Action of swim-up and caffeine on equine frozen sperm

Alves, Natália de Castro; Diniz, Soraia de Araújo; Viegas, Rodrigo Novaes; Arigoni, Ana Luiza; Freitas, Marina Morra; Lana, Ângela Quintão; Lagares, Monique de Albuquerque

doi:10.1590/1984-3143-AR2022-0056

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ORIGINAL ARTICLE • Anim. Reprod. 19 (4) • 2022 • https://doi.org/10.1590/1984-3143-AR2022-0056 copy

Action of swim-up and caffeine on equine frozen sperm

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Cryopreservation of equine semen is crucial to semen commercialization. However, it reduces sperm motility and longevity. Thus, sperm selection methods and addition of motility-activating substances to sperm, such as caffeine, may improve sperm quality of equine frozen semen. The objective of the current work was to evaluate the effects of caffeine on recovery and quality parameters of frozen-thawed sperm subjected to swim-up selection to be used in intracytoplasmic sperm injection (ICSI) in assisted reproductive techniques. Stallion semen were frozen and after thawing different caffeine concentrations were added to the samples performing four treatments control (no caffeine), 3, 5, and 7.5 mM caffeine. Sperm kinematic and motility were assessed by computer-assisted sperm analysis (CASA). Then, the four treated samples were submitted to the swim-up sperm selection, and the number of recovered sperm and morphology were evaluated at four times 20, 40, 60, and 80 min. The swim-up increased the recovery proportion of normal morphology sperm without (80.1±1%) or with caffeine addition (3mM: 81.2±1%, 5mM: 79.9±1% and 7.5 mM 78.9±1%) compared to the thawed semen (70±2%). However, the addition of 5 mM caffeine induced an increase in sperm motility (38.9±2.8 vs. 32.6±3.4%, P<0.05), and sperm recovery after swim-up (7.9x10⁶ vs. 3.4x10⁶ sperm/ml, P<0.05) compared to the control. The addition of 5 mM caffeine to frozen-thawed equine semen before swim-up selection improved sperm motility and increased the sperm recovery rate while not decreasing the percentage of morphologically normal sperm. Thus, caffeine addition to frozen-thawed equine semen before swim-up selection has potential clinical application in improving sperm quality for use in ICSI.

Keywords:
spermatozoa; cryopreservation; freezing; sperm selection; stallion

Treatment (mM caffeine)	TM (%)	VAP (µm/s)	VSL (µm/s)	VCL (µm/s)	BCF (Hz)	STR (%)	LIN (%)	WOB (%)	ALH (µm)
0	32.6 ± 3.4^b	23.0 ± 1.3^a	17.2 ± 1.1^a	31.4± 1.5^ab	7.4 ± 0.3^b	72.6 ± 1.3^a	52.8 ± 1.3^a	71.8 ± 1.4^a	2.1 ± 0.1^a
3	27.2 ± 1.9^b	21.2 ± 0.9^a	15.9 ± 0.8^a	29.6± 1.0^b	7.6 ± 0.3^b	73.3± 1.1^a	52.2 ± 1.4^a	70.7 ± 1.1^a	2.1 ± 0.1^a
5	38.9 ± 2.8^a	24.3 ± 1.1^a	18.4 ± 0.9^a	33.5± 1.3^ab	8.0 ± 0.3^ab	74.3± 1.1^a	53.7 ± 1.5^a	71.7 ± 1.1^a	2.2 ± 0.1^a
7.5	33.6 ± 2.8^ab	24.9 ± 1.6^a	18.5 ± 1.3^a	35.3± 1.9^a	8.6 ± 0.3^a	72.9 ± 1.3^a	50.8 ± 1.7^a	68.9 ± 1.4^a	2.1 ± 0.1^a

Sperm morphology (%)
Treatment	Normal	Midpiece	Bent tail	Head	Proximal CD	Distal CD
Thawed semen	70.0 ± 2.1^b	7.2 ± 3.8^ab	9.0 ± 2.4^a	5.4 ± 4.3^a	4.4 ± 3.7^a	4.0 ± 4.2^a
0 mM	80.1 ±1.0^a	6.5 ±2.1^b	1.1 ± 1.1^b	5.5 ± 2.7^a	4.0 ±2.9^a	2.8 ± 2.1^a
3 mM	81.2 ±1.0^a	8.1 ±3.5^ab	1.0 ± 1.9^b	5.1 ± 2.2 ^a	2.7 ±2.3^a	1.9 ± 1.7^a
5 mM	79.9 ± 1.0^a	10.4 ± 3.6^a	1.2 ± 1.8^b	3.8 ± 2.8 ^a	2.9 ± 2.6^a	1.8 ± 2.1^a
7.5 mM	78.9 ± 1.0^a	9.3 ± 4.0^ab	0.6 ± 1.1^b	4.5 ± 2.9 ^a	3.9 ± 2.4^a	2.8 ± 2.6 ^a

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[1] *Corresponding author: nataliac@vetufmg.edu.br; mlagares@ufmg.br

Caffeine (mM)	Recovered sperm (× 10⁶ sperm/mL)
Caffeine (mM)	20 min	40 min	60 min	80 min	Mean
0	3.5±1.1	4.9±2.0	3.5±1.2	1.7±0.4	3.4±0.7^b
3	3.4±0.8	3.7±1.2	10.1±4.7	5.7±2.4	5.7±1.4^ab
5	8.2±3.7	9.7±4.2	5.9±2.7	7.7±4.1	7.9± 1.7^a
7.5	3.5±0.9	5.4±2.2	6.7±3.1	5.8±2.3	5.3± 1.1^ab
Mean	4.6±1.6	5.9±2.4	6.5±2.9	5.2±2.3