Suprazero cooling rate, rather than freezing rate, determines post thaw quality of rhesus macaque sperm

Abstract

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10(6) sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to -110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.

Keywords: Cryopreservation; Lipid peroxidation; Rhesus; Sperm.

Figure 1

Representative fluorescent images of rhesus sperm labeled with membrane lipid probe, BODIPY^581/591 C₁₁. The images were obtained at 100x magnification in a Zeiss Axio Imager M1 microscope. (A) phase-contrast image of rhesus sperm. Red fluorescence represents overall incorporation of the non-oxidized probe into the cell (B and D); Green fluorescence represents oxidization of membrane incorporated probe (C); Merged images in which areas of LPO (lipid peroxidation) appear yellow (D). Arrows indicate one sperm with high LPO as distinguished from sperm with low oxidation state.

Figure 2

Thermocouple measurements of cooling and freezing rates of various foam raft holders floating on the surface of liquid nitrogen. The slow initial suprazero cooling rate combined with medium subzero rate (SM) averaged 0.5ºC/min above zero and 45ºC/min below zero. The slow initial suprazero cooling rate combined with fast subzero rate (SF) rate averaged 0.5ºC/min above zero and 93ºC/min below zero.

Figure 3

Thermocouple measurements of cooling and freezing rates of various foam raft holders floating on the surface of liquid nitrogen. The medium initial suprazero cooling rate combined with medium subzero rate (MM) rate averaged 45ºC/min above and 42ºC/min below zero. The fast initial suprazero cooling rate combined with fast subzero rate (FF) rate averaged 93ºC/min above and 87ºC/min below zero.

Figure 4

Comparison of rhesus sperm quality parameters between fresh and frozen sperm. Sperm were exposed to five different cryopreservation treatments: Fresh, slow initial suprazero cooling rate combined with medium subzero rate (SM), slow initial suprazero cooling rate combined with fast subzero rate (SF), medium initial suprazero cooling rate combined with medium subzero rate (MM), and fast initial suprazero cooling rate combined with fast subzero rate (FF). Bars bearing different superscripts differ significantly. Sperm cell quality in response to cryopreservation was different from fresh controls; frozen sperm demonstrated significantly different motility, membrane integrity, and LPO (P < 0.0001).

Figure 5

Membrane integrity measured by propidium iodide (PI) exclusion for rhesus sperm exposed to five different cryopreservation treatments: Fresh, slow initial suprazero cooling rate combined with medium subzero rate (SM), slow initial suprazero cooling rate combined with fast subzero rate (SF), medium initial suprazero cooling rate combined with medium subzero rate (MM), and fast initial suprazero cooling rate combined with fast subzero rate (FF). Slow supra zero cooling resulted in more membrane-intact sperm cells compared to medium and fast rates. Cellular response to all cryopreservation treatments was different from fresh controls. Bars bearing different superscripts differ significantly (P < 0.0001).

Figure 6

Comparison of total motility (TM) and progressive motility (PM) between fresh and frozen sperm. Sperm were exposed to five different cryopreservation treatments: Fresh, slow initial suprazero cooling rate combined with medium subzero rate (SM), slow initial suprazero cooling rate combined with fast subzero rate (SF), medium initial suprazero cooling rate combined with medium subzero rate (MM), and fast initial suprazero cooling rate combined with fast subzero rate (FF). All treatments except for fresh were grouped and subsequently averaged into a “frozen” category for analysis. Bars bearing different superscripts differ significantly; TM was different from PM across all treatments (P < 0.05) and frozen sperm demonstrated significantly reduced total and progressive motility compared to fresh semen (P < 0.0001).

Figure 7

Total (TM) and progressive motility (PM) response of rhesus sperm exposed to five different cryopreservation treatments: Fresh, slow initial suprazero cooling rate combined with medium subzero rate (SM), slow initial suprazero cooling rate combined with fast subzero rate (SF), medium initial suprazero cooling rate combined with medium subzero rate (MM), and fast initial suprazero cooling rate combined with fast subzero rate (FF). Medium and fast supra zero cooling rates resulted in decreased TM and PM post thaw compared to slow treatments. All cryopreservation treatments were different from fresh controls (P < 0.0001); TM was different from PM across all treatments (P < 0.05). Bars bearing different superscripts differ significantly (P < 0.001).

Figure 8

Lipid peroxidation (LPO) measured by BODIPY^581/591 C₁₁ for rhesus sperm exposed to five different cryopreservation treatments: Fresh, slow initial suprazero cooling rate combined with medium subzero rate (SM), slow initial suprazero cooling rate combined with fast subzero rate (SF), medium initial suprazero cooling rate combined with medium subzero rate (MM), and fast initial suprazero cooling rate combined with fast subzero rate (FF)using flow cytometery. Frozen sperm resulted in increased LPO compared to fresh controls (P<0.001). Slow supra zero cooling resulted in less LPO compared to medium or fast rates. Bars bearing different superscripts differ significantly (P < 0.05).