Phillip H. Purdy

Effect of Adding Cholesterol to Bull Sperm Membranes on Sperm Capacitation, the Acrosome Reaction, and Fertility

Abstract When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival

Acceptable fertility using cryopreserved ram sperm is currently only achieved using laparoscopic intrauterine insemination. Improving the cryosurvival of ram sperm may permit greater fertility rates using more practical techniques. This study was conducted to determine if treating ram sperm with six different cyclodextrins pre-loaded with cholesterol (CLC), prior to cryopreservation increases sperm cryosurvival and if this technology can be used with neat semen. Subsequent experiments evaluated how adding CLC to sperm affected sperm cholesterol content, sperm osmotic tolerance limits, sperm post-thaw survival after incubation and the capacity of sperm to bind to zona pellucidae of cattle and sheep oocytes. Sperm treated with 2-hydroxypropyl-beta-cyclodextrin prior to cryopreservation exhibited greater percentages of motile sperm (62%) compared to the control (no CLC treatment) samples (43%, P<0.05), after thawing. In addition, samples treated with methyl-beta-cyclodextrin exhibited percentages of motile and viable sperm similar to samples treated with 2-hydroxypropyl-beta-cyclodextrin. Other CLC-treated samples were similar to the control. The CLC concentration that optimized sperm cryosurvival was 2mg CLC/120 x 10(6) sperm for both methyl-beta- and 2-hydroxypropyl-beta-cyclodextrin when added to neat semen prior to cryopreservation. Addition of 2mg CLC not only maintained greater percentages of motile sperm compared to the control samples, but maintained greater percentages of motile sperm during a 3h incubation after thawing. In addition, 2-hydroxypropyl-beta-cyclodextrin pre-loaded with cholesterol maintained greater percentages of viable sperm (33%), than control sperm (18%; P<0.05). Treating ram sperm with CLC increased the sperm cholesterol content>1.9-fold and although some cholesterol was lost from the sperm during cooling and cryopreservation, the cholesterol content remained greater in CLC-treated sperm after cooling and after thawing than in control sperm (P<0.05). In addition, CLC-treated sperm maintained greater percentages of motile sperm through a wide range of osmotic solutions (150 and 425 mOsm) while control sperm lost motility in solutions outside a more narrow range (270 to 370 mOsm). Greater numbers of CLC-treated sperm bound to zona pellucida than control sperm (P<0.05), although number of sperm binding cattle and sheep oocytes, was similar (P>0.05). In conclusion, treating ram sperm with CLC increases sperm cryosurvival rates and sperm longevity after thawing. It also increases the cholesterol content, osmotic tolerance, and zona-binding capabilities of sperm. Finally, CLCs can be added to neat semen, making this technology feasible for practical application using current cryopreservation techniques for ram semen.

The fertility of ram sperm held for 24 h at 5°C prior to cryopreservation.

Diluted ram sperm can be held for 24h at 5 degrees C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n=3 in experiment 1 and n=6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 x 10(6)sperm/mL, in one step, with a Tris-egg yolk-glycerol diluent. In experiment 1, diluted samples were cooled to 5 degrees C over 2h, and then divided. Sperm in one fraction were loaded into 0.5mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 degrees C for 24h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n=196) using controlled internal drug releasing devices (CIDR) for 12d and at CIDR removal each ewe was administered PMSG (500IU in experiment 1 and 350IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P<0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 degrees C for 24h prior to cryopreservation without altering sperm fertility.

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.

Membrane Modification Strategies for Cryopreservation

Cell membranes can be modified using cyclodextrins loaded with lipids or unilamellar liposomes. Lipid choice can greatly influence the organization of the targeted membrane and result in a cell that is more capable of surviving cryopreservation due to altered membrane-phase transition properties or membrane reorganization that may alter the normal physiologic processes of the treated cell. The protocols described here explain the preparation of the cyclodextrins and liposomes, impact of the amount and type of lipids, and general principles for treating cells using either of these technologies.