The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. L-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).
Sloths are animals that suffer with the destruction and fragmentation of forests. They experience a low population growth rate and need to be studied further for the preservation of the species. The objective of this study was to contribute data relevant to the reproductive physiology of this species, selecting a semen collection method and evaluating seminal characteristics that have never before been described in the literature. Fifteen Bradypus tridactylus males were captured in Manaus, Brazil. Nine of them were captured during the first half of 2004 (Group 1) and the others during the second half (Group 2). The animals were anesthetized with an i.m. injection of a combination of ketamine (10 mg/kg) and xylasine (1 mg/kg). Semen was collected by electroejaculaton using a rectal probe designed for domestic cats. Electrostimulations were given with a 0-100 mA/0-12 V variable electrostimulator in sequences of three progressive intensities, with ten repetitions at each intensity and variation of 10 mA between them. They started with 20 mA and peaked at 60 mA. Each stimulus lasted about 3 s. It was not possible to define the best intensity of stimulus to use and ejaculation could take place at any time of the stimulation (Fisher's exact test). Sperm motility and vigor were immediately analyzed. Sperm count was determined in a Neubauer chamber at a 1:50 (v:v) dilution in formol-saline. Morphology was examined at the same dilution. Fresh semen smears were made and stained using Spermac Stain� (Minit�b, Tiefenbach, Germany) protocol for a better evaluation of the spermatozoa acrosome and midpiece. In both methods 200 cells were counted for morphological evaluation. All animals ejaculated approximately 30 �L to 90 �L of semen. In some ejaculates the semen was too thin and flowed down the penis, so that the volume effectively collected was not sufficient for a complete spermiogram. Spermatozoa presented a wide variety of defects, and some physical characteristics differed (not significantly) between samples collected during the first and second halves of the year. Motility and vigor were very low, the sperm did not show forward progression, only oscillatory movement. However, a high percentage (80%) of spermatozoa were moving. The concentration in Group 1 ranged from 5000 spermatozoa/mm3 to 685 500 spermatozoa/mm3 (mean � 218 571.4 � 242 499.4). Sperm concentation was not assessed in Group 2. The morphology of the head could be elongated or squared, or the head could have a base narrower than the apex. The tail showed a unique feature: the midpiece narrowed abruptly, forming a nip in its transition to the tail. This was similar in appearance to the segmental aplasia of the mitochondrial sheath, but it was considered normal because it was observed in all spermatozoa. Although further studies are necessary to standardize the semen evaluation of sloths and to define the best protocol for electroejaculation, this pioneering study has shown the characteristics of sloth spermatozoa and the possibility of collecting semen throughout the electroejaculation process in this species. This work was supported by Fapesp 03/07457-4.
The aim of this study was to compare the viability of in vitro-produced bovine embryos following quick freezing in ethylene glycol (EG) and subsequent dilution of EG by either a two- or a three-step procedure. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=544) were selected 7 and 9 days after insemination and randomly distributed to one of three EG equilibration treatment groups. Embryos were exposed to 10% EG for 10min, and then to 17%, 22% or 28% EG for 30s (respectively referred to as EG 17, EG 22 and EG 28). In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25-mL straws which were then heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. The thawed embryos of the EG 17, EG 22 and EG 28 groups were randomly assigned to one of two EG dilution procedures. Two-step dilution consisted of transfer of embryos into PBS+0.2% BSA+0.3M sucrose solution for 3min, and then PBS+0.2% BSA for 3min. Three-step dilution consisted of transfer of embryos into PBS+10% EG+0.2% BSA+0.3M sucrose for 3min, PBS+0.2% BSA+0.3M sucrose for 3min, and then PBS+0.2% BSA for 3min. Embryos were co-cultured on a granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the Table. No significant differences were found between the two- and three-step dilution procedures (P>0.05) for in vitro-produced bovine embryos cryopreserved by quick freezing. This project was supported by FAPESP (01/11266-4). Table 1 In vitro re-expansion and hatching rates (%) of rapidly frozen embryos after two- or three-step dilution
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