In vitro fertilisation with chilled-stored ram spermatozoa

Theriogenology 41:302, 1994 IN VITRO FERTIUSATION WITH CHII I FO-STORED RAM SPERMATOZOA. T. Stojanov, S.J. Robinson, S.L Rhodes, J.K. O'Brien, G. Evans & W.M.C. Maxwell Department of Animal Science, University of Sydney, NSW, 2006. Australia The in vivo fertilising capacity of ram spermatozoa decreases with chilled storage, but can be maintained for up to 10 days (Salamon et a/.,1979: Anim. Reprod. Sci. 2, 373-385). This study examined the effect of chilled storage on the in vitro fertilising capacity of ram spermatozoa. Immature oocytes were cultured in TCM-199 (M-5017; Sigma, St Louis, MO) + 10% foetal bovine serum (Multiser~M: Cytosystems P/L, Castle Hill, NSW) and LH (Lutropin-V: Vetrepharm, London, Ontario), FSH (Folltropin-V: Vetrepharm) and oestradiol (Sigma). Pooled semen from 4 rams was diluted 4 fold with a Tris-based medium and stored at 5°C for either 0, 7 or 14 days. The diluted fresh and stored semen was either washed twice (centrifugation, 100g, 6 min., resuspended in Hepes-SOF+3mg/ml BSA) [Wash], or layered onto a Percoll (Sigma) gradient, centrifuged (1000g, 15 min.) and washed as above [Percoll]. The sperm were then resuspended in fertilisation medium (bicarbonate-buffered SOF+20% sheep serum 2 to the desired concentrations (Expt. 1" 1 x 106 sperm/ml, Expt. 2:1 x 10N or 3 x 108 sperm/ml). After 22 hr culture, cumulusexpanded oocytes were placed in 501~1 droplets of the sperm suspensions under oil at 39 ° C in 5% CO 2 in air. Eighteen hours later, presumptive zygotes were fixed, stained with aceto-orcein and examined for fertiUsation (Table 1). Table 1: Effect of sperm conce~b~dion, preparation and storage time on in vilro f e r t U ~ . Sperm Conc. (sperm/ml)

Methodof Storage Preparation Time(Days)

1 x 10e

Wash (Expt.1)

1 x 10e

Percoll (Expt.1)

No. Oocytes

0 (Fresh) 7 14 0 7

92 153 200 130 170

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1 x 10e

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Fertilisation Polyspermic2, RateI, % % 65.0a 18.3b 6.0¢ 84.6d 31.8° 20.Of

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3.5 0 0 5.5 0 0

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Percoll

0

80

85.0°

14.7"

(Expt.2)

7

100

36.0 b

2.8 a

14 105 19.4~ 0b 3 x 106 Percoll 0 73 98.6d 52.8c (Expt.2) 7 148 43.9b 10.8° 14 128 21.1° 0b ~roportions in columns and within experimentswitll Oifferentsuperscripts differ (P
302

Copyright © 1994 Butterworth-Heinemann