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.
Keywords:
Bull; Semen; Freezing; Botubov®; Triladyl®
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a,bc Different letters on the bars indicate statistically significant differences (P < 0.0001).BCON (control): semen added only Botubov® extender; BIGF100: Botubov® + 100 ng/mL of IGF-I; BIGF200: Botubov® + 200 ng/mL of IGF-I; BINS150: Botubov® +150 µUI/mL of insulin; BINS200: Botubov® + 200 µUI/mL of insulin; BASS1: Botubov® +100 ng/mL of IGF-I + 150 µUI /mL of Insulin; BASS2: Botubov® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.
a,bc Different letters on the bars indicate statistically significant differences (P ( 0.05). BCON (control): semen added only Botubov® extender; BIGF100: Botubov® + 100 ng/mL of IGF-I; BIGF200: Botubov® + 200 ng/mL of IGF-I; BINS150: Botubov® +150 µUI/mL of insulin; BINS200: Botubov® + 200 µUI/mL of insulin; BASS1: Botubov® +100 ng/mL of IGF-I + 150 µUI /mL of Insulin; BASS2: Botubov® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.
a,bc Different letters on the bars indicate statistically significant differences (P ( 0.0001). TCON (Control): semen added only Triladyl®; TIGF100: Triladyl® + 100 ng/mL of IGF-I; TIGF200: Triladyl® + 200 ng/mL of IGF-I; TINS150: Triladyl® + 150 µUI/mL of insulin; TINS200: Triladyl® + 200 µUI/mL of insulin; TASS1: Triladyl® + 100 ng/mL IGF-I + 150 µUI/mL insulin; TASS2: Triladyl® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.
a,bc Different letters on the bars indicate statistically significant differences (P ( 0.05). TCON (Control): semen added only Triladyl®; TIGF100: Triladyl® + 100 ng/mL of IGF-I; TIGF200: Triladyl® + 200 ng/mL of IGF-I; TINS150: Triladyl® + 150 µUI/mL of insulin; TINS200: Triladyl® + 200 µUI/mL of insulin; TASS1: Triladyl® + 100 ng/mL IGF-I + 150 µUI/mL insulin; TASS2: Triladyl® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.
a,bc Different letters on the bars indicate statistically significant differences (P ( 0.05). TCON (Control): semen added only Triladyl®; TIGF100: Triladyl® + 100 ng/mL of IGF-I; TIGF200: Triladyl® + 200 ng/mL of IGF-I; TINS150: Triladyl® + 150 µUI/mL of insulin; TINS200: Triladyl® + 200 µUI/mL of insulin; TASS1: Triladyl® + 100 ng/mL IGF-I + 150 µUI/mL insulin; TASS2: Triladyl® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.
TCON (Control): semen added only Triladyl®; TIGF100: Triladyl® + 100 ng/mL of IGF-I; TIGF200: Triladyl® + 200 ng/mL of IGF-I; TINS150: Triladyl® + 150 µUI/mL of insulin; TINS200: Triladyl® + 200 µUI/mL of insulin; TASS1: Triladyl® + 100 ng/mL IGF-I + 150 µUI/mL insulin; TASS2: Triladyl® + 200 ng/mL of IGF-I + 200 µUI/mL of insulin.