025) when it was used with ejaculated spermatozoa, and the percentage of high-quality embryos (64.4 versus 50.3%, P = 0.006) when epididymal spermatozoa were used. These results may suggest that both sperm maturity and oocyte quality play a role in oocyte activation. However. this study is to be continued to confirm these findings.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

Objective: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm.Design: Laboratory study.Setting: Fertility/assisted fertilization center.Patient(s): Couples undergoing surgical sperm retrieval for ICSI (n = 204).Intervention(s): Application of calcium ionophore A23187 for AOA.Main Outcome Measure(s): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (Subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups.Result(s): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA.Conclusion(s): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected. (Fertil Steril (R) 2009;92:131-6. (C)2009 by American Society for Reproductive Medicine.)