Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates

Abstract

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P<0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P<0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P<0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.

Ultrastructural description of fresh and frozen/thawed sperm derived from collared peccaries (Pecari tajacu Linnaeus, 1,758)

“…Finally, the samples were packed into 0.25 ml plastic straws (IMV Technologies, L'Aigle, France) that were placed horizontally in an insulated box for 5 min, at 5 cm above N 2 vapors, and then plunged into liquid N 2 for storage at −196°C, after a fast freezing rate of −40°C/min. After one week, frozen straws were thawed by immersion in a water bath at 37°C for 1 min (Silva et al, ). Samples were evaluated as described for fresh semen.…”

Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c)

“…One epididymis was flushed with 5 ml of a Tris-based extender [3.028 g Tris-hydroxymethyl-aminomethane, 1.78 g monohydrated citric acid, and 1.25 g d -fructose dissolved in 100 ml of ultrapure water (Tris; 295 mOsm/l)] (Castelo et al ., 2010a). The other epididymis was flushed using 5 ml ACP-116c (ACP ® , ACP Biotecnologia, Fortaleza, Brazil; 307 mOsm/l) (Silva et al ., 2013). This product is composed of dehydrated coconut water and pH regulators.…”

“…Thawing rate is another important factor that can affect sperm quality (Andrabi, 2009). Currently, peccaries’ ejaculated sperm samples have been thawed at temperatures varying from 37°C for 1 min (Castelo et al ., 2010b) to 70°C for 8 s (Silva et al ., 2013), but no information is available on thawing their epididymal sperm. Given the need to better understand appropriate conditions for sperm preservation and thawing, we aimed to establish a protocol for freezing and thawing epididymal sperm of collared peccaries by comparing the effectiveness of Tris-based and ACP-116c extenders on post-thaw sperm quality.…”

Theoretical investigation of a novel microwave antenna aided cryovial for rapid and uniform rewarming of frozen cryoprotective agent solutions

“…Cryodevices play an important role in cryopreservation, storage, and the following rewarming processes [1,2]. Research shows that the properties of the cryoprotective agents (CPAs) and the cells in cryopreservation systems are correlated with the external environment [3].…”