Live rats resulting from injection of oocytes with spermatozoa freeze‐dried and stored for one year

Abstract

This study was designed to examine whether rat spermatozoa after freeze-drying and 1-year storage can participate in full-term development following intracytoplasmic sperm injection (ICSI). Cauda epididymal spermatozoa from Crlj:Wistar rats were frozen in liquid nitrogen (LN 2 ), first dried for 14 hr at 0.37 hPa and then for 3 hr at 0.001 hPa. The dried spermatozoa were stored for 1 year in a desiccator at þ258C, or in a refrigerator at þ48C, or in LN 2 at À1968C. Controls consisted of sperm that had only been frozen and stored in LN 2 . After being stored, spermatozoa were sonicated to dissociate the sperm tail and were injected into oocytes from superovulated Slc:SD rats. The respective fertilization rates of oocytes injected with frozen sperm, or with freeze-dried sperm stored at þ25, þ4, and À1968C were 79%, 75%, 70%, and 73%. However, the corresponding cleavage rates of injected oocytes were 63%, 1%, 38%, and 36%. After transfer of >80 zygotes of each group into recipients, the respective percentages of full-term normal offspring resulting from frozen sperm or from freeze-dried sperm stored at þ25, þ4, and À1968C were 36%, 0%, 7%, and 14%. These results demonstrate that the storage temperature significantly influenced the likelihood of term development of rats produced by injection of oocytes with freezedried spermatozoa. Chromosomal analysis of the rat spermatozoa in the ICSI oocytes indicated that chromosomal aberration in freeze-dried spermatozoa stored at þ258C (100%) occurred more frequently than in frozen control spermatozoa (41%) and freezedried spermatozoa stored at À1968C (35%), and the frequency of chromosomal aberrations in freezedried spermatozoa stored at þ48C (65%) was the intermediate. In conclusion, rat spermatozoa freezedried and stored at þ48C for 1 year are capable of participating in full-term development after ICSI. Mol. Reprod. Dev. 75: 890-894, 2008.

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

“…A prolonged storage of FD spermatozoa at ambient temperatures may induce such alterations and/or DNA damage. We have previously reported that one-year storage of FD rat spermatozoa at a room temperature has resulted in a higher incidence of chromosomal aberrations compared to those stored in LN 2 or refrigerator [7]. This has been accompanied with great reduction of cleavage rate after injection of FD sperm stored at +25 ْ◌C (1%) compared to 36-38% after injection of FD sperm stored at +4 or -196 ْ◌C.…”

“…Intracytoplasmic injection of freeze-dried spermatozoa has been resulted in successful production of live offspring in mouse [1,[4][5][6]32], rat [3,7,33] and rabbit [2], and blastocyst-stage embryos in cattle [8,9] and pig [10]. However, long-term storage of FD spermatozoa at room temperature has not yet been achieved in any mammalian species.…”

“…After thawing in water bath at 37 ْ◌C for 30 sec, content of 0.5-mL straw was layered on the top of percoll density gradient consisting of 2 mL of 45% percoll above 2 mL of 90% percoll and centrifuged for 20 min at 700 g. The sperm pellet was resuspended in freeze-drying buffer and washed twice for 5 min each at 300 g. The sperm suspension (3-5 x 10 6 sperm/mL) was divided to 200 µL aliquots in 5-mL volume glass vials (no.2; Maruemu, Osaka, Japan), and then frozen by plunged into LN 2 of 1-cm depth. The frozen samples were 6 transferred onto the shelf (-30 ْ◌C) of programmable freeze-dryer (ALPHA 2-4; Christ, Harz, Germany), and dried primary for 14 h at 0.37 hPa and secondary for 3 h at 0.001 hPa, as described previously [7].…”

“…Offspring derived from the intracytoplasmic injection of FD spermatozoa have been reported firstly in mouse [1], followed by rabbit [2] and rat [3]. To date, preservation of FD spermatozoa for extended periods (≥1 year) was valid only at a refrigeration temperature in mouse [4, 5, and 6], rabbit [2] and rat [7]. On the other hand, blastocyst development after ICSI with FD sperm heads was reported in large domestic animals including cattle [8,9] and pig [10].…”

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

“…As one of the cryopreservation methods in mammalian spermatozoa, freeze-drying has been applied in mouse [1][2][3][4][5][6][7][8], rat [9,10], rabbit [11], bull [12], boar [13,14] and human [15] spermatozoa. An advantage in freeze-drying is to store the spermatozoa without the use of liquid nitrogen for a long period.…”