153 IN VITRO CULTIVATION OF CAPRINE EMBRYOS PRODUCED IN VIVO

Independent of embryo stage, when grade III embryos are transferred, they usually provide poor pregnancy rates. The aim of this study was to compare the effect of different in vitro cultivation media on the development of in vivo derived grade III caprine embryos. Twelve adult crossbreed goats from the Animal Breeding and Reproduction Laboratory of Maringá State University were used for this experiment. The goats were not pregnant or lactating. After embryo collection, embryos were classified regarding their development and quality, according to the International Embryo Transfer Society manual. Morulae classified as grade III were used in this experiment. The embryos were cultivated in 2 different media: Holding Plus medium (Bioniche Animal Health, Belleville, Ontario, Canada) or PBS plus 10% of bovine fetal serum (Nutricell, Campinas, Brazil) in 38.5°C waterbath with water circulation for 24 hours. After 24 h, the embryos were morphologically assessed. The percentage of embryos that developed during the cultivation period was calculated to evaluate the effectiveness of culture media. The embryos that produced new cell divisions and changed from grade III (poor quality) to grade II or grade I (excellent or good) were considered developed embryos. Chi-square was used to determine statistical differences between media. In the present work, the rate of embryos that developed in Holding Plus medium (Bioniche Animal Health) was 75%, and with PBS plus 10% bovine fetal serum was 40% of the total cultivated embryos. Table 1.Embryo development assessment after in vitro cultivation process

Radioimmunoassay of bovine placental lactogen using recombinant and native preparations: determination of fetal concentrations across gestation

Concentrations of bovine placental lactogen (bPL) were determined in fetal plasma samples by twelve double-antibody competitive radioimmunoassay systems (RIA I–XII) based on either recombinant bPL (non-glycosylated) or native bPL (glycosylated). Both preparations were used as standard and tracer, and for primary antisera production. The minimum detection limit measured by these RIA varied from 0.02 to 0.6 ng bPL mL–1. The coefficients of correlation of different bPL RIA systems were up to 90% (P < 0.0001) when each RIA was tested against the average values of all twelve RIA systems. All developed RIA were used to investigate the incidence of different bPL isoforms in bovine fetal serum samples (n = 71). Fetal concentrations ranged from 11.8 to 35.7 ng mL–1 at the third month and from 1.1 to 13.5 ng mL–1 at the ninth month of gestation. They tended to decrease with advancing gestation. In general, those RIA systems that used recombinant bPL as the standard measured higher values than those using the native bPL preparation. These differences decreased toward the end of gestation (P < 0.05), suggesting a lower rate of glycosylation. Our results provide evidence of different glycosylated isoforms of bPL in fetal serum at different gestation periods.

The Study of the Frequency of Medium Changing in the Fabricating Tissue-Engineered Bone

Marrow mesenchymal cells contain stem cells and can regenerate tissue. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However there is room for improvement in the culture methods; and here, we examined the optimal frequency of medium changing. Marrow cells were collected from the femur of a Fisher 7 week-old male rat. At 2 weeks after primary culture in a standard medium (MEM containing 15% bovine fetal serum), the cultures were trypsinized to prepare a cell suspension and divided into two groups, with or without addition of dexamethasone (Dex) to the osteogenic medium. To investigate optimal frequency, we further divided into 5 sub-groups; no changing (M0), 1 time/week (M1), 2/week (M2), 3/week (M3), and everyday (M7). After 2 weeks of subculture, the tissue was harvested and then ALP activity and calcium and DNA contents measured. In both of the Dex groups, there was significantly high ALP activity in the higher frequency group; but there was no significant DNA content. Also, in the Dex(+)group, there was a significant increase of calcium content in only the M3 and M7 sub-groups. Thus, we showed that the osteogenic potential of cultured bone is cultivated by increasing the frequency of medium changing.

Effects of the addition of glutathione during maturation on in vitro fertilisation of prepubertal goat oocytes

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a ubiquitous intracellular free thiol that improves development of the male pronucleus at fertilisation and has also been implicated in promoting the development of preimplantation embryos. The objective of this study was to evaluate the effects of adding GSH or cysteine to the in vitro maturation medium on intracellular GSH amounts after in vitro maturation and fertilisation of prepubertal goat oocytes. Oocytes were matured in TCM199 medium supplemented with 10% bovine fetal serum, 1 mg/ml 17β-estradiol, 10 μg/ml o-FSH, 10 μg/ml LH and 50 mg/ml gentamicin. In vitro maturation medium was completed with two independent treatments: GSH at different concentrations (0, 0.25, 0.50 and 1.00 mM) and L-cysteine at different concentrations (0, 150, 300, 600 and 900 μM). After 27 h of culture at 38.5 °C in 5% CO2 in air, the nuclear stage was evaluated. Simultaneously, another sample of oocytes was frozen and the intracellular GSH level was evaluated with spectrophotometric methodology. Oocytes were inseminated with fresh semen (2-3 × 106 sperm/ml) in TALP medium supplemented with 1 mg/ml hypotaurine. Oocytes were fixed at 20 h post-insemination to evaluate the in vitro fertilisation. Oocytes matured in 1.00 mM GSH-supplemented medium exhibited higher amounts of intracellular GSH (3.23 pmol per oocyte). The percentage of normal fertilisation (17-27%) was similar for the treatment groups. In conclusion, the addition of 1.00 mM GSH to the maturation medium could be a useful method for increasing the intracellular GSH levels of prepubertal goat oocytes. However, this increase was not associated with a higher normal fertilisation rate of prepubertal goat oocytes.

Effect of the age of the pupal holotissue on the nutritional quality of artificial diets for Trichogramma spp. (Hymenoptera: Trichogrammatidae)

The nutritional quality of artificial diets composed of pupal holotissues of Diatraea saccharalis (Fabr.) (Lep.: Crambidae) from three different age classes (1-2, 3-4 and 5-6 days old) were tested for rearing Trichogramma galloi Zucchi and T. pretiosum Riley (Hym.: Trichogrammatidae) in vitro. Pupal holotissues were added to egg yolk, bovine fetal serum, lactoalbumine hydrolysate and preservatives, and offered to the parasitoids into artificial eggs. The quality of these diets in supporting the development of both parasitoids was evaluated by assessing the acceptance and parasitism of the artificial host eggs, larval and pupal survival, size, parasitism capacity and longevity of the in vitro-reared females, and the presence of deformed adults. Diets composed of 5-6 d old pupal holotissues did not support the larval development of both parasitoids as well as reduced the acceptance and/or parasitism of artificial eggs by T. galloi and T. pretiosum. The factors affecting the nutritional quality of pupal holotissues collected from different developmental stages are discussed.

Successful development in vitro of hamster 8-cell embryos to ‘zona-escaped’ and attached blastocysts: assessment of quality and trophoblast outgrowth

The peri-implantation development involves zona escape (hatching) of blastocysts and their attachment and proliferation. These events are difficult to studyin vivo, so in this study hamster 8-cell embryos were cultured through the hatched and attached blastocyst stages using different formulations of hamster embryo culture medium (HECM)-2. Supplementation of succinate, amino acids, vitamins (inositol, pantothenate, choline chloride) and bovine serum albumin (BSA) to HECM-2 supported 100% development of ‘zona-escaped’ blastocysts. In this medium (designated as hatching, i.e. HECM-2h) all blastocysts invariably deflated and escaped from focally lysed zonae, which underwent complete dissolution. In their presence, pre-morula stage embryos also escaped from zonae. Omission of BSA from HECM-2h failed to support zona escape whereas that of vitamins reduced zona escape (34.0% 7.0). Blastocysts with the potential to undergo zona escape in HECM-2h were of high quality as they had a higher mean cell number (MCN) than the MCN of those developing in BSA-free HECM-2h (35.2 1.6 v. 24.3 1.1). Cell allocation (i.e. trophectoderm to inner cell mass ratio) in blastocysts remained unaltered in both media (2.6 0.2 v. 2.7 0.2). Supplementation of 10% bovine fetal serum (BFS) to HECM-2h was detrimental to the development of blastocysts (22.3% 7.4) and none of them underwent zona escape. Interestingly, BFS was required either as a supplement to the medium or as a coating on dishes for azonal blastocysts to attach (≥70%) and exhibit trophoblast (TB) outgrowth (30.3 × 103 2.9 × 103 µm2 at 48 h). These results show that HECM-2h supports maximal development of zona-escaped blastocysts with the potential to attach and exhibit TB outgrowth, and there is a developmental stage-specific requirement for serum during peri-attachment in hamster development.

Primary culture of prepubertal human testicular cells isolated from testes collected at necropsy

The aim of the present work was to develop a method for maintaining prepubertal human testicular cells in culture. Seven pairs of testes of boys who died of causes unrelated to endocrine or metabolic diseases were obtained at necropsy. Histology of the testes was normal. Testes were digested with collagenase and dispersed cells were seeded in multi-well dishes in the presence of 5% bovine fetal serum. After the first day, cells were cultured for five days in serum-free medium in the presence or absence of 918 pmol/l insulin. At the end of culture, microscopic examination showed healthy looking cells with characteristics compatible with pre-Sertoli cells; peritubular cells were identified by immunocytochemistry. In the presence of insulin, cells were able to secrete either testosterone or estradiol into the medium, as well as to reveal aromatase activity. In order to study the effect of the time elapsed between death and beginning of cultures, steroidogenic activity was related to this post mortem time. It was found that, in the presence of insulin, cells obtained from testes with less than 24 h of post mortem time secreted testosterone (64±7.2 pmol/106 cells·24 h, mean±sd) while cells obtained from testes with more than 24 h of post mortem time did not secrete testosterone. With long post mortem times, aromatase activity under insulin increased from non-detectable to 35 pmol/106 cells·24 h. Time course studies showed that cells with capacity to secrete testosterone increase this secretion gradually up to day 10 of culture, while those with detectable aromatase activity showed increments in this activity during the first week of culture. It is concluded that it is possible to maintain cultures of prepubertal human testicular cells obtained at necropsy. In order to maintain testosterone secretion capacity, the time between death and initiation of culture should not exceed 24 h.