Membrane Damage during Dilution, Cooling and Freezing‐Thawing of Boar Spermatozoa Packaged in Plastic Bags

Abstract

Summary The objective of the present investigation was to determine the degree of membrane damage in boar spermatozoa during the course of a cryopreservation procedure, including thawing. Ejaculates from four fertile Swedish Yorkshire boars were frozen under controlled conditions in Teflon®‐plastic bags (2.5 ml) using 3% glycerol as cryoprotectant. Membrane integrity was monitored using supravital flourescent dyes and confirmed by scanning electron microscopy. The results show that cooling to +5° C significantly affected the permeability of the plasmalemma. Supercooling (‐6° C) during the freezing program did not further deteriorate the integrity of the spermatozoal membrane. The thawing procedure however, dramatically increased the frequency of spermatozoa with damaged plasma membranes. Thus particular attention must be paid on designing a better thawing procedure when dealing with boar semen frozen in plastic bags.

First report on in vivo fertility trial of frozen thawed boar semen in India#

“…Sperm motility was assessed subjectively under a microscope (Olympus, BX 51 FT, Japan) equipped with 35°C microscope stage and phase contrast optics at a magnification of 400X. Before artificial insemination, frozen thawed semen was also evaluated for live sperm by Eosin-Nigrosin staining, acrosomal status by Nigrosin-Eosin-Giemsa staining (Tamuli and Watson, 1994), sperm plasma membrane integrity by Carboxyfluorescein Diacetate and Propidium Iodide staining (Ortman and Rodriguez-Martinez, 1994), hypo-osmotic swelling test of spermatozoa in 100 mOsm (Jeyendran et al, 1984), mitochondrial membrane potentiality of live spermatozoa by JC-1 staining (Cossarizza et al, 1993), lipid peroxidation in sperm by BODIPY C-11 staining (Aitken et al, 2007) and integrity of sperm DNA by Acridine Orange staining (Thuwanut et al, 2008).…”

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art

“…Boar spermatozoa has been processed in plastic straws of different volumes (0.25 to 5 mL, Johnson et al 2000), in flattened 5 mL straws (Weitze et al 1987), in metal or in plastic bags of various types and constitution (Bwanga et al 1991, Mwanza & Rodriguez-Martinez 1993, Ortman & Rodriguez-Martinez 1994Eriksson & RodriguezMartinez 2000, Saravia et al 2005. The latter developed, denominated "FlatPacks™" proven equally good or better than 0.25 mL straws in terms of sperm cryosurvival despite of the fact that they held 5 mL of semen (an entire dose for cervical AI, 5 billion spermatozoa), thus waiving the need of pooling innumerable straws when thawing.…”

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa

“…In both situations, water moves across the membrane towards the side with higher osmolarity, e.g. extracellularly during cooling or in the dehydrated cytoplasm during thawing 6 . These phenomena are apparently not present during sperm transport in vivo 4 , or when epididymal spermatozoa are processed (handled and cryopreserved) 7 .…”

“…They cause, however, extensive cell death among processed boar ejaculated spermatozoa 8 . The high dehydration occurring during slow cooling accumulates solutes intracellularly (a cause for cell death) while the compensating quick transport of water during thawing causes a substantial rate of membrane and organelle damage that leads to impairment of cell function, a reduced lifespan and, eventually, also cell death 6 .…”