Domestic Cat Oocytes Research Articles

91IMPACT OF PROPYLENE GLYCOL V. ETHYLENE GLYCOL ON OOCYTE MATURATION, SPINDLE INTEGRITY, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO IN THE DOMESTIC CAT

A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25°C or 0°C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5°C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135–141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti-α-tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25°C) or low (1.5M PrOH at 25°C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5×105 motile sperm mL−1 ) in Ham’s F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey’s multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P<0.05) in 3.0M PrOH at 0°C (29.3±8.3%; mean±SD), 3.0M EG at 25°C (33.7±8.9%), and 0°C (29.4±11.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P<0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.8±8.6%), except 1.5M PrOH at 25°C (20.7±10.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P<0.05) in 0.75M PrOH (32.1±4.1%), 1.5M EG (33.4±4.0%), and 3.0M PrOH (29.3±3.8%) compared to control (50.1±4.0%) or 1.5M PrOH (50.6±4.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P<0.05) in 1.5M EG (16.5±7.4%) and 3.0M PrOH (14.9±7.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25°C (32.5±7.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25°C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

Development of in vitro matured, in vitro fertilized domestic cat embryos following cryopreservation, culture and transfer

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 °C in 5% O 2, 5% CO 2, 90% N 2 for 24 h. In Experiment 1, after IVM/IVF, on Day 2 ( n=56), Day 4 ( n=48) and Day 5 ( n=42) of IVC, embryos were equilibrated for 10 min at 22 °C in HEPES (15 mM) Tyrode’s (HeTy) with 1.4 M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 °C/min to −6.0 °C and held for 10 min. After seeding, cooling resumed at 0.3 °C/min to −30 °C and after a 10 min hold, straws were plunged into liquid nitrogen (LN 2). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (±S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152±19, Day 4=124±20, Day 5=121±24) than in controls (Day 2=141±25, Day 4=169±21, Day 5=172±19). In Experiment 2, embryos frozen on Day 2 ( n=68), Day 4 ( n=49) or Day 5 ( n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6±2.4), 4 (mean=12.2±3.7) and 6 (mean=12.0±1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.

Timing of nuclear maturation of nonstored and stored domestic cat oocytes

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O2 atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10°C for 16–24h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O2 atmosphere (a humidified gas atmosphere of either 5% CO2 in air or 5% CO2:5% O2:90% N2) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17–18, 20–21, 24–26, 28–30, 33–34 or 42–45h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P>0.05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO2 in air versus 5% CO2:5% O2:90% N2) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17–18h of culture was 36.1% and did not significantly increase (P>0.05) over time up to 28h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42–45h of culture. Therefore, we conclude that two “waves” of nuclear maturation of cat oocytes can be distinguished. The first wave takes place within 26h and it is likely that most oocytes of this wave mature by 17–18h; the second wave occurs after 28–30h of IVM. It can be assumed that this double wave may reflect the presence of two oocyte populations with two different degrees of “prematuration” which require different lengths of IVM.

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus–oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco’s modified Eagle medium (DMEM) alone as a control group or supplemented with 100ng/ml IGF-I. After culturing for 32–34h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.

Gonadotropin exposure, salt storage and storage duration affect penetration of domestic cat oocytes by homologous spermatozoa

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2–3 weeks) immature oocytes, or salt-stored (2–3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2–3 weeks, 2–3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2–3 weeks of storage than after 2–3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2–3 weeks or 2–3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.

In vitro maturation, fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat’s reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 °C for a short period (1–6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus–oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage ( P<0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected ( P>0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower ( P<0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 °C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts.

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of ICSI nonactivated (106/129, 82.2% vs 42/102, 41.2%). After 7 d in vitro culture, the frequency of in vitro development of cleaved embryos to morula stage was higher (P<0.001) in the ICSI activated group (37/106, 34.9%) that the ICSI nonactivated (2/102, 4.8%), while the development to blastocyst was not different (0 vs 7/106, 6.6%; P=0.088). We conclude that a higher percentage of oocytes will undergo meiotic maturation in SOF than in TCM 199. We also conclude that embryos can be produced with ICSI of in vitro matured oocytes using frozen-thawed epididymal cat semen.

Development of embryos produced by intracytoplasmic sperm injection of domestic cat oocytes

Development of embryos produced by intracytoplasmic sperm injection of domestic cat oocytes

Development to blastocysts of domestic cat oocytes matured and fertilized in vitro after prolonged cold storage.

Four experiments determined the kinetics of in vitro maturation and fertilization of cat oocytes and the effects of prolonged cold storage of ovaries before oocyte recovery on in vitro maturation/in vitro fertilization (IVM/IVF) success. Domestic cat ovaries were collected at ovariohysterectomy and stored at 4 degrees C in PBS until oocyte recovery and culture in Eagle's minimal essential medium (EMEM) containing FSH, LH, oestradiol and BSA for maturation. In Expt 1, meiotic maturation was assessed at 0, 12, 24, 38 and 48 h of culture. After 24 h, > 61% of oocytes were in telophase I or metaphase II. In Expt 2, oocytes were recovered from ovaries stored for 24, 48 or 72 h and cultured in EMEM for 24 h. There was no difference among groups (P > 0.05) in the ability to achieve nuclear maturation (mean +/- SEM, 57.1 +/- 5.3%, 60.4 +/- 5.4%, 55.4 +/- 15.1% for 24, 48 and 72 h, respectively). Fertilization and embryo development after insemination at 16, 24, 32, 40 and 48 h of culture were examined in Expt 3. Of 98 oocytes inseminated at 32 h, 69% cleaved, 59% developed into morulae and 13% into blastocysts, more (P < 0.05) than those oocytes inseminated at earlier and later times. Development to blastocysts occurred after insemination at 16 (1.2%), 24 (9.1%) and 32 (13.3%) h of culture, but not after insemination at 40 or 48 h. Expt 4 involved cold storage of ovaries for 24, 48 or 72 h before oocyte recovery and insemination at 32 h of culture (the optimal time measured in Expt 3). Compared with storage for 24 h, fertilization success was lower (P < 0.05) in the 48 and 72 h groups, and, although 9.1% of inseminated oocytes from the 24 h storage group developed to blastocysts, none (P < 0.05) achieved this stage after 48 or 72 h of storage. These results indicate that domestic cat oocytes reach nuclear maturity by 24 h in culture and can be fertilized and develop to blastocysts optimally after insemination at 32 h. Oocytes recovered from ovaries stored at 4 degrees C for up to 72 h are capable of reaching telophase I or metaphase II in vitro. However, only oocytes stored within the ovary for 24 h produce blastocysts, indicating that the ability to achieve nuclear maturation is an inadequate indicator of fertilization and developmental competence.

Enhancing zona penetration by spermatozoa from a teratospermic species, the cheetah (Acinonyx jubatus).

Cheetahs (Acinonyx jubatus) produce poor quality ejaculates that can limit the efficiency of standard assisted reproduction including artificial insemination (AI) and in vitro fertilization (IVF). The purpose of this study was to: (1) further study sperm-oocyte interaction in this teratospermic species by examining the ability of malformed sperm to interact with various oocyte barriers; and (2) assess the potential of zona piercing for assisting IVF in a teratospermic felid. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Semen was collected by electroejaculation from six male cheetahs and ejaculates were processed for IVF. Sperm aliquots from each ejaculate were assessed for a sperm motility index (SMI) over time. Zona-intact (ZnIn-SS) oocytes (n = 78) and ZnPd-SS oocytes (n = 74) were coincubated with spermatozoa in vitro for 6 h. The proportion of morphologically abnormal spermatozoa per ejaculate was high for all males (range 81.5% to 95.9%). SMI values at 0 and 6 h were variable, ranging from 50 to 75 and 0 to 40, respectively. Spermatozoa from all ejaculates bound to and penetrated the outer zona pellucida of ZnIn-SS and ZnPd-SS oocytes similarly (P > 0.05). The proportion of oocytes containing spermatozoa within the inner zona layer and the average number of spermatozoa per oocyte in this region were greater (P < 0.05) for the ZnPd-SS than ZnIn-SS oocytes (39.2% and 1.0 versus 12.8% and 0.2, respectively). Although zona piercing enhanced sperm penetration, there was no increase (P > 0.05) in pleiomorphic spermatozoa penetrating the inner zona pellucida or PVS.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

In vitro and in vivo development of domestic cat oocytes following intracytoplasmic sperm injection or subzonal insemination

Objective: To study the fusiogenic ability of human spermatozoa and oocytes.Design: Retrospective study of 3,027 oocytes included in a program of subzonal insemination (SUZI).Setting: Assisted fertilization program in an academic research environment.Patient(s): Couples with characterized male factor infertility or previous unexplained IVF failures.Intervention(s): Subzonal insemination (SUZI) was performed after study of the sperm pathology. The number of microinjected spermatozoa was controlled.Main Outcome Measure(s): Fertilization and polyspermia rates were analyzed according to the number of microinjected spermatozoa and to the indication of SUZI.Result(s): The fertilization rate increased linearly between one and three microinjected spermatozoa. Above this number, the rate plateaued around 25%. Polyspermia was correlated with the number of microinjected spermatozoa (r = 0.97). The fusiogenic ability of motile sperm cells was dependent on the semen characteristics and the sperm pathology. Observed diploid and polyspermia rates did not differ from the calculated probability of microinjecting only one or at least one fertilizing spermatozoon into the perivitelline space, respectively.Conclusion(s): Data support the hypothesis that a physiologic block at the human oolemma is absent. The post-SUZI fertilization rate also can be explained by the probability of finding one fertilizing spermatozoon among those that were microinjected and by the limited number of sperm heads allowed to decondense in the ooplasm.

Zona pellucida piercing enhances zona penetration by spermatozoa from normospermic and teratospermic domestic cats.

Poor quality ejaculates appear associated with reduced zona penetration and poor fertilization in vitro in some felid species or populations. Even morphologically normal sperm from these teratospermic ejaculates are compromised in ability to penetrate the zona pellucida and fertilize oocytes. A domestic cat model and zona piercing were used to study the function of normal and malformed sperm. Male cats naturally producing different proportions of morphologically normal sperm (group I, > 60%; group II, 45-55%; group III, < 40%) were identified. Electroejaculates were subjected to sperm washing (WS) and half of these to swim-up (SU) processing following WS, and all samples were assessed for a sperm motility index (SMI) over time. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Control (nonmanipulated) SS oocytes (n = 430) and ZnPd-SS oocytes (n = 430) were coincubated with sperm in vitro. The proportion of morphologically normal sperm/ejaculate differed (P < 0.05) among groups averaging 68.8% for group I, 53.5% for group II, and 34.7% for group III. After SU processing, the percent normal sperm among groups was similar (P > 0.05; range, 70.8-80.2%). Average SMI for all groups was high (> 70) at 0 hour and decreased over time, but the descent of slope was greater (P < 0.05) for group III than groups I and II. The proportion of oocytes penetrated was not influenced (P > 0.05) by sperm-processing treatment. For all groups, inner zona penetration was greater (P < 0.05) in ZnPd-SS than SS oocytes (group I, 52.3% vs. 34.9%; group II, 37.9% vs. 20.3%; group III, 32.0% vs. 17.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17β and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.

Pregnancy rate and survival in culture of bovine IVF embryos frozen in various cryoprotectants and thawed in a one step system

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 °C in 5% O2, 5% CO2, 90% N2 for 24 h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 °C in HEPES (15 mM) Tyrode’s (HeTy) with 1.4 M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 °C/min to −6.0 °C and held for 10 min. After seeding, cooling resumed at 0.3 °C/min to −30 °C and after a 10 min hold, straws were plunged into liquid nitrogen (LN2). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (±S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152±19, Day 4=124±20, Day 5=121±24) than in controls (Day 2=141±25, Day 4=169±21, Day 5=172±19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6±2.4), 4 (mean=12.2±3.7) and 6 (mean=12.0±1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.

Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay.

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4 degrees C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 x 10(6) sperm/ml) were preincubated for 1.0 h (38 degrees C, 5% CO2 in air) and then co-incubated (2 x 10(5) sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P greater than 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 +/- 5.4%; leopard cat, 38.6 +/- 2.8%) or stored ZP (domestic cat, 32.4 +/- 4.2%; leopard cat, 27.6 +/- 2.3%). Sperm incubated in protein-free medium (TLP-PVA) were less capable (P less than 0.05) of ZP penetration (domestic cat, 14.6 +/- 5.9%; leopard cat, 7.9 +/- 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 +/- 5.9%; leopard cat, 58.4 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)