Oocyte Cleavage Research Articles

379 EFFECT OF SUPPLEMENTARY INOSITOL 1,4,5-TRIPHOSPHATE INJECTION ON FERTILIZATION AND EMBRYO DEVELOPMENT AFTER BOVINE ICSI

During fertilization in mammals, sperm membrane-bound phospholipase C zeta induces breakdown of ooplasmic membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2), which leads to the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 induces intracellular Ca2+ oscillations ([Ca2+]i), which trigger inactivation of maturation-promoting factor (MPF) and mitogen-activated kinase (MAPK), resulting in decondensation of the sperm head, resumption of meiosis, extrusion of a second polar body, cortical granule exocytosis, formation of pronuclei (PN), and entry into the first cell cycle. In bovine ICSI, injection of a single spermatozoon into an oocyte does not consistently induce [Ca2+]i oscillations, as was observed in IVF, and this may, at least in part, explain the low rates of fertilization and embryo development. Although IP3 has been used as a powerful activator for nuclear transferred zygotes or parthenogenetic oocytes, few studies have evaluated the effect of IP3 injection on normal fertilization and embryo development after ICSI. The objective of this study was to determine the effect of injecting IP3 during bovine ICSI on fertilization and embryo development in vitro. Chemically defined media (CDM) were used throughout (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585–596). The injection volume of IP3, which was dissolved in calcium- and magnesium-free PBS, was approximately 0.01 nL, and the total dose for injection along with a spermatozoon was about 250 �M, which was determined by the 2PN formation rate in preliminary experiments. Semen from 3 bulls was used to produce embryos in 5 replicates. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22–23 h under 5% CO2 in air at 38.5�C, and oocytes with a first polar body were used for ICSI. Motile sperm from frozen–thawed semen were used for sperm injection, with or without IP3 in a 50-�L drop of GMOPS medium, with a piezo-driven pipet of 7–8 �m inner diameter. After ICSI, presumptive zygotes were cultured in CDM-1 for 3 days, and then in CDM-2 for 5 days at 39�C under 5% CO2/5% O2/90% N2. Cleavage and blastocyst development were evaluated at the end of each culture period. Data were subjected to Fisher's exact test. Cleavage in control and IP3 groups was 36.4 and 50.0%, respectively (P < 0.05). Respective blastocyst rates per oocyte were 5.5 and 13.0% (P > 0.05). This study showed that injection of IP3 during the sperm injection process improved cleavage of bovine oocytes after ICSI.

Fertilization and Development In Vitro of Bovine Oocytes Following Intracytoplasmic Injection of Heat-Dried Sperm Heads

This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120 degrees C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25 degrees C for 7-10 days following drying at 50 and 56 degrees C than at 90 and 120 degrees C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50 degrees C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7-10 days at 4 degrees C than 25 degrees C after drying at 50 degrees C for 8 h. Longer storage (7 days-12 mo) of heat-dried spermatozoa at 4 degrees C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.

Repeat Ovum Pick‐up and In Vitro Embryo Production from Adult Ewes with and without FSH Treatment

Ovum pick-up (OPU) was performed three times on adult ewes after synchronization with (n = 4) or without (n = 4) FSH treatment to investigate the effects of FSH treatment on the number of ovarian follicles, oocytes recovered, oocyte quality and development in vitro. FSH treatment increased the number of ovarian follicles (85 vs 162) and oocytes recovered (33 vs 91), although recovery rate was similar for ewes with and without FSH (91/162, 56.2% and 33/85, 38.8% respectively). Of the oocytes recovered, those classified as grades I and II were similar between ewes with (78/91, 85.7%) and without FSH treatment (27/33, 81.8%). The number of ovarian follicles was similar after repeated OPU for ewes treated with FSH, but for ewes not treated with FSH the number of ovarian follicles decreased with repeated OPU. The number of oocytes recovered decreased for FSH-treated ewes only, while the oocyte recovery rate and proportion of oocytes classified as grades I and II were not affected by repeated OPU. Oocyte cleavage (46/78, 58.9% and 19/24, 79.2%) and blastocyst formation (35/46, 76.1% and 12/19, 63.2% respectively) were similar for ewes with and without FSH treatment. The number of ovarian follicles varied between ewes (p < 0.05) although the number of oocytes recovered and oocyte development in vitro were similar between ewes.

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 μM ionomycin (5 min, Group 1) alone, ionomycin + 1.9 mM 6-dimethylaminopurine (DMAP, 3 h, Group 2), and ionomycin + 10 μg/ml cycloheximide (CHX, 3 h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater ( P < 0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19–22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater ( P < 0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater ( P < 0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed ( P < 0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.

Effect of methyl-β-cyclodextrin treatment of pig spermatozoa on in vitro fertilization and embryo development in the absence or presence of caffeine

A series of experiments were carried out to develop a new method to reduce pig polyspermic fertilization and produce more normal embryos, in vitro. Experiment 1 determined the effect of methyl-β-cyclodextrin (MCD) treatment during cryopreservation on sperm acrosome reaction and sperm fertilization. Compared to the non-MCD-treated control, MCD treatment increased the percentage of acrosome-reacted spermatozoa at thawing and 2 h after incubation in fertilization medium ( P < 0.01). Treatment with MCD also increased ( P < 0.05) sperm-penetration rate, number of spermatozoa in oocytes, and fertilization efficiency in the caffeine-free fertilization medium. Experiment 2 was designed to examine the effect of withdrawal of caffeine (caffeine-free) from fertilization medium on fertilization parameters and early embryo development. Using MCD-treated spermatozoa, there was no difference in sperm-penetration rate, oocyte cleavage rate, and blastocyst formation rate between the caffeine-free and caffeine-supplemented groups. However, polyspermic fertilization rate was lower, and fertilization efficiency and blastocyst cell number were higher in the caffeine-free group compared to the caffeine-supplemented group ( P < 0.05). Experiment 3 studied the effect of caffeine and different concentrations of spermatozoa on fertilization parameters. Sperm-penetration rate did not differ between the caffeine-free and the caffeine-supplemented groups at different sperm concentrations. Caffeine and sperm concentration had an effect on the number of spermatozoa in oocytes and on the polyspermic fertilization rate ( P < 0.002). Caffeine also affected fertilization efficiency ( P < 0.05). In conclusion, treating spermatozoa with MCD and withdrawing caffeine from fertilization medium may provide a new method to produce a large number of normal embryos, in vitro.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times ( P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation ( P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation ( P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased ( P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher ( P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.

304 PRODUCTION OF PORCINE EMBRYOS OF A PREDETERMINED SEX AFTER IN VITRO FERTILIZATION OF IN VITRO-MATURED OOCYTES WITH SEX-SORTED FROZEN-THAWED BOAR SPERM

Pre-sexed embryos and offspring have been produced after IVF and embryo transfer (ET) with sex-sorted frozen-thawed sperm in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95). The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a predetermined sex and that these embryos could be successfully nonsurgically transferred. Ovaries were collected from abattoir slaughtered gilts (n = 138) and selected COCs were matured in vitro (Long et al. 1999 Theriogenology 51, 1375–1390). Sperm were collected from a mature boar and diluted with Androhep (1:3, semen:Androhep; Minitube, Verona, WI, USA), stained with H33342, and separated into X and Y sperm using a SX MoFlo (Cytomation, Inc., Fort Collins, CO, USA). Sex-sorted sperm were cryopreserved in 0.5 mL straws using the Westendorf protocol modified for sorted sperm (Bathgate, unpublished). Thawed sperm (Y sperm only) were prepared for IVF by centrifugation (300g, 10 min) through a Porcipure gradient (Nidacon Int. AB, Gothenburg, Sweden), and washed (centrifugation 300g, 10 min) in mTALP-PVA. For IVF, COCs were denuded and groups of 100 oocytes were transferred to 200-μL drops of mTALP-PVA (Long et al. 1999) and incubated with 5,000 motile sperm for 4–6 (Short) or 18–20 h (Long) . Presumptive zygotes were washed and transferred to 100-μL drops of mNCSU-23 (Long et al. 1999) and cultured until Day 4 (Day 0 = IVF) in humidified 5% CO2, 6% O2, 89% N2. Oocyte cleavage was assessed 48 h post-insemination, and on Day 4 selected morulae were transferred to recipient sows (n = 7 Large White × Landrace; 65 morulae per sow) nonsurgically using a Firflex catheter (Magapor, Zaragoza, Spain). Sex of remaining embryos was confirmed by PCR and restriction analysis (Cong et al. 1993 Hum. Mol. Genet. 2 1187–1191). Data from three replicates were arc sin transformed and analyzed by ANOVA. Oocyte cleavage was similar after Short (724/1547; 46.8%) or Long (598/1528; 39.1%) co-incubation. Resort analysis showed sperm to be &gt;91% purity, and all sexed morulae were of the predicted sex (16/16). Delayed return to estrus (&gt;23 days) was observed in 5 recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine IVP and ET protocols are required to enable full in vivo development. This work was supported by XY, Inc., Fort Collins, CO, USA.

346 BENEFICIAL EFFECTS OF ACETYL-l-CARNITINE TREATMENT DURING IVM ON POST-FERTILIZATION DEVELOPMENT OF BOVINE OOCYTES IN VITRO

The lower competence of in vitro-matured oocytes for post-fertilization development is attributed to the lack of physiological factors in in vitro maturation (IVM) that regulate maturation events which occur exclusively in the cytoplasm of oocytes. It has been found recently that mitochondrial function plays an important role in regulation of oocyte developmental competence via metabolic regulation of energy production. Acetyl-l-carnitine (ALC) is known to enhance fatty acid oxidation and energy production in the mitochondria, and to exert enhancing effects on cellular proliferation and survival. In this experiment, we examined the effects of ALC on IVM and post-fertilization development of bovine oocytes. Cumulus-oocyte complexs (COCs) were aspirated from 2-5 mm follicles of ovaries from a slaughterhouse. COCs were cultured in IVM medium (mSOFaa+estradiol+hCG+BSA) with or without ALC (10 mM) for 24 h at 39�C under 5% CO2 in air, and then fertilized according to the conventional method. After 6 h of insemination, presumptive zygotes were freed from cumulus cells by repeated pipetting and cultured in mSOFaa with 1% FCS at 39�C under 5% CO2, 5% O2, and 90% N2. At 48 h post-fertilization, the rates of cleaved embryos were assessed. The cleaved embryos were transferred to mSOFaa with 5% FCS and cultured for additional 6 days at 39�C under 5% CO2, 5% O2, and 90% N2. The percentages of embryos developing to the blastcyst stage were assessed on Days 6, 7, and 8 (fertilization = Day 0), and the data were analyzed for statistically significant differences with the t-test. For examinination of mitochondrial organization in oocytes at different maturation stages, oocytes were stained for active mitochondria with MitoRed (1 �M in IVM medium for 2 h at 37�). When COCs were matured in medium without (control) or with ALC, although the rates of post-fertilization cleavage of oocytes were 60% to 70% despite the presence or absence of ALC, ALC significantly (P &lt; 0.05) increased the rates of cleaved embryos forming blastcysts on Days 6, 7, and 8 (30%, 36%, 40%) compared with those in the control (13%, 21%, 34%). We next examined effects of ALC treatment during IVM on active mitochondria distribution in oocytes. In 75% of immature oocytes, active mitochondria localized in the periphery of the oocytes (peripheral type). After 24 h of IVM without ALC, while 17% of oocytes remained in a peripheral type, 44% showed some migration of active mitochondria toward the center of the oocytes (semiperipheral type) and 39% presented a diffused distribution of active mitochondria in the whole oocyte cytoplasm (diffused type). On the other hand, in ALC treated oocytes, 60% of the oocytes presented a diffused type, 25% exhibited a semiperipheral type, and 15% had still maintained a peripheral distribution. These results provide the first evidence that ALC treatment during IVM of bovine oocytes enhances their post-fertilization development to the blastcyst stage and enhances the frequency of oocytes that exhibit an extensive relocation (diffused type) of active mitochondria to the inner oocyte cytoplasm.

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P &gt; 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P &lt; 0.05) and declined faster (P &lt; 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P &lt; 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P &gt; 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0-93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro , and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 +/- 2.8 blastocysts per lamb.

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex‐sorted Frozen‐thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.

Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes

Objective: To evaluate the effects of zona pellucida changes following the cryopreservation of bovine oocytes using two different techniques. Design: Prospective randomised study. Setting: University research facility and IVF research laboratory, NURTURE, University of Nottingham, UK. Intervention: 508 bovine oocytes were collected from abattoir-derived ovaries and cryopreserved by slow freezing using standard straws and a new vitrification method using our self-constructed cryoloops. After thawing, the oocytes were inseminated by ICSI and standard IVF. Main outcome measures: Oocyte degeneration and oocyte cleavage. Results: Cleavage rates after IVF in fresh oocytes was higher (49.5%) than the cryopreservation groups (slow freezing: 15.4% and vitrification: 25.8%), whereas after ICSI, the rates were similar (fresh: 17.3%, slow freezing: 9.4%, vitrification: 16.0%). Conclusions: Zona pellucida changes after oocyte cryopreservation significantly reduce the embryo formation rates after IVF. ICSI bypasses this complication and in selected species it could be an appropriate method of insemination.

Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection

The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 μM ionomycin for 5 min (ionomycin); (2) exposure to 5 μM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3 h culture in 1.9 mM 6-dimethylaminopurine (DMAP – ionomycin + DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin + DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin + DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly ( P < 0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin + DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher ( P < 0.05) than those of the ionomycin + DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin + DMAP for the production of viable blastocysts and calves.

Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA.

The transcription factor TFIIA is encoded by two genes, TFIIAalphabeta and TFIIAgamma. In higher eukaryotes, the TFIIAalphabeta is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAbeta subunit starts at Asp 278. We found that a cleavage recognition site (CRS), a string of amino acids QVDG at positions -6 to -3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAbeta greatly affect degradation of the beta-subunit. We propose that cleavage and subsequent degradation fine-tune the amount of TFIIA in the cell and consequently the level of transcription.

Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development.

Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.

Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22–24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 μg/ml and three sperm concentrations: 0.5×10 6, 1.5×10 6, and 4.5×10 6 ml −1; 10 μg/ml of heparin and 1.5×10 6 ml −1 of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52–54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18–20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased ( P<0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm ( P<0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5×10 6 ml −1) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 μg/ml) and sperm concentrations (4.5×10 6 ml −1). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility.

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.

Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats.

We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, androgen-binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke-exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.