Testicular sperm preservation and embryo development following intracytoplasmic sperm injection into ooplasm of domestic cats

Abstract

Testicular tissue is an important sperm source of dead or castrated animals. Optimal preservation of testicular tissue by cold storage and cryopreservation would be beneficial for future embryo production. This thesis composes of 4 parts as follows: EXP.1 aimed to examine the effect of cold storage on plasma membrane integrity, DNA integrity and fertilizing ability of testicular spermatozoa. Intact testes were cold stored in Dulbecco’s Buffered Saline (DPBS) at 4ºC for 1 - 7 days. Sperm membrane and DNA integrities were examined by Ethidium homodimer-1 and TUNEL staining, respectively. Spermatozoa from cold stored testes were injected into cat oocytes to evaluate their fertilizing ability, indicated by chromatin configuration of injected oocytes which were stained by DAPI at 18 h after injection. The data showed that, in the first 4 days, the number of spermatozoa with intact plasma membrane (alive spermatozoa) was comparable to non-preserved control (Day 0) (P > 0.05). After cold storage for 7 days, viability of preserved testicular spermatozoa remained up to approximately 50%, while the incidence of DNA fragmented spermatozoa was approimately1 %. However, duration of cold storage did not significantly impact on sperm DNA integrity and fertilizing ability (P > 0.05). EXP.2 aim to (1) compare the efficiency of DPBS and HEPES-containing medium (HM) to protect sperm membrane integrities during cold storage (2) examine the effects of bovine serum albumin (BSA) concentration on sperm membrane integrity during cold storage (3) examine the DNA integrity and fertilizing ability of spermatozoa cold stored within HM supplemented with 1.6 % BSA for 1 week. Spermatozoa were extracted before cold storage in DPBS and HEPES-contained medium (HM) for 7 days. Sperm plasma membrane was daily examined by Ethidium homodimer-1. The protective efficiencies of DPBS and HM on sperm membrane integrity were not significant different (P > 0.05), though HM provided the better result than DPBS. Testicular spermatozoa were then cold stored within HM supplemented with BSA at different concentrations (0.4, 0.8 and 1.6 %) for 1 week. The number of alive testicular spermatozoa from HM supplemented with 0.4 % BSA was lower than that of 0.8 % and 1.6 % through 1 week of storage. Due to HM with 1.6 % BSA gave the best result it was therefore selected to be a preserving medium for assessment of DNA integrity (by TUNEL assays) and in vitro embryo development after intracytoplasmic sperm injection (ICSI). Although DNA integrity of testicular spermatozoa stored in HM supplemented with 16% BSA significantly declined after cold stored for 7 days, the embryos produced by ICSI with chilled spermatozoa could develop in vitro to cleavage (44.7 %) morula (13.6 %) and blastocyst (7.3 %) stages at the similar rates with those of fresh spermatozoa (P > 0.05). EXP.3 aimed to (1) compare the protective efficiency on sperm membrane and DNA integrities of 4 different cryoprotectants including glycerol, ethylene glycol, 1, 2-propanediol and dimethyl sulphoxide (2) compare the effects of 2 different freezing techniques on sperm membrane and DNA integrities (3) determine gamete activation up to 18 h after ICSI with spermatozoa recovered from cryopreserved testicular tissues (4) evaluate in vitro development of embryos produced by ICSI with spermatozoa recovered from cryopreserved testicular tissues. The result revealed that at the same concentration of cryoprotectants, 5 % (v/v), testicular tissue cryopreservation by 2-step freezing with glycerol maintained the best sperm membrane integrity. This parameter was comparable to that of spermatozoa from non frozen tissues. Cryopreservation did not impact on sperm DNA integrity (irrespective of cryoprotectants and freezing techniques). Spermatozoa from frozen testicular tissues can fertilize cat oocytes without external oocyte activation. However, male and female activation occurred in an asynchronous and independent manner. The percentages of cleavage (32.7 %), morula (6.5 %) and blastocyst (4.4 %), and also the blastocyst cell number of embryos produced by ICSI with cryopreserved testicular spermatozoa were comparable to non-crypreserved control (P > 0.05). EXP.4 aimed to study in vitro and in vivo development of frozen cleaved embryos (2- to 8 cell stage) derived by ICSI with spermatozoa from frozen-thawed testicular tissues. The result revealed that ICSI with frozen-thawed testicular spermatozoa yielded the lower cleavage rate than conventional IVF. Frozen-thawed ICSI embryos developed to morula (22.6 %) and blastocyst (21.3 %) stage at the lower percentages than that of non-frozen ICSI embryos (45.2 and 38.7 %, respectively, P < 0.05). However, the blastocyst cell numbers of frozen and non-frozen ICSI embryos were not different. After transfer of frozen-thawed embryos produced by ICSI with cryopreserved testicular tissues into the oviducts of eCG/hCG treated recipient cats, 3 out of 7 queens were pregnant. Only one of these cats delivered 2 male kittens on day 64 after transfer. This study showed that cold storage and cryopreservation of testicular tissue could prolong storage time of spermatozoa for subsequent embryo production. Frozen embryos produced by ICSI with spermatozoa from cryopreserved testicular tissues can develop in vitro and in vivo. In addition, this is the first report of the births of kittens after transfer of frozen-thawed embryos produced by ICSI with sperm recovered from cryopreserved testicular tissues. This study will be the fundamental knowledge for development of gamete preservation and embryo production for endangered wild felid conservation.