Elisângela Mirapalheta Madeira

Evaluation of amides and centrifugation temperature in boar semen cryopreservation

Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P<0.05) to 5% methylformamide (MF; 43.2+/-2.4%) and 3% glycerol (GLY; 38.1+/-2.3%), with no significant difference between MF and GLY. Sperm membrane integrity was higher (P<0.05) for DMA than for MF or GLY (50.9+/-1.9, 43.3+/-2.5, and 34.5+/-2.8%, respectively). Sperm membrane integrity was higher in DMF (47.9+/-2.1%) than in glycerol (34.5+/-2.8%, P<0.05), but was similar to other treatments (P>0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P<0.05). For sperm motility and membrane integrity, 5% DMA exceeded (P<0.05) 3% DM, with greater membrane integrity than 3% DMF (P<0.05). In both experiments, sperm motility and membrane integrity were superior at 15 degrees C versus 24 degrees C (P<0.05), with no interaction between centrifugation temperature and treatments (P>0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.

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.

Selection of porcine oocytes in vitro through brilliant cresyl blue staining in distinct incubation media.

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB-) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB- oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB-; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB-. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB-, ReproPel/BCB+, ReproPel/BCB- and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB- (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB- (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB- (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

Avaliação de diferentes crioprotetores intra e extracelulares na criopreservação de sêmen de touros

The experiment was designed as 3 x 2 factorial design, with three internal cryoprotectants, glycerol (GLY), etileneglycol (EG) and dymethilformamide (DMF) and two external, egg yolk (YOLK) and density low lipoproteina (LDL). The motility at thawing for GLY-YOLK (53.9±1.96) was higher than other treatments (P 0.05), but were higher than the others (P<0.05). The evaluation of membrane integrity through hypoosmotic swelling test (HOST) indicate that DMF-YOLK (13.6±1.30) and DMF-LDL (9.8±0.78) were different (P<0.05) and lower than the others (P<0.05). The use of ethylene glycol associated to egg yolk can be a viable alternative to the use of glycerol in bull semen freezing protocols.