Estrous Sheep Serum Research Articles

Serum supplementation during in vitro fertilization of sheep oocytes influences blastocyst quality through the differential abundance of mRNA transcripts.

Incubation with estrous sheep serum (ESS) is required to induce in vitro capacitation of spermatozoa during in vitro fertilization of small ruminants. However, the effect of adding different serum concentrations in the fertilization media on the quality of resulting blastocysts has not yet been studied. Here, 298 sheep oocytes were co-incubated with capacitated spermatozoa with either 10% or 2% ESS. There were no differences between treatments in cleavage (10% ESS: 63.81 ± 5.87% and 2% ESS: 45.31 ± 5.87%) and blastocyst rates (10% ESS: 20.83 ± 2.12% and 2% ESS: 15.93 ± 2.12%). Nonetheless, in vitro-produced blastocysts from the 10% ESS treatment showed a higher transcript abundance of mRNAs involved in apoptosis (ITM2B and BCL2), antioxidant defence (GPX1) and growth-related imprinting (IGF2R). Our data suggest that ESS supplementation during in vitro fertilization can influence the quality of sheep embryos at later stages of development by increasing the transcription of developmentally important genes.

Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes.

High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets’ content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3–7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1+ and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase− spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Effect of different media additives on capacitation of frozen–thawed ram spermatozoa as a potential replacement for estrous sheep serum

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin–hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin–hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen–thawed ram spermatozoa. Only the medium supplemented with heparin–hypotaurine–BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin–hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.

Blastocyst rate of in vitro embryo production in sheep is affected by season

It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes.

Treatment of ovine oocytes with certain water-soluble vitamins during in vitro maturation (IVM)

The main aim of this study was to evaluate the effects of water-soluble vitamins (singly or in combination), on in vitro maturation (IVM) and subsequent embryonic development of sheep oocytes. Cumulus oocyte complexes (COCs) were evaluated in three experiments. Experiment 1: COCs were matured in modified synthetic oviduct fluid maturation medium (mSOFM), with or without various protein supplements (polyvinyl alcohol (PVA)+mSOFM: defined maturation medium, DMM; BSA+mSOFM: semi-defined maturation medium; SDMM and FCS+mSOFM: undefined maturation medium; UDMM). Experiment 2: COCs were matured in mSOFM with or without various protein supplements containing 1× minimal essential medium (MEM) vitamins. Experiment 3: COCs were matured in SDMM with the addition of certain water-soluble vitamins (pantothenate, riboflavin, and thiamin), singly or in combination. The COCs were co-incubated with epididymal sperm of rams in SOFF (synthetic oviduct fluid fertilization) media, with 10% heat-inactivated estrous sheep serum for 22–24h. Embryos were cultured in a SOFC1 (culture 1) medium for 48h, followed by culture in a SOFC2 (culture 2) medium for 6 days. The addition of BSA-V to mSOFM (SDMM) improved the mean blastocyst formation, compared to other treatments. The presence of 1× MEM vitamins in SDMM increased the blastocyst formation rate, compared with DMM, UDMM, and mSOFM. The presence of single vitamin (pantothenate) in the SDMM improved blastocyst rate formation, compared with SDMM+thiamin; SDMM+pantothenate plus thiamin; SDMM+riboflavin plus thiamin; and SDMM+all three vitamins together. This is the first report to show the stimulatory effect of certain single vitamins on the in vitro development of matured ovine oocytes.

334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytes Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01

Laparoscopic ovum pick-up and in vitro production of sika deer embryos: Effect of season and culture conditions

Amongst the 200 deer subspecies worldwide, more than 40 are considered as endangered. In vitro embryo production may represent an efficient way to produce and disseminate offspring from sparse remaining individuals in these species. With a view to establishing a method of in vitro embryo production, we assessed the ovarian response after hormonal stimulation (oFSH), oocyte yield following laporoscopic ovum pick-up (LOPU) and oocyte developmental competence according to seasonal reproductive status in sika deer ( Cervus nippon nippon). Twelve adult sika deer hinds were allocated between two groups and submitted weekly to oFSH follicular growth stimulation followed by LOPU. Hinds in Group A ( n = 6) were treated first during the breeding season (5 weeks), and then during the non-breeding season (3 weeks). Hinds in Group B ( n = 6) were submitted to similar procedures but in the reverse order (treated first during the non-breeding season). Cumulus–oocytes complexes (COC) recovered from Group B were allowed to mature in vitro for 24 h in TCM-199 medium supplemented with oFSH, goat follicular fluid and 100 μM cysteamine. In vitro fertilization was performed with frozen/thawed semen in SOFaa medium supplemented with 20% estrous sheep serum and presumptive zygotes were cultured in the presence or absence of ovine oviductal epithelial cell monolayer (oOEC) in SOFaa–BSA medium. Mean number of follicles aspirated per hind per session decreased significantly between breeding and non-breeding season in Group A (9.8 ± 0.7 versus 3.2 ± 0.7, mean ± S.E.M., respectively, P < 0.001) but did not change between the non-breeding and the subsequent breeding season in Group B (5.3 ± 0.7 and 5.7 ± 0.7, respectively, P > 0.05). Irrespective of the season, good quality COC with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Whereas development to the blastocyst stage did not occur in SOF medium alone, high development rates to the blastocyst stage were observed in oOEC co-culture regardless of season (22% and 34% of total oocytes in co-culture during non-breeding and breeding season, respectively).

115 RELATIONSHIP BETWEEN IN VIVO FERTILITY OF MANCHEGA EWES LAPAROSCOPICALLY INSEMINATED WITH FROZEN - THAWED RAM SEMEN AND HETEROLOGOUS CALF IN VITRO FERTILIZATION

Cryopreservation may damage the fertilizing ability of spermatozoa. For this reason, it is important to assess the sperm before artificial insemination. In vitro fertilization (IVF) tests are the most suitable for assessing overall sperm function during fertilization. The aim of this work was to study whether a relationship exists between heterologous calf in vitro fertilization and in vivo fertility in Manchega sheep, with the purpose of predicting in vivo fertility of males before using their semen. Frozen–thawed sperm from 4 Manchega rams was used to laparoscopically inseminate 481 ewes of the same breed. Sperm was cryopreserved in a TRIS-yolk-glycerol extender. A minimum of 10 females were laparoscopicly inseminated per ram. These same straws were used for IVF (4 replicates per male). Domestic calf ovaries were collected at a slaughterhouse. Inmature cumulus–oocyte complexes (COCs) were aspirated and matured in vitro in TCM-199 with 10 ng mL-1 EGF and 100 �M cysteamine. After 24 h, zona-intact mature oocytes were incubated in synthetic oviduct fluid supplemented with 10% estrous sheep serum. Thawed spermatozoa were co-incubated with the oocytes (one million per well) for 40 h, and the cleavage rate was asessed. A regresion analysis was performed. The in vivo fertility ranged from 25.00 to 62.50%. Two rams had a fertility under 30.00% and the other ones over 55.00%. The in vitro fertility ranged from 42.50 to 58.50%. The in vivo fertility was not related to the in vitro fertility (P = 0.17). This could be due to the low number of males used in this work. Heterologous calf in vitro fertilization tests cannot be used to predict in vivo fertility of ram semen since no relationship was found between both variables. Nevertheless, these results are preliminary and we are working with more rams and replicates to obtain more information. This work was funded by INIA and Consejeria de Agricultura de Castilla-La Mancha. Garcia-Alvarez enjoyed a studentship from the INIA

In vitro maturation of caprine oocytes in different culture media

Oocytes were aspirated from caprine ovaries, washed and cultured in TCM-199 containing penicillin, streptomycin and 10, 15, or 20% of fetal bovine serum (FBS), estrous sheep serum (ESS) or estrous goat serum (EGS). After 24–26 h culture, oocytes were separated from cumulus and corona cells by hyaluronidase and by passing through a fine pipette, fixed in acetic alcohol, stained with aceto orcein and observed under a phase-contrast microscope for evidence of maturation. High maturation rates (74–94%) were observed in all concentrations of the three different sera examined. No significant difference was observed between different concentrations and among different sera. Almost no maturation (4%) was observed in the medium lacking protein supplement. In conclusion, these sera with the concentrations examined can be substituted for one another for in vitro maturation of caprine oocytes.

Dose-dependent effect of heparin on fertilizing ability of goat spermatozoa

Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 °C in 5% CO 2 in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes.

Binding of oviductal specific proteins to the membrane of bovine spermatozoa

Amongst the 200 deer subspecies worldwide, more than 40 are considered as endangered. In vitro embryo production may represent an efficient way to produce and disseminate offspring from sparse remaining individuals in these species. With a view to establishing a method of in vitro embryo production, we assessed the ovarian response after hormonal stimulation (oFSH), oocyte yield following laporoscopic ovum pick-up (LOPU) and oocyte developmental competence according to seasonal reproductive status in sika deer (Cervus nippon nippon). Twelve adult sika deer hinds were allocated between two groups and submitted weekly to oFSH follicular growth stimulation followed by LOPU. Hinds in Group A (n = 6) were treated first during the breeding season (5 weeks), and then during the non-breeding season (3 weeks). Hinds in Group B (n = 6) were submitted to similar procedures but in the reverse order (treated first during the non-breeding season). Cumulus–oocytes complexes (COC) recovered from Group B were allowed to mature in vitro for 24 h in TCM-199 medium supplemented with oFSH, goat follicular fluid and 100 μM cysteamine. In vitro fertilization was performed with frozen/thawed semen in SOFaa medium supplemented with 20% estrous sheep serum and presumptive zygotes were cultured in the presence or absence of ovine oviductal epithelial cell monolayer (oOEC) in SOFaa–BSA medium. Mean number of follicles aspirated per hind per session decreased significantly between breeding and non-breeding season in Group A (9.8 ± 0.7 versus 3.2 ± 0.7, mean ± S.E.M., respectively, P < 0.001) but did not change between the non-breeding and the subsequent breeding season in Group B (5.3 ± 0.7 and 5.7 ± 0.7, respectively, P > 0.05). Irrespective of the season, good quality COC with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Whereas development to the blastocyst stage did not occur in SOF medium alone, high development rates to the blastocyst stage were observed in oOEC co-culture regardless of season (22% and 34% of total oocytes in co-culture during non-breeding and breeding season, respectively).

Estrous sheep serum as a potent agent for ovine IVF: Effect on cholesterol efflux from spermatozoa and the acrosome reaction

The aim of the present study was to evaluate the effect of heat-inactivated estrous sheep serum (ESS) on sheep IVF. When the capacitation and the fertilization media contained 20% ESS, a fertilization rate of 85% was achieved. The beneficial effect of ESS on sheep IVF was further demonstrated since the fertilization rate was null when ESS was omitted during sperm capacitation and fertilization. Estrous sheep serum supported both sperm capacitation and fertilization as shown by the results of experiments in which it was omitted during one of these steps: sperm capacitation in serum-free medium resulted in delayed sperm-oocyte penetration, while fertilization in serum-free medium significantly decreased the percentage of fertilized oocytes. To investigate the influence of serum on sperm ability to undergo the acrosome reaction, salt-stored follicular sheep oocytes were inseminated, and the acrosomal status of spermatozoa attached to zonae was examined by electron microscopy after a 4-h period of coincubation. Quantitative analysis on thin sections demonstrated that fewer acrosome-reacted spermatozoa were observed when the capacitation and insemination steps were carried out in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS (0.08, [0; 0.34], (median, range)/100μm zona vs 1.32, [0.90; 2.28]/100μm zona; P < 0.01). Since a higher number of spermatozoa attached to the zona surface in DM-H medium, the proportion of acrosome-reacted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 54%, [25%; 100%]; P < 0.01) in the absence of serum. These results indicate that in our IVF system the development of the acrosome reaction depended on serum. Sperm cholesterol efflux during in vitro capacitation was measured on [ 3H] cholesterol labeled spermatozoa resuspended in DM-H or DM-H-SS medium. A time-dependent cholesterol removal was observed in the presence of serum (60 ± 5%, mean ± SD, after 5 h), whereas it was limited to 14 ± 3 % in DM-H medium; hence addition of serum to the capacitation medium efficiently supports cholesterol efflux, which is thought to be a key-event in the capacitation process.

Assessment of fertilizing ability of goat spermatozoa by in vitro fertilization of cattle and sheep intact oocytes

Intacy oocytes of cattle and sheep were used to study factors that affect the in vitro fertilizing (IVF) ability of goat spermatozoa in vitro. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature and spermatozoa were then resuspended in medium Talp plus serum, and were incubated further for 1 h at 39°C in 5% CO 2 in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until oocytes were matured. In Experiment 1, the pattern of sperm penetration in matured cattle, sheep and goat oocytes was studied using either residual or standard experimental levels of calcium in the IVF medium. In Experiment 2, the same pattern was studied now by evaluating the sperm penetration rate in cattle oocytes under increasing concentrations of heparin in the IVF medium and then comparing them with those obtained using sheep and goat oocytes. As in Experiment 2, in Experiment 3 the pattern of sperm penetration was studied using increasing concentrations of caffein in the IVF medium. Gametes were incubated for 18 h, and penetration rates were assessed by the presence of pronuclei and sperm tail in the oocyte cytoplasm. The results indicate that 1) the sperm penetration rate in intact cattle, sheep and goat oocytes follows a pathway that depends on calcium; 2) heparin improves the sperm penetration rate in cattle, sheep and goat oocytes following a comparable pattern; and 3) caffeine depresses the sperm penetration rate in cattle, sheep and goat oocytes, but the pattern of inhibition depends on the genetic origin of the oocytes. The results also suggest that cattle and sheep intact oocytes can be used to assess the fertilizing ability of goat spermatozoa.