Gissele Rambo

Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker

The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35mL petri dishes in a CO(2) incubator, (2) 35mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2+/-10.1 and the IVP rate was 90.5%. The PSP differed across all systems (P=0.0006): 15.5+/-0.5 for flasks, 6.3+/-0.4 for CO(2), and 3.9+/-0.4 for bags. The IVP rate for flasks (95.0%) was greater (P=0.01) than for CO(2) and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO(2) for three boars (P>0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4+/-6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater (P<0.0001) for oocytes frozen in cryotubes (7.0+/-0.3% and 95.8%, respectively) than those frozen in OPS (3.7+/-0.3% and 83.4%, respectively), with no differences found for three boars (P>0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.

Inseminação artificial intra-uterina em leitoas com sêmen criopreservado com dimetilacetamida e glicerol

Este estudo teve por objetivo avaliar o uso da dimetilacetamida (DMA) e de glicerol na criopreservacao de semen suino sobre as taxas de concepcao e fertilizacao in vivo, utilizando o metodo de inseminacao artificial pos-cervical. Foram sincronizadas 60 leitoas pre-puberes e inseminadas com o uso de semen congelado com glicerol 3% (30 femeas) e DMA 5% (30 femeas). O metodo de inseminacao utilizado foi o pos-cervical, com concentracao de 1 x 109 espermatozoides vivos por dose. Apos 36 a 40h da inseminacao, as femeas foram abatidas, sendo realizada a contagem de corpos hemorragicos (CH) nos ovarios. Foi realizada a lavagem dos ovidutos das femeas, verificando o numero de estruturas recuperadas (oocitos e embrioes), calculando-se as taxas de concepcao e fertilizacao. A media de CH nas femeas do grupo glicerol 3% nao diferiu (P>0,05) daquelas do grupo DMA 5% (10,4 x 10,2, respectivamente). Nao houve diferenca (P>0,05) nas taxas de recuperacao de estruturas entre os grupos glicerol 3% (68,9%) e DMA 5% (66,9%). Os resultados obtidos nos grupos glicerol 3% e DMA 5% para as taxas de concepcao (73,3 x 76,6%) e fertilizacao (48,6 x 59,4%) nao apresentaram diferenca (P>0,05). Conclui-se que nao ha diferencas nas taxas de concepcao e fertilizacao in vivo utilizando-se semen congelado com o uso de dimetilacetamida ou de glicerol.