Sperm cryopreservation of a live-bearing fish, the platyfish Xiphophorus couchianus

Abstract

The objectives of this study were to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio, as well as somatic relationships of body length, body weight, and testis weight to sperm density in the platyfish Xiphophorus couchianus. Sperm motility and survival duration after thawing were significantly different between cryopreservation with dimethyl sulfoxide (DMSO) and glycerol, with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) across a range of 240-300 mOsm/kg. Samples cooled from 5 to -80 degrees C at 25 degrees C/min yielded the highest post-thaw motility, although no significant difference was found for cooling rates across the range of 20-30 degrees C/min. In addition, the highest motility after thawing was found in samples equilibrated from 10 to 30 min with 14% glycerol and cooled at 25 degrees C/min. The post-thaw motility declined rapidly with use of 10% glycerol and cooling at 5 degrees C/min across the equilibration range of 10 min to 2h. Sperm motility with a dilution ratio of sperm to extender of 1:10 was not different at 10 min after thawing with those samples at greater dilutions, but declined significantly from Day 1 after thawing and showed lower survival duration when stored at 4 degrees C. However, the additional dilution of sperm solutions with HBSS (300 mOsm/kg) immediately after thawing significantly slowed the decline of motility and prolonged the duration of survival. Based on the above findings, the highest average sperm motility (78+/-3 %) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsm/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 25 degrees C/min from 5 to -80 degrees C before plunging into liquid nitrogen, and thawed at 40 degrees C in a water bath for 7 s. If diluted within 5 h after thawing, sperm frozen by the above protocol retained continuous motility for 15 days when stored at 4 degrees C.

Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: Male-to-male variation in post-thaw motility and production of F1 hybrid offspring

“…Therefore, sperm cryopreservation and artificial insemination with cryopreserved sperm present significant challenges in live-bearing fishes compared to fishes with external fertilization. Thus far, sperm cryopreservation in live-bearing fishes has been reported in two Xiphophorus species, X. helleri and X. couchianus (Huang et al, 2004a,b,c; Yang et al, 2006, 2007b), but currently live young have only been produced from cryopreserved sperm of X. helleri (Yang et al, 2007b). …”

“…For X. couchianus , sperm cryopreservation has been studied using post-thaw motility as the evaluation indicator (Huang et al, 2004a), but evaluation of artificial insemination with cryopreserved sperm has not been reported. For X. helleri , sperm suspended in hypertonic extender (500 mOsmol/kg) were osmotically immobilized (they are motile when held in isotonic buffer), and the immobilization of sperm was able to extend retention of motility during refrigerated (non-frozen) storage compared to storage of non-immobilized sperm in isotonic solutions, and did not affect the motility of sperm after thawing (Yang et al, 2006).…”

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

“…In previous studies on sperm cryopreservation in this genus, two species, Xiphophorus helleri and Xiphophorus couchianus, have been investigated [14,16]. High motility after thawing (40-78%) was obtained using glycerol as cryoprotectant and Hanks' balanced salt solution (HBSS) at 310-320 mOsmol/kg as the extender.…”

Pregnancy Outcome of Intracytoplasmic Sperm Injection in Relation to Duration of Cryopreservation of Spermatozoa Obtained through Testicular Sperm Extraction

“…In addition, the assisted reproductive technology outcome appears not to have been affected by the duration of period of cryopreservation. However, there is no sufficient data about the effect of cryopreservation duration on the pregnancy outcome [13]. Thus, the present study was designed to analyze pregnancy outcome in patients underwent ICSI procedure in relation to duration of cryopreservation of the sperms.…”