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ALMEIDA, F.S. Effect of insulin on bovine semen cryopreservation on in vitro embryo production [Efeito da insulina na criopreservação do sêmen bovino sobre a produção in vitro de embriões]. 2020. 64 f. Dissertação (Mestrado em Ciências) Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2020. Insulin is an anabolic hormone capable of acting in the protection of sperm cells to minimize cryopreservation insults, also acts in the production of in vitro embryos (PIV) bringing benefits in embryonic development due to its effect antiapoptotic and mitogenic. Thus, the present study aims to evaluate the effects of insulin addition to the means of cryopreservation of semen on the potential of PIV of embryos. For this, two ejaculates of six bulls (n=12) were used, each ejaculate was distributed in two treatments for cryopreservation: Control group (CO): semen diluted in Triladyl medium (n = 12) and Insulin Group (IN): semen diluted in Triladyl medium supplemented with insulin (150 μIU/mL, n = 12).Two semen reeds from each match of each treatment were thawed, homogenized and submitted to percoll gradient selection. Sperm obtained after Percoll were evaluated for motility, vigor, (acridine orange), plasma membrane and acrosomal integrity, mitochondrial potential and DNA fragmentation, and after they were used for in vitro fertilization (IVF) of embryos. For PIV, four replications were performed, and ovaries follicles from commercial slaughterhouse were aspirated and the cumulative-oocyte complexes that presented layers of compact and oocyte cumullus cells with homogeneous cytoplasm were selected The oocytes were submitted to the maturation process in vitro (IVM) (22h/38.5C/5%CO2) and IVF performed shortly after maturation. Matured oocytes were distributed into two groups, one group received semen CO (n=1587) and the other semen IN (n= 1580). After fertilization the oocytes were incubated (18h/38.5C/5%CO2) and the presumed zygotes were transferred to in vitro cultivation following the order of treatments and incubated for 8 days (38.5oC/5%CO2/5%O2/90%N2). The IVF response was determined by the cleavage rate on the third day of cultivation. On day 8, the number of blastocyst and the development rate were evaluated in relation to presumed zygotes. The data were submitted to variance analysis by the mixed procedure (PROC MIXED) of the SAS version 9.3 program. In all statistical analyses, the significance level considered was P≤0.05. Cleavage rate was not affected by the addition of insulin to the cryopreservation dilution of semen (64.21±2.04 x 63.62±1.69%), but insulin was noted to have caused a reduction in blastocyst rates (CO: 24.06±2.69% x IN: 20.04±2.54%) and embryonic development (CO: 35.81±3.23% x IN: 30.21±3.37%). On the other hand, bull effects were found for cleavage rates (ranging from 54.19±2.34 to 71.91±2.39%), blastocyst (ranging from 6.76±0.97 to 36.18±2.22%) and embryonic development (ranging from 12.32±1.53 to 50.12±2.04%), but these changes were not related to variations in sperm quality. In conclusion, insulin added to sperm cryopreservation extender has no beneficial effect on embryo production rates in vitro.

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