Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).

In vitro fertilization with cryopreserved inbred mouse sperm.

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term.

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6J x SJL/J)F(1) mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.

Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.

In an attempt to optimize a method of cryopreserving spermazoa from mice bearing mutations, we investigated the effect on motility of temperature for the collection of mouse sperm and the rate of thawing after freezing. Comparison among samples of sperm collected from both the caudae epididymides and vas deferens placed directly in a cryoprotectant of 3% skim milk and 18% raffinose equilibrated at 37, 23, and 3 degrees C showed no difference in the number of viable sperm harvested. Concentration and motility was highest after collection at 37 degrees C (22.3 x 10(6) sperm/ml with 80% motility) combined with rapid thawing at 37 degrees C (2.9 x 10(6) sperm/ml with 84% motility in the swim-up fraction). The fertilization capacity of sperm collected and thawed at 37 degrees C was analyzed in vitro and no difference was observed between the cryopreserved sperm and the control (91 and 89%, respectively). Transfer of in vitro fertilized embryos to pseudopregnant recipients resulted in 37% implantation at Day 10 of pregnancy and 38% live births at term.

Diluting lidocaine and mepivacaine in balanced salt solution reduces the pain of intradermal injection.

BACKGROUND AND OBJECTIVES: Intradermal injection of local anesthetics prior to percutaneous needle insertion is often painful. This study evaluated the effect of diluting lidocaine and mepivacaine with balanced salt solution on perception of pain on intradermal injection. METHODS: Twenty healthy volunteers were each intradermally injected with six solutions in random order. These solutions were: normal saline (NS), 0.9% benzyl alcohol in NS, 0.2% lidocaine in NS, 0.2% lidocaine in balanced salt solution, 0.2% mepivacaine in NS, and 0.2% mepivacaine in balanced salt solution. Discomfort of each injection was reported on a 0-2 pain scale. The degree of anesthesia at each site was evaluated by pinprick every minute for 20 minutes. RESULTS: Benzyl alcohol and lidocaine and mepivacaine in balanced salt solution caused the least injection pain. However, mepivacaine in NS and NS alone caused the most pain. The anesthetic effect of benzyl alcohol was judged adequate for only 4 minutes whereas both lidocaine and mepivacaine in either NS or balanced salt solution gave adequate anesthesia for at least 15 minutes. CONCLUSIONS: The dilution of lidocaine and mepivacaine with balanced salt solution produces a solution that is both painless on injection and of moderate duration.