Impact of adding different concentrations of IGF-I and insulin to the semen extender on bull sperm quality post-cryopreservation

ABSTRACT This study aimed to evaluate the addition of different concentrations of IGF-I and insulin to egg yolk-based extender to improve bovine semen cryopreservation. Two experiments were developed to evaluate the effects of the additives in two commercial extenders, Botubov® (Experiment 1) and Triladyl® (Experiment 2), both with the same design. Three ejaculates from four bulls (n = 12) were used. Each ejaculate was divided into seven equal fractions for dilution (60x106 spermatozoa/mL) in the following treatments: CON: extender only; IGF100: IGF-I 100ng/mL; IGF200: IGF-I 200ng/mL; INS150: insulin 150µUI/mL; INS200: insulin 200µUI/mL; ASS1: IGF-I 100ng/mL + insulin 150µUI/mL; ASS2: IGF-I 200ng/mL + insulin 200µUI/mL. Semen was cryopreserved by an automated system. Post-thawed sperm were evaluated regarding motility by CASA (Computer-assisted sperm analysis), and membranes by fluorescent probes (H342, PI, FITC-PSA and JC-1). For Botubov® extender, INS150 was more efficient in preserving total and progressive motility, VCL, BCF, plasma and mitochondrial membranes. A similar response was seen when insulin was added to the Triladyl® extender, INS150 was more efficient in preserving sperm motility, plasma membrane integrity and mitochondrial potential. Thus, the addition of insulin 150µUI/mL, regardless of the composition of the extender, contributes to better preserving bovine sperm from the cryopreservation effects. RESUMO Este estudo teve o objetivo de avaliar a adição de diferentes concentrações de IGF-I e insulina a diluidores, à base de gema de ovo, para melhorar a criopreservação do sêmen bovino. Dois experimentos foram desenvolvidos para avaliar os efeitos dos aditivos em dois diluidores comerciais: Botubov® (Experimento 1) e Triladyl® (Experimento 2), ambos com o mesmo delineamento. Foram utilizados três ejaculados de quatro touros (n=12). Cada ejaculado foi dividido em sete frações para diluição (60x106espermatozoides/mL), nos seguintes tratamentos: CON: somente diluidor; IGF100: 100ng/mL de IGF-I; IGF200: 200ng/mL de IGF-I; INS150: 150µUI/mL de insulina; INS200: 200µUI/mL de insulina; ASS1: 100ng/mL de IGF-I + 150µUI/mL de insulina; ASS2: 200ng/mL de IGF-I + 200µUI/mL de insulina. O sêmen foi criopreservado por sistema automatizado. Após a criopreservação, o sêmen foi avaliado quanto à motilidade espermática por CASA e quanto às membranas espermáticas (plasmática, acrossomal e mitocondrial) por sondas fluorescentes (H342, PI, FITC-PSA e JC-1). Par