RESUMO
Two experiments were carried out to examine in vitro quality and in vivo fertility of rabbit semen diluted in ultra-high temperature (UHT) skim milk. In the first experiment, pooled ejaculates of 10 adult rabbits were divided in three aliquots. Each aliquot was diluted in saline solution, TrisC or UHTm extender and kept at room temperature for 24 h. Sperm quality assessment was performed during all the incubation periods. In the second experiment, 27 adult rabbit does were inseminated with semen incubated for 5 h. Embryo recovery was performed 96 h after insemination. Results showed that treatments diluted in UHTm registered the highest values of spermatozoon with total motility, intact and functional plasma membrane and greater number of embryos recovered in rabbit does. We conclude that UHT skim milk would be a good extender for improved intra-uterine insemination in rabbits and to keep sperm cells for several hours at room temperature.
Assuntos
Temperatura Alta , Leite , Preservação do Sêmen/métodos , Espermatozoides , Animais , Membrana Celular , Feminino , Fertilização , Inseminação Artificial , Masculino , Coelhos , Análise do Sêmen , Contagem de Espermatozoides , Motilidade dos EspermatozoidesRESUMO
Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in bovine follicular fluid; when added to maturation culture media, it affects oocyte competence (depending on the type and concentration of LA used). To date, little is known about the effective level of incorporation of LA and there is apparently no information regarding its esterification into various lipid fractions of the oocyte and its effect on neutral lipid storage. Therefore, the objective was to assess the uptake and subcellular lipid distribution of LA by analyzing incorporation of radiolabeled LA into oocyte polar and neutral lipid classes. The effects of various concentrations of LA on the nuclear status and cytoplasmic lipid content of bovine oocytes matured in vitro was also analyzed, with particular emphasis on intermediate concentrations of LA. Neutral lipids stored in lipid droplets were quantified with a fluorescence approach. Linoleic acid at 9 and 43 µM did not affect the nuclear status of oocytes matured in vitro, and 100 µM LA inhibited germinal vesicle breakdown, resulting in a higher percentage of oocytes arrested at the germinal state (43.5 vs. 3.0 in controls; P < 0.05). Bovine oocytes actively incorporated LA from the maturation medium (83.4 pmol LA per 100 oocytes at 22 hours of incubation; P < 0.05) and metabolized it mainly into major lipid classes, e.g., triacylglycerols and phospholipids (61.1% and 29.3%, respectively). Supplementation of the maturation medium with LA increased triacylglycerol accumulation in cytoplasmic lipid droplets at all concentrations assayed (P < 0.05). In conclusion, LA added to a defined maturation medium at concentrations that did not alter the nuclear status of bovine oocytes matured in vitro (9 and 43 µM) improved their quality by increasing the content of neutral lipids stored in lipid droplets. By directing the free fatty acid (LA) to triacylglycerol synthesis pathways and increasing the degree of unsaturation of membrane phospholipids, the oocyte was protected from lipotoxic effects (with an expectation of improved cryotolerance).
Assuntos
Bovinos , Ácido Linoleico/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Animais , Radioisótopos de Carbono , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Células Cultivadas , Meios de Cultura , Citoplasma/química , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos não Esterificados/metabolismo , Feminino , Marcação por Isótopo , Ácido Linoleico/metabolismo , Lipídeos/análise , Oócitos/ultraestrutura , Triglicerídeos/metabolismoRESUMO
The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P<0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1 microL, 61.3% 0.5 microL and 80.5% control, P<0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P<0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification-warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.