Sperm cryopreservation in Windsnyer boars; principles, technique, and updated outcomes

Thema, Mamonene Angelinah; Mphaphathi, Masindi Lottus; Ledwaba, Mahlatsana Ramaesela; Nedambale, Tshimangadzo Lucky

doi:10.1590/1984-3143-AR2022-0100

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SHORT COMMUNICATION • Anim. Reprod. 20 (3) • 2023 • https://doi.org/10.1590/1984-3143-AR2022-0100 copy

Sperm cryopreservation in Windsnyer boars; principles, technique, and updated outcomes

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The domestic pig breeds are in danger of extinction whereas the erosion of their gene pool is a serious concern because they significantly contribute to the rich biodiversity. Overall aim of this study was to determine the protocol for preserving the semen of the Windsnyer boars for conservation. A total of 18 ejaculates (6 replications/boar) were collected from three Windsnyer boars of proven fertility with the use of hand-gloved approach method, twice per week. Boars semen were pooled and extended with Beltsville Thawing Solution [(BTS) IMV Technologies, France], held at 18°C for 3 hours and centrifuged. The sperm pellet was re-suspended with Fraction A (20% egg yolk + BTS) and cooled at 5°C for 1 hour. Following cooling, semen was divided and diluted into different cryoprotectants (ethylene glycol, glycerol, propanediol, ethylene glycol + glycerol + propanediol) at equal contribution to make the total concentrations [4, 8, 12 and 16% and the 0% (control; without cryoprotectant)] and loaded into 0.25 mL straws. Two cryopreservation methods (liquid nitrogen vapour and controlled rated) were used to cryopreserve the semen straws. Semen straws were thawed at different temperatures (5, 18, 37 and 40°C) and evaluated for sperm motility, viability, and morphology traits. Post-thawed sperm total motility (36.0±5.3) and live normal sperm (49.5±8.3) percentages were recorded to be higher in the treatment supplemented with 16% glycerol (P<0.05). The highest sperm total motility percentage was recorded at 40°C (26.8±3.2) thawing temperature for liquid nitrogen vapour treatment (P<0.05). In conclusion, 16% glycerol was found to be the suitable cryoprotectant concentration for semen cryopreserved with liquid nitrogen vapour method and thawed at 40°C.

Keywords:
glycerol; propanediol; ethylene glycol; cryopreservation; Windsnyer boars

Treatment	CPA concentration (%)	Sperm viability and morphology trait
Treatment	CPA concentration (%)	Live normal (%)	Dead (%)	Head abnormality (%)		Proximal droplet (%)	Distal droplet (%)	Coiled tail (%)
CPA free	0	22.3±5.8^b	77.7±6.4^a		0±0^b	0±0^b	0±0^b	0±0^b
Glycerol	4	29.0±7.6^b	71.0±7.6^b		0±0^b	0±0^b	0±0^b	0±0^b
	8	23.0±9.3^c	77.0±9.6^a		0±0^b	0±0^b	0±0^b	0±0^b
	12	28.4±4.7^b	71.0±6.9^b		0.2±0.4^a	0.2±0.4^a	0.2±0.4^a	0±0^b
	16	17.0±6.6^c	81.5±5.3^a		0.5±0.5^a	0.5±0.5^a	0.5±0.5^a	0±0^b
Propanediol	4	32.1±6.7^b	67.9±7.2^b		0±0^b	0±0^b	0±0^b	0±0^b
	8	28.7±3.2^b	71.3±3.3^b		0±0^b	0±0^b	0±0^b	0±0^b
	12	29.0±3.5^b	70.4±3.9^b		0.3±0.5^b	0.3±0.5^a	0±0^b	0±0^b
	16	49.5±8.3^a	50.5±7.5^c		0±0^b	0±0^b	0±0^b	0±0^b
Ethylene glycol	4	25.7±9.5^b	74.1±9.5^b		0.2±0.4^a	0±0^b	0±0^b	0±0^b
	8	21.7±6.9^c	78.1±6.9^a		0.2±0.4^a	0±0^b	0±0^b	0±0^b
	12	35.5±9.5^b	64.5±12.9^b		0±0^b	0±0^b	0±0^b	0±0^b
	16	34.7±9.1^b	65.3±9.1^b		0±0^b	0±0^b	0±0^b	0±0^b
EG + GLY + PDO	4	28.8±4.4^b	67.5±4.7^b		0±0^b	0.7±1.0^a	0.7±0.8^a	2.3±0.5^a
	8	20.7±5.2^c	78.5±5.1^a		0±0^b	0.5±0.5^a	0±0^b	2.0±2.2^a
	12	21.8±3.5^c	68.7±8.3^b		0±0^b	1.2±0.7^a	0.7±1.0^a	3.3±0.8^a
	16	30.4±6.2^b	65.5±6.1^b		0±0^b	0.9±0.8^a	0.8±1.3^a	2.3±1.4^a

Sperm motility trait	CPA free	CPA
	CPA free	Ethylene glycol				Glycerol				Propanediol
	0%	4%	8%	12%	16%	4%	8%	12%	16%	4%	8%	12%	16%
TM (%)	8.0±1.8^b	14.0±2.4^b	9.0±3.9^b	12.0±2.9^b	9.0±4.6^b	18.0±8.2^b	19.0±6.2^b	12.0±3.9^b	36.0±5.3^a	16.0±8.0^b	6.0±2.7^b	23.0±5.1^b	19.0±8.1^b
PM (%)	4.0±1.8^b	8.0±3.1^b	8.0±2.9^b	8.0±4.5^b	2.0±2.0^b	8.0±6.4^b	13.0±6.2^b	8.0±5.2^b	19.0±7.4^a	8.0±3.2^b	5.0±2.6^b	16.0±8.4^a	9.0±7.1^b
RAP(%)	1.0±1.3^b	4.0±4.1^b	4.0±3.2^b	5.0±6.1^b	0.3±0.7^b	4.0±4.1^b	8.0±3.2^b	2.0±2.2^b	10.0±6.9^a	3.0±2.6^b	1.0±1.1^b	9.0±9.0^b	9.0±7.5^b
MED(%)	4.7±1.8^b	5.0±3.2^b	5.0±4.2^b	5.0±5.7^b	5.7±2.5^b	9.0±6.7^b	7.8±3.4^b	6.0±3.6^b	19.0±11.7^a	5.0±2.8^b	4.0±3.5^b	9.0±5.4^b	5.0±4.6^b
SLW (%)	2.3±1.7	5.0±2.8	0±0	2.0±1.9	3.0±2.7	5.0±4.8	3.2±2.4	4.0±5.2	7.0±6.1	8.0±5.2	1.0±1.1	5.0±5.4	5.0±2.7
STC (%)	92.0±1.9^a	86.0±2.4^b	91.0±3.9^ab	88.0±2.9^ab	91.0±4.5^ab	82.0±8.2^b	81.0±6.2^b	86.0±3.9^b	64.0±5.3^c	84.0±8.0^b	94.0±2.7^a	77.0±5.1^c	81.0±8.1^b
BCF(Hz)	19.0±9.8^a	13.0±4.8^b	21.0±3.6^a	21.0±9.2^a	15.0±5.4^a	14.0±6.1^b	21.0±6.9^ab	15.0±6.5^a	16.0±5.7^a	9.0±6.7^b	18.0±3.6^a	18.0±5.4^a	12.0±9.5^b

Sperm viability and morphology trait	Cryopreservation method
	Liquid nitrogen vapour				Slow freezing
	5°C	18°C	37°C	40°C	5°C	18°C	37°C	40°C
Live normal (%)	37.7±4.2^bc	45.3±8.9^b	44.2±8.9^b	65.4±4.9^a	26.5±6.6^c	35.2±10.2^bc	31.7±4.2^bc	34.8±13.3^bc
Dead (%)	60.8±14.2^b	53.0±9.5^c	52.5±9.5^c	32.0±5.4^d	73.5±6.2^a	63.8±10.8^b	65.3±5.5^b	62.5±13.1^b
Head abnormal (%)	0.5±0.3^b-d	0.5±0.5^a	1.0±0.9^a	1.2±0.4^a	0±0^b	0.3±0.5^a	0.8±0.9^a	0.7±0.8^a
Proximal droplet (%)	0.4±0.8^a	0.5±0.5^a	0.7±0.5^a	0.3±0.5^a	0±0^b	0.3±0.5^a	0.7±1.0^a	0.8±0.8^a
Distal droplets (%)	0.3±0.8^a	0.5±0.5^a	0.5±0.5^a	0.3±0.5^a	0±0^b	0.2±0.5^a	0.8±0.9^a	0.6±0.8^a
Coiled tail (%)	0.3±0.8^a	0.2±0.4^a	1.1±0.9^a	0.8±0.4^a	0±0^b	0.2±0.4^a	0.7±1.0^a	0.6±0.8^a

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[1] *Corresponding author: masindim@arc.agric.za