Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C

Pessoa, G.A.; Martini, A.P.; Trentin, J.M.; Minela, T.; Rubin, M.I.B.

doi:10.1590/1678-4162-11986

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Veterinary Medicine • Arq. Bras. Med. Vet. Zootec. 72 (06) • Nov-Dec 2020 • https://doi.org/10.1590/1678-4162-11986 copy

Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C

[Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 10⁶ sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.

Keywords:
semen; equine; cooling

	Defects
Treatments	Acrossoma	Head	Middle Piece	Tail	Normal
Control	1.43 ± 0.31^a	2.2 ± 0.28^bc	2.31 ± 0.37^ab	14.13 ± 0.76b	78.39 ± 0.97b
Centrifuged	2.16 ± 0.39^a	3.07 ± 0.39^a	2.92 ± 0.39^a	19.43 ± 1.17a	72.4 ± 1.51c
Glass wool	0.80 ± 0.17^b	1.62 ± 0.32^ab	1.14 ± 0.21^c	7.78 ± 0.99b	88.63 ± 1.22a
Sperm Filter	1.52 ± 0.28^ab	2.55 ± 0.24^c	1.99 ± 0.3^b	14.05 ± 0.92c	79.87 ± 0.99b
P Value	0.0296	0.0055	0.0004	<0.0001	< 0.0001

Time	Control	Centrifuged	Sperm Filter	Glass wool	P value
0h	70.0± 2.8^b	71.1 ± 2.78^b	73.8 ± 3.14^b	82.2 ± 1.9 ^a	0.0015
24h	35.1 ± 2.81^c	39.2 ± 2.4b^c	43.3 ± 2.4^b	51.6 ± 2.1^a	0.0001
48h	16.38 ± 2.8^b	18.33 ± 2.94^b	25.0 ± 2.02^a	27.2 ± 1.9^a	0.001

Time	Control	Centrifuged	Sperm Filter	Glass wool	P value
0h	66.6 ± 3.0^b	69.1 ± 3.1^b	70.7 ± 2.2^b	76.2 ± 2.7^a	0.001
24h	41.7 ± 2.7^b	44.2 ± 1.9^b	44.9 ± 1.9^ab	48.2 ± 2.7^a	0.02
48h	21.2 ± 3.2^b	24.6 ± 1.3^b	25.6 ± 2.4^b	31.8 ± 2.2^a	0.008

Time	Control	Centrifuged	Sperm Filter	Glass wool	P Value
0h	56.1± 2.5^b	55.4 ± 2.4^b	57.4 ± 2.6^b	64.4 ± 1.9 ^a	0.007
24h	35.0 ± 2.4^c	34.7 ± 3.5^c	36.8 ± 2.7^ab	41.8 ± 2.5^a	0.03
48h	19.55 ± 2.3^b	18.2± 2.8^b	24.0 ± 2.2^ab	27.3 ± 2.3^a	0.04

			Defect
Groups	Acrossoma	Head	Middle piece	Tail	Normal
Centrifuged	1.24 ± 0.19^a	2.73 ± 0.36^a	1.6 ± 0.25^a	19.87 ± 1.13^a	78.39 ± 0.97^b
Androcoll-E	0.65 ± 0.12^b	1.41 ± 0.29^b	0.91 ± 0.13^a	7.52 ± 0.89^b	89.5 ± 1.1^a
Glass wool	0.71 ± 0.14^b	1.6 ± 0.38^b	1.4 ± 0.34^a	9.1 ± 0.81^b	86.8 ± 1.28^a
P Value	0.02	0.03	0.22	0.001	0.001

Treatment’
Time	Centrifuged	Androcoll-E	Glass wool	P value
Total motility (%)
0h	63.3 ± 2.1^b	85.0 ± 2.9^a	78.5 ± 2.2^a	0.001
24h	36.6 ± 2.5^b	48.0 ± 2.9^a	46.2 ± 2.4^a	0.002
48h	16.2 ± 2.6^b	35.3 ± 2.4^a	27.0 ±2.1^a	0.001
Hyposmotic swelling test (%)
0h	63.9 ± 3.2^b	72.6 ± 1.7^a	74.2 ± 2.2^a	0.001
24h	38.6 ± 2.6^b	48.4 ± 2.1^a	42.1 ± 2.3^ab	0.02
48h	20.3 ± 1.7^b	32.4 ± 2.1^a	28.9 ± 1.9^a	0.03
MTT absorbance (nm)
0h	0.692 ± 0.03^b	0.885 ± 0.04^a	0.873 ± 0.04^a	0.005
24h	0.569 ± 0.04^b	0.683 ± 0.04^a	0.656 ± 0.03^a	0.04
48h	0.377 ± 0.03^b	0.564 ± 0.03^a	0.452 ± 0.02^ab	0.02
	CFDA/PI (%)
0h	51.8 ± 2.0^b	66.85 ± 1.9^a	65.4 ± 2.6^a	0.001
24h	30.9 ± 2.3^b	45.35 ± 2.6^a	43.0 ± 3.1^a	0.001
48h	17.4 ± 3.6^b	34.9 ± 1.6^a	24.9 ± 3.2^b	0.01
	Eosin-nigrosin (%)
0h	48.6 ± 1.65^b	62.7 ± 2.1^a	62.8 ± 1.8^a	0.001
24h	31.5 ± 1.5^b	43.0 ± 2.2^a	38.5 ± 2.5^a	0.002
48h	16.1 ± 2.1^c	37.6 ± 2.1^a	29.5 ± 2.2^a	0.03

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