Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health

Nguyen, Hai Thanh; Do, Son Quang; Athurupana, Rukmali; Wakai, Takuya; Funahashi, Hiroaki

doi:10.1590/1984-3143-AR2022-0127

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ORIGINAL ARTICLE • Anim. Reprod. 20 (3) • 2023 • https://doi.org/10.1590/1984-3143-AR2022-0127 copy

Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Up to now, the definitive conclusion of the positive effects of rapid transient thawing at higher temperatures for shorter durations has not been obtained yet and is still under discussion due to some contradictory findings and limited assessment of post-thawed parameters. The purpose of the current study was to evaluate the effectiveness of rapid thawing in water at 70 °C by using various post-thawed parameters of frozen bull spermatozoa. Experiment 1, monitoring the change of temperature inside frozen bull straw thawed in water at different temperatures. Experiment 2, evaluation of various post-thawed characteristics of frozen bull spermatozoa thawed in water at different temperatures by using a computer-assisted sperm analysis, flow cytometry and immunocytochemistry. The time it took for the temperature inside the straw to warm up to 15 °C was nearly twice as faster when the straw was thawed in 70 °C water compared with 39 °C. Although there were differences among bulls, viability, motility, and mitochondrial membrane potential of spermatozoa thawed at 70 °C for 8 seconds and stabilized at 39 °C for 52 seconds were significantly higher than those of controls (thawed at 39 °C for 60 seconds) at 0 and 3 h after thawing. Just after thawing, however, there were no differences in acrosome integrity and distribution of phospholipase C zeta1, whereas mitochondrial reactive oxygen species production was significantly lower in spermatozoa thawed at 70 °C. From these results, we conclude that rapid thawing at 70 °C and then stabilization at 39 °C significantly improves viability, motility and mitochondrial health of bull spermatozoa rather than conventional thawing at 39 °C. The beneficial effect of rapid transient thawing could be due to shorter exposure to temperatures outside the physiological range, consequently maintaining mitochondrial health.

Keywords:
bull semen; cryopreservation process; phospholipase C zeta1 (PLCZ1); temperature of thawing

Time (sec) after plunged into thawing water	Temperature of water in which frozen straws were thawed at
	39 °C		70 °C
	Temperature (°C) in straw	Warming rate (°C/sec)	Temperature (°C) in straw	Warming rate (°C/sec)
0	-196.0	-	-196.0	-
2	-45.2 ± 5.6^B	75.4 ± 2.8^b	-28.9 ± 3.9^A	83.6 ± 1.9^a
4	-10.8 ± 1.0^B	17.2 ± 2.3	-5.3 ± 0.9^A	11.8 ± 1.6
6	-6.3 ± 0.7^B	2.2 ± 0.6^b	11.9 ± 2.3^A	8.6 ± 1.3^a
8	-4.7 ± 0.4^B	0.8 ± 0.4^b	37.2 ± 3.2^A	12.6 ± 1.7^a
10	2.2 ± 1.5^B	3.4 ± 0.7	47.7 ± 2.3^A	5.3 ± 0.9
12	10.0 ± 2.0	3.9 ± 1.0	-	-
14	20.2 ± 1.8	5.1 ± 1.0	-	-
16	26.3 ± 1.0	3.1 ± 1.2	-	-

The change sphere of temperature	Estimated average time (sec) to take the change of temperature inside straws thawed at
The change sphere of temperature	39 °C	70 °C
-196 to 0 °C	9.6 ± 0.3^A	5.1 ± 0.2^B
-196 to 15 °C	13.0 ± 0.4^A	6.5 ± 0.2^B
-196 to 37 °C	39.8 ± 0.3^A	7.6 ± 0.2^B
-196 to 39 °C	43.8 ± 0.8^A	8.2 ± 0.3^B

Thawing protocols	Bull	Viability (0 h, %)	Viability (3 h, %)	High MMP (0 h, %)	High MMP (3 h, %)
CT39	A	57.7 ± 4.2^c	40.7 ± 0.4^c	46.9 ± 1.5^d	41.5 ± 0.4^bc
	B	73.4 ± 0.4^ab	69.7 ± 0.7^a	82.5 ± 1.1^a	65.1 ± 1.7^a
	C	65.2 ± 1.9^bc	54.3 ± 0.6^b	55.1 ± 2.2^bc	38.1 ± 0.5^c
	D	36.5 ± 0.9^d	27.9 ± 1.3^d	49.8 ± 2.2^cd	25.2 ± 1.1^d
	Total	58.2 ± 3.1	48.1 ± 3.3	58.6 ± 3.1	42.4 ± 3.1
RT70	A	65.1 ± 4.6^bc	43.2 ± 1.2^c	52.6 ± 2.2^cd	45.2 ± 0.5^b
	B	77.2 ± 0.5^a	73.9 ± 0.5^a	83.3 ± 1.2^a	69.4 ± 1.4^a
	C	68.8 ± 1.2^ab	58.8 ± 1.1^b	61.3 ± 1.6^b	41.5 ± 1.7^bc
	D	37.4 ± 0.9^d	27.5 ± 1.5^d	47.9 ± 1.9^cd	26.1 ± 0.6^d
	Total	62.1 ± 3.3	50.8 ± 3.7	61.3 ± 3.0	45.5 ± 3.3
P among bulls		<0.01	<0.01	<0.01	<0.01
P between thawing protocols		<0.05	<0.01	<0.05	<0.01
P TPS x bulls		0.62	0.06	0.09	0.44

Thawing protocols	Bull	Total motility (%)	Progressive motility (%)	Rapid progressive motility (%)	VSL (μm/s)	VCL (μm/s)	VAP (μm/s)	ALH (μm)	BCF (Hz)
CT39	A	43.9 ± 2.0^bc	38.8 ± 2.3^cd	32.2 ± 2.7^cd	58.2 ± 4.9^ab	195.9 ± 6.5^ab	73.8 ± 1.6	4.0 ± 0.2^abc	8.8 ± 1.0^b
	B	69.2 ± 2.2^a	57.1 ± 3.8^ab	49.0 ± 3.9^ab	48.8 ± 5.3^b	207.0 ± 10.7^ab	73.2 ± 5.8	4.3 ± 0.2^ab	6.6 ± 0.4^b
	C	44.2 ± 1.6^bc	36.4 ± 1.2^cd	30.0 ± 1.7^cd	65.2 ± 5.0^ab	192.2 ± 9.4^ab	78.8 ± 4.8	3.3 ± 0.2^c	11.5 ± 0.2^a
	D	34.5 ± 1.7^d	27.8 ± 1.9^d	22.8 ± 1.7^d	62.6 ± 2.8^ab	198.1 ± 3.4^ab	77.8 ± 2.5	3.9 ± 0.1^abc	8.4 ± 0.3^b
	Total	47.9 ± 2.8	40.0 ± 2.5	33.5 ± 2.4	58.7 ± 2.5	198.3 ± 3.9	75.9 ± 2.0	3.9 ± 0.1	8.8 ± 0.4
RT70	A	50.9 ± 2.8^b	45.1 ± 2.9^bc	38.1 ± 2.4^bc	64.8 ± 3.4^ab	203.1 ± 7.5^ab	81.0 ± 2.7	4.0 ± 0.2^abc	9.0 ± 0.9^ab
	B	75.8 ± 1.7^a	67.1 ± 4.9^a	55.0 ± 4.5^a	52.0 ± 2.3^b	218.8 ± 4.6^a	82.9 ± 2.7	4.4 ± 0.2^a	7.1 ± 0.3^b
	C	48.6 ± 2.5^b	42.2 ± 3.0^c	34.5 ± 2.4^cd	69.9 ± 3.3^a	191.6 ± 8.5^ab	83.6 ± 3.3	3.5 ± 0.1^bc	11.6 ± 0.7^a
	D	35.7 ± 1.6^cd	27.5 ± 1.8^d	21.9 ± 2.2^d	58.4 ± 3.2^ab	184.1 ± 6.7^b	78.2 ± 4.6	3.5 ± 0.2^bc	8.4 ± 0.3^b
	Total	52.8 ± 3.2	45.5 ± 3.3	37.4 ± 2.8	61.3 ± 2.0	199.4 ± 4.2	81.4 ± 1.7	3.8 ± 0.1	9.0 ± 0.4
P among bulls		<0.01	<0.01	<0.01	<0.01	<0.05	0.74	<0.01	<0.01
P between thawing protocols		<0.01	0.01	0.06	0.36	0.84	<0.05	0.73	0.65
P TPS x bulls		0.48	0.38	0.59	0.55	0.34	0.65	0.50	0.97

Thawing protocols	Bull	Total motility (%)	Progressive motility (%)	Rapid progressive motility (%)	VSL (μm/s)	VCL (μm/s)	VAP (μm/s)	ALH (μm)	BCF (Hz)
CT39	A	29.9 ± 0.8^c	24.2 ± 1.3^b	20.9 ± 1.5^bc	50.4 ± 3.0^ab	161.1 ± 8.6^ab	73.2 ± 4.1^ab	2.9 ± 0.1^ab	12.5 ± 0.7^ab
	B	51.4 ± 1.5^b	42.7 ± 3.0^a	35.6 ± 3.1^a	48.6 ± 3.1^ab	159.8 ± 15.2^ab	67.3 ± 4.1^ab	3.3 ± 0.3^a	12.7 ± 0.7^ab
	C	28.4 ± 0.9^c	22.5 ± 0.9^bc	18.9 ± 1.6^bcd	56.4 ± 4.8^a	164.4 ± 11.6^ab	76.2 ± 4.7^a	2.5 ± 0.3^b	15.1 ± 0.6^a
	D	20.6 ± 1.4^d	14.2 ± 1.2^d	10.6 ± 1.6^d	40.9 ± 3.7^b	134.5 ± 8.3^b	61.1 ± 7.3^b	2.7 ± 0.3^b	10.8 ± 0.4^b
	Total	32.6 ± 2.5	25.9 ± 2.3	21.5 ± 2.1	49.1 ± 2.8	154.9 ± 5.8	69.4 ± 2.7	2.9 ± 0.2	12.8 ± 0.4
RT70	A	34.0 ± 1.2^c	29.2 ± 1.0^b	24.8 ± 1.8^b	57.2 ± 7.1^ab	171.2 ± 11.8^ab	77.9 ± 5.6^ab	3.2 ± 0.2^ab	13.6 ± 1.0^ab
	B	57.3 ± 2.0^a	48.9 ± 2.6^a	38.3 ± 3.2^a	51.0 ± 7.3^ab	180.1 ± 6.9^a	81.6 ± 2.6^ab	3.5 ± 0.3^a	13.0 ± 0.3^ab
	C	30.7 ± 1.1^c	26.3 ± 1.3^b	21.6 ± 1.1^bc	62.6 ± 3.2^a	161.3 ± 7.0^ab	80.0 ± 3.7^a	2.6 ± 0.2^b	14.2 ± 0.5^a
	D	21.8 ± 1.2^d	15.4 ± 1.9^cd	12.3 ± 2.1^cd	46.6 ± 6.5^b	151.7 ± 5.4^ab	67.0 ± 6.0^b	2.6 ± 0.2^b	11.0 ± 0.6^b
	Total	36.0 ± 2.8	29.9 ± 2.7	24.3 ± 2.2	54.3 ± 3.2	166.1 ± 4.4	76.6 ± 2.5	3.0 ± 0.1	12.9 ± 0.4
P among bulls		<0.01	<0.01	<0.01	<0.05	<0.05	<0.05	<0.01	<0.01
P between thawing protocols		<0.01	<0.01	0.07	0.15	0.12	<0.05	0.71	0.72
P TPS x bulls		0.30	0.55	0.96	0.97	0.64	0.70	0.96	0.54

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[1] *Corresponding author: hirofun@okayama-u.ac.jp