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

Pinto, S.C.C.; Souza, F.A.; Arruda, R.P.; Alves, M.B.R.; Batissaco, L.; Garcia-Oliveros, L.N.; Passarelli, M.S.; Carneiro, L.C.; Celeghini, E.C.C.

doi:10.1590/1678-4162-12946

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Veterinary Medicine and Technology and Inspection of Animal Products • Arq. Bras. Med. Vet. Zootec. 75 (05) • Sep-Oct 2023 • https://doi.org/10.1590/1678-4162-12946 copy

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

[Impacto da adição de diferentes concentrações de IGF-I e insulina ao diluidor de sêmen sobre a qualidade espermática bovina após a criopreservação]

Authorship SCIMAGO INSTITUTIONS RANKINGS

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^®

Sperm characteristics	Bulls
Sperm characteristics	1	2	3	4
Motility (%)	75.0±5.0	78.33±2.88	71.66±2.88	73.33±2.88
Vigor (1-5)	2.83±0.76	2.66±0.28	2.83±0.38	3±0
Concentration (x10⁶ sptz/mL)	215±80.46	470±268.37	169±56.712	570±247.84
Major defects (%)	7.66±0.57	6.66±2.08	6.33±2.08	4±2.64
Minor defects (%)	5.33±3.05	8.33±0.57	6.33±2.88	6±1.73
PIAIHP (%)	79.66±5.03	85.33±2.08	77.66±9.71	78±8.66

Parâmetros	BCON	BIGF100	BIGF200	BINS150	BINS200	BASS1	BASS2	p
VCL (μm/s)	127.75±7.7^b	134.18±12.6^b	124.51±10.0^b	152.16±10.2^a	132.39±11.2^b	135.42±13.3^b	129±10.5^b	≤0.05
VSL (μm/s)	86.43±8.0	82.79±5.9	77.74±7.3	89.72±8.1	81.07±7.2	84.74±7.7	80.77±5.9	≥0.05
VAP (μm/s)	99.68±9.2	95.94±7.5	89.98±8.2	98.23±9.8	94.88±8.6	97.25±8.6	93.18±6.5	≥0.05
LIN (%)	62.42±2.07	62.51±3.26	62.39±3.26	62.73±2.83	61.22±2.48	63.10±2.49	63.50±1.92	≥0.05
STR (%)	84.49±2.96	85.83±1.26	83.21±4.20	85.74±3.42	82.71±2.61	84.92±3.15	84.37±3.19	≥0.05
WOB (%)	71.95±1.67	72.28±2.54	72.14±2.70	72.47±1.92	71.54±1.70	72.18±2.08	73.08±1.76	≥0.05
ALH (μm)	2.44±0.10^b	2.43±0.20^b	2.21±0.16^b	2.81±0.16^a	2.40±0.14^b	2.42±0.18^b	2.36±0.18^b	≤0.05
BCF (Hz)	22.89±0.97^ab	21.64±1.14^bc	21.01±1.17^c	23.60±1.14^a	22.46±0.87^abc	22.85±1.17^ab	22.0±1.15^bc	<0.0001

Parameters	BCON	BIGF100	BIGF200	BINS150	BINS200	BASS1	BASS2
AI (%)	78.04±1.57	70.13±1.82	78.42±3.23	81.13±1.38	79.96±2.28	76.71±3.93	75.92±3.52

Bull	Treatment	SM (%)	TM (%)	PM (%)	VCL (µm/s)	VSL (µm/s)	VAP (µm/s)	LIN (%)	STR (%)	WOB (%)	ALH (µm)	BCF (Hz)
1	BCON	36.67± 5.77	47.77± 14.72	39.10± 13.19	128.43± 7.45	101.47± 12.57	114.20± 13.27	67.16± 3.59	88.77± 1.32	75.57± 3.09	2.47± 0.06	24.67± 1.69
	BIGF100	33.33± 5.77	31.23± 8.27	22.93± 9.40	124.67± 13.77	82.57± 19.31	95.57± 17.27	65.60± 8.33	85.87± 4.78	76.23± 5.47	2.30± 0.10	21.93± 1.50
	BIGF200	30.00± 10.00	34.87± 14.43	26.27± 8.73	127.67± 8.09	87.27± 1.56	98.53± 3.75	68.47± 3.12	88.63± 1.76	77.27± 2.15	2.20± 0.17	22.53± 2.17
	BINS150	56.67± 5.77	64.37± 9.99	47.50± 3.66	157.57± 10.06	114.77± 6.65	112.70± 8.88	70.40± 0.60	91.53± 6.81	75.97± 5.46	2.67± 0.12	25.43± 1.24
	BINS200	46.67± 11.55	47.67± 20.75	33.50± 11.52	136.53± 6.39	85.47± 7.06	106.43± 15.88	62.57± 3.07	85.60± 2.86	73.07± 1.96	2.47± 0.12	23.27± 0.23
	BASS1	35.00± 8.66	40.73± 14.07	34.17± 10.73	140.73± 7.79	95.33± 14.53	107.30± 14.96	67.53± 6.85	88.73± 1.19	76.03± 6.88	2.43± 0.23	23.27± 2.51
	BASS2	31.67± 2.89	27.10± 4.06	21.60± 5.36	132.97± 4.81	88.97± 12.44	101.43± 9.34	66.73± 7.06	87.40± 4.29	76.20± 4.50	2.40± 0.00	22.17± 1.19
2	BCON	45.00± 5.00	57.23± 7.48	45.07± 6.10	148.73± 13.54	99.13± 8.76	116.97± 11.57	61.77± 2.16	84.87± 1.01	72.77± 3.03	2.67± 0.23	24.47± 1.42
	BIGF100	40.00± 17.32	37.53± 23.83	30.50± 21.06	168.47± 11.42	99.63± 6.18	117.30± 3.87	59.33± 5.37	84.87± 2.72	69.80± 4.69	2.83± 0.25	24.70± 1.75
	BIGF200	33.33± 5.77	37.57± 11.64	29.00± 8.23	151.63± 4.49	92.13± 3.19	108.20± 3.21	60.77± 3.21	85.17± 2.33	71.40± 2.08	2.67± 0.21	22.97± 1.10
	BINS150	58.33± 7.64	69.77± 17.15	51.83± 10.45	175.40± 8.26	103.80± 3.48	119.87± 4.57	61.30± 6.35	85.43± 2.80	71.63± 5.02	3.10± 0.17	25.63± 0.81
	BINS200	48.33± 2.89	56.53± 6.79	44.93± 9.32	162.10± 6.48	99.33± 6.31	117.20± 2.76	61.43± 5.97	84.73± 3.35	72.40± 4.12	2.70± 0.20	24.53± 0.55
	BASS1	38.33± 7.64	44.67± 17.38	36.10± 13.67	169.77± 17.28	100.73± 12.14	116.27± 8.98	59.67± 8.37	86.43± 4.49	68.83± 6.63	2.83± 0.38	25.97± 1.50
	BASS2	31.67± 12.58	39.10± 19.05	31.43± 16.98	152.73± 19.55	92.77± 6.37	106.13± 6.11	61.30± 7.43	87.43± 1.24	70.10± 7.94	2.77± 0.49	25.03± 3.86
3	BCON	40.00± 10.00	59.00± 19.63	49.33± 18.92	111.93± 10.72	73.23± 2.51	82.90± 2.60	63.60± 1.51	88.33± 0.31	72.00± 1.67	2.17± 0.12	21.50± 0.72
	BIGF100	41.67± 2.89	48.73± 5.65	42.73± 5.89	108.77± 11.52	74.57± 7.67	83.47± 7.92	68.57± 2.30	89.27± 1.18	76.80± 1.85	1.93± 0.15	20.50± 2.00
	BIGF200	48.33± 10.41	57.50± 3.42	51.77± 6.86	108.67± 7.17	71.77± 5.92	81.60± 6.51	66.30± 1.51	88.33± 0.59	75.03± 1.55	1.97± 0.06	20.77± 0.21
	BINS150	56.67± 7.64	73.67± 13.32	63.13± 5.25	126.27± 3.70	77.93± 1.18	84.60± 2.72	67.23± 1.75	89.80± 0.44	74.87± 1.56	2.43± 0.06	22.27± 0.12
	BINS200	51.67± 2.89	57.00± 7.40	51.33± 5.49	110.50± 3.44	73.47± 6.37	82.07± 5.19	66.40± 3.70	85.60± 2.86	74.17± 2.46	2.00± 0.00	21.27± 0.25
	BASS1	50.00± 10.00	63.73± 3.03	55.87± 2.42	107.30± 9.44	72.03± 5.59	81.00± 6.64	67.20± 0.82	88.90± 0.66	75.53± 0.93	1.93± 0.15	21.53± 0.50
	BASS2	46.67± 7.64	56.60± 5.64	48.43± 5.42	101.77± 16.18	67.93± 10.24	77.20± 10.39	66.77± 2.11	87.83± 1.89	76.07± 2.00	1.87± 0.21	21.30± 1.47
4	BCON	41.67± 18.93	46.30± 32.54	39.73± 29.44	121.93± 7.58	71.92± 16.62	81.33± 10.40	57.17± 10.00	84.23± 7.22	67.47± 6.31	2.47± 0.21	20.93± 1.21
	BIGF100	41.67± 22.55	38.23± 27.45	29.83± 21..32	134.83± 33.76	74.40± 6.66	87.43± 9.42	56.53± 8.42	85.20± 1.81	66.30± 9.04	2.67± 0.81	19.43± 2.72
	BIGF200	23.33± 5.77	22.00± 24.39	17.53± 18.28	110.10± 3.12	59.80± 19.84	71.60± 16.91	54.03± 16.95	82.00± 9.89	64.87± 13.91	2.00± 0.26	17.80± 3.12
	BINS150	50.00± 17.32	54.93± 25.28	45.63± 21.08	149.43± 20.27	84.93± 5.16	82.07± 15.77	55.30± 6.63	81.90± 5.38	67.40± 3.91	3.07± 0.55	21.07± 1.22
	BINS200	41.67± 18.93	43.50± 29.70	32.53± 24.16	120.43± 6.15	66.03± 16.11	80.50± 13.91	54.47± 10.86	81.30± 6.12	66.53± 8.41	2.43± 0.21	20.77± 0.92
	BASS1	26.67± 5.77	29.67± 20.13	23.30± 15.68	123.90± 4.01	70.90± 12.68	84.43± 8.62	58.00± 12.58	83.53± 6.91	68.30± 8.84	2.50± 0.35	20.63± 0.95
	BASS2	26.67± 5.77	30.80± 13.47	24.73± 13.15	128.53± 30.20	73.43± 1.60	87.97± 7.08	59.20± 13.21	83.90± 7.88	69.93± 9.70	2.43± 0.67	19.50± 0.72

Bull	Treatment	PI (%)	IA (%)	HMP (%)	PIAIHP (%)
1	BCON	45.00±23.94	78.33±18.99	44.17±22.75	32.83±13.32
	BIGF100	31.83±14.22	66.00±16.04	22.33±16.65	21.67±17.18
	BIGF200	29.33±10.42	76.00±19.98	25.83±9.78	20.50±3.77
	BINS150	59.00±11.00	80.17±13.53	52.33±13.16	56.33±12.50
	BINS200	41.33±18.66	73.67±21.33	39.83±17.96	39.83±17.96
	BASS1	27.50±6.08	65.83±15.00	41.17±24.57	26.17±5.20
	BASS2	17.83±5.80	74.17±28.29	41.17±40.90	15.83±3.25
2	BCON	45.67±12.66	80.33±6.21	52.00±19.92	45.00±13.48
	BIGF100	39.50±17.39	74.83±9.22	38.50±17.76	37.83±16.60
	BIGF200	30.83±12.39	75.83±8.08	31.17±12.34	29.33±11.24
	BINS150	65.50±12.13	82.17±7.52	55.50±17.30	61.50±15.88
	BINS200	50.00±10.58	84.00±6.14	47.83±11.41	54.00±17.50
	BASS1	32.50±15.80	78.50±19.22	41.83±6.01	31.17±15.63
	BASS2	32.83±8.69	71.17±3.01	33.67±12.35	31.67±9.83
3	BCON	32.67±7.37	80.00±4.36	32.33±15.12	30.83±7.08
	BIGF100	31.83±3.18	70.33±24.95	28.00±4.09	26.50±4.58
	BIGF200	41.50±6.50	88.00±2.60	45.67±4.65	39.17±7.94
	BINS150	48.33±9.45	84.33±5.03	41.00±11.27	46.33±8.50
	BINS200	40.33±5.35	79.67±3.25	37.67±5.11	35.67±0.29
	BASS1	43.83±16.43	84.67±2.57	41.67±15.82	40.67±17.47
	BASS2	38.83±3.62	86.33±4.19	38.83±4.25	35.83±1.89
4	BCON	27.67±14.98	73.50±5.27	30.17±18.82	27.00±16.09
	BIGF100	22.83±12.09	63.33±14.05	28.50±4.77	21.00±11.32
	BIGF200	24.17±17.62	73.83±9.45	27.17±16.65	21.83±17.82
	BINS150	46.50±15.88	77.83±5.20	39.67±17.06	45.00±16.53
	BINS200	34.17±15.73	82.50±12.32	33.17±16.44	29.50±17.76
	BASS1	30.83±17.25	77.83±11.62	29.67±17.92	23.50±14.41
	BASS2	24.33±8.02	71.17±8.25	30.17±13.48	21.50±9.26

Parameters	TCON	TIGF100	TIGF200	TINS150	TINS200	TASS1	TASS2
AI (%)	81.70±3.21	81.58±1.82	80.45±3.50	87.47±3.89	82.29±2.07	83.91±3.71	83.62±2.33

Bull	Treatment	SM (%)	PI (%)	IA (%)	HMP (%)	PIAIHP (%)
1	TCON	46.67±5.77	42.50±7.47	76.33±14.75	80.17±9.67	40.83±9.00
	TIGF100	48.33±5.77	37.67±9.57	77.17±13.14	81.67±9.50	37.67±9.57
	TIGF200	40.00±5.00	23.50±10.00	78.50±18.03	67.33±14.07	23.17±9.75
	TINS150	65.00±0.00	48.17±12.85	87.00±9.84	85.17±5.80	48.17±12.85
	TINS200	58.33±7.64	50.83±6.83	78.83±5.25	80.17±4.19	48.33±7.11
	TASS1	45.00±0.00	31.50±4.27	84.33±14.01	75.17±10.30	31.33±4.25
	TASS2	38.33±7.64	34.00±15.52	84.50±12.62	62.83±14.92	33.50±15.31
2	TCON	40.00±0.00	54.50±10.40	85.67±9.07	66.83±19.21	50.00±10.04
	TIGF100	53.33±5.77	40.83±4.91	80.83±11.45	70.50±16.50	40.50±5.22
	TIGF200	45.00±0.00	34.00±9.99	78.00±15.22	67.83±13.33	33.00±9.73
	TINS150	60.00±5.00	55.00±5.77	95.83±4.37	86.33±0.58	54.50±4.92
	TINS200	55.00±5.00	46.33±6.66	85.00±8.67	80.17±8.28	45.67±7.29
	TASS1	43.33±14.43	32.33±10.56	85.33±6.90	79.00±4.44	32.00±9.99
	TASS2	36.67±11.55	49.50±20.66	77.67±20.89	62.67±13.58	36.67±8.22
3	TCON	26.67±5.77	18.83±10.25	88.67±9.29	64.50±43.75	16.50±12.58
	TIGF100	35.00±8.66	21.67±17.16	86.00±3.12	78.00±18.21	21.00±16.09
	TIGF200	30.00±5.00	23.83±15.25	90.67±5.48	73.33±27.43	23.50±15.26
	TINS150	48.33±2.89	27.17±2.02	89.83±6.79	77.33±26.84	27.00±2.00
	TINS200	40.00±0.00	19.33±7.25	86.67±9.39	64.83±40.18	17.50±6.06
	TASS1	33.33±2.89	15.50±10.04	92.00±8.05	75.67±18.76	14.83±9.65
	TASS2	26.67±7.64	15.83±7.65	83.33±13.32	58.33±24.10	15.50±7.37
4	TCON	25.00±5.00	19.00±9.34	76.17±14.77	59.83±36.30	19.00±9.34
	TIGF100	23.33±15.28	6.67±4.25	82.33±13.29	42.00±35.38	6.67±4.25
	TIGF200	25.00±13.23	11.67±10.75	74.67±21.68	39.83±33.14	10.8310.05±
	TINS150	40.00±15.00	22.00±8.72	77.17±14.34	57.83±22.68	21.67±8.50
	TINS200	33.33±20.21	13.50±10.64	78.67±14.22	63.50±38.67	13.17±10.91
	TASS1	20.00±0.00	8.50±8.79	74.00±16.82	29.50±30.63	8.17±8.69
	TASS2	25.00±8.66	9.00±0.87	89.00±19.05	28.83±23.96	8.83±1.15

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E-mail: abmvz.artigo@gmail.com

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[1] *⁽Corresponding author: celeghin@usp.br