Birth of Live Mice Resulting from Oocytes Fertilized in Vitro with Cryopreserved Spermatozoa1

Abstract

The objective of this study was to develop a reliable method to cryopreserve mouse spermatozoa, using in vitro fertilization (IVF) of oocytes, development of resultant zygotes into blastocysts, and the birth of live young after embryo transfer as criteria of sperm survival. Epididymal sperm were frozen in a raffinose-glycerol solution containing either egg yolk (EY) or BSA. Sperm frozen in the presence of EY yielded higher survivals than those frozen in the presence of BSA; when used for IVF, the former resulted in more blastocysts (26% vs. 5%). Comparison between one-step and stepwise dilution of the cryoprotectant revealed no differences in sperm viability as assayed by a vital stain. Nevertheless, IVF using frozen sperm recovered by one-step or stepwise dilution resulted in 33% and 17% blastocyst formation, respectively. When embryos produced with cryopreserved sperm were transferred into recipients, pregnancies (12 of 15 recipients) and the birth of live young (57 pups from 167 embryos transferred) resulted. These results were comparable to those obtained from transferral of embryos produced by IVF with fresh sperm (17 pregnancies from 19 recipients, 87 live young from 197 embryos). Thus, this study has resulted in the development of a simple and efficient cryopreservation procedure for mouse sperm.

Age-related Yields of Mouse Sperm and Oocytes

“…Accordingly, a sperm with a high circular but not forward motility was scored as "1". Out of these, the percentage of moving a spermatozoon was estimated and accordingly the motility score was reduced, a simplified procedure according to [8,18,19]. E.g., in a sample with a very high forward motility just 75% of all spermatozoa were moving the score of "3" was reduced to 75%, i.e.…”

“…However, an in vitro-fertilization required to recover a line from frozen spermatozoa is complex [7,8] the success is often influenced externally, e.g. genetic background, mutation, osmotic stress, batches of the culture media used, environment, etc.…”

Mouse Spermatozoa Ctyopreservation.

“…[10] 35-36 -** -** Okuyama et al [9] 42 -** -** Takeshima et al [12] 34-36 -45 Nakagata & Takeshima [15] 71-95 -51 Tada et al [16] 85 57 -ICR (CD-1) Storey et al [25] 33-58 -** -** <hybrid> B6CF1 (C57BL/6 X BALB/c) Yokoyama et al [11] 19-37 -17-75 B6C3F1 (C57BL/6N X C3H/He) Nakagata & Takeshima [15] 59 -56 B6D2F1 (C57BL/6J X DBA/2J) Sztein et al [20] 89-93 37 38 Songsasen and Leibo [21] 64-73 -** -** Songsasen and Leibo [22] 61 -42 Songsasen et al [23] 26-39 -24-47 M.m.musculus Nakagata et al [18] 48-63 -24-51 *PZD oocytes were inseminated with frozen-thawed spermatozoa with low motility. **Fertilized oocytes were not transferred.…”

“…Since that time, numerous researchers have reported successful mouse sperm cryopreservation by means of various procedures [15][16][17][18][19][20][21][22][23][24][25][26]. Nevertheless, the in vitro fertilization rates and rate of development to fetus and live offspring after transfer of embryos derived from frozen spermatozoa varies considerably among the different research groups (Table 1).…”

Cryopreservation and Transport of Mouse Spermatozoa at -79.DEG.C..

“…At present, cryopreservation of spermatozoa for long periods is usually carried out at -196°C in liquid nitrogen (LN 2 ). Since the first reports of successful mouse sperm cryopreservation [8,15,18], mouse spermatozoa have also been successfully stored at -196°C [6,14,16], as have various other species [2]. If frozen spermatozoa PAN Inc. (Tokyo, Japan).…”