A comparison of 1- and 2-cell ova production by F2 50% Meishan versus F1 White line gilts

The objective of this study was to compare recovery of pronuclear and 2-cell ova from F2 50% Meishan (MX) gilts versus F1 White line (L42) gilts. Sexually mature MX and L42 gilts were allocated across 2 treatments: Super (MX:n=9; L42:n=10) and Control (MX:n=6; L42:n=5) in a 2 x 2 factorial experiment. Allyl trenbolone (AT) was used to synchronize estrus in all gilts. Super gilts were given pregnant mare serum gonadotropin (PMSG: 1250 IU) at 24 h after AT withdrawal. Eighty-five hours after PMSG administration, all Super gilts received 750 IU of human chorionic gonadotropin (hCG). Super gilts which exhibited estrus within 24 h of hCG administration (MX-Super: n=6; L42-Super: n=5) and all Control gilts were bred naturally to Line 3 boars at 12 and 24 hours after the onset of estrus. Ova were recovered from Super gilts between 60 and 64 h after hCG and Control gilts at 48 h after the onset of estrus. All 1- and 2-cell ova were centrifuged at 15000 x g and observed using differential interference contrast microscopy. The mean ovulation rate was greater (P<0.05) for both MX-Super and L42-Super gilts in comparison to their respective Control groups. No differences were detected in the mean ovulation rate (P>0.38) or the mean number of 1- and 2-cell ova recovered (P>0.50) between MX-Super and L42-Super gilts. The proportion of 1- and 2-cell ova which exhibited visible pronuclei or nuclei was also similar among MX-SUPER and L42-SUPER gilts. This study demonstrates that MX gilts respond/perform comparably to L42 gilts with respect to estrus synchronization, superovulation, ova yield, and the ease of visibility of pronuclei or nuclei in the ova.     

Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Development potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.     

Effects of Zeranol upon luteal maintenance and fetal development in peripubertal gilts

Eighty gilts were utilized to determine whether zeranol implants could maintain hCG-induced corpora lutea (CL) in peripubertal gilts and to examine the effects of a Zeranol implant on fetal development. Crossbred gilts (171+/-0.3 days of age, 109.1+1.4 kg) were blocked by weight and ancestry to control (n=40) or treatment (n=40) groups. To induce ovulation and CL maintenance, treated gilts received 500 IU of hCG i.m. and a Zeranol ear implant (Ralgro, 36 mg; day 0). All gilts were checked once daily for estrus with a mature boar from days 3-58 of the experiment. On day 42, treated gilts received two 10 mg injections of Lutalyse (PGF(2)alpha) spaced 6 h apart. Treated gilts not displaying estrus within 7 days of PGF(2)alpha received two additional 10 mg of PGF(2)alpha spaced 6 h apart on day 49. On days 44-58, gilts detected in estrus were inseminated twice, 24 h apart with pooled semen via AI. Blood samples were obtained on days 0, 7, 18 and 42 and analyzed for serum progesterone (P(4)). Bred gilts were slaughtered on days 58-62 of gestation. Ovulation, as determined by serum concentrations of P(4) on day 7 of the experiment, was induced by hCG in 79.5% of treated gilts. Zeranol implants, however, failed to increase (P>0.05) the proportion of gilts available for breeding (treated, 21/39; control, 18/40). Of gilts inseminated on days 44-58, 16/21 treated gilts and 16/18 control gilts were pregnant at slaughter on days 58-62 of gestation. Number of fetuses (7.5 versus 12), fetal weight (83 versus 121 g), fetal length (117 versus 132 mm) and fetal survival (45% versus 78%) were reduced (P<0.001) by Zeranol implants. These data indicate that treatment of peripubertal gilts with a 36 mg Zeranol implant did not increase the proportion of gilts available for breeding while causing deleterious effects upon the fetuses.     

Morphology of porcine cumulus-oocyte-complexes depends on the stage of preovulatory maturation

The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5% (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8% of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4%; P < 0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7% of COCs with expanded cumulus; P < 0.01), and 34 h after hCG, 98.3% of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2%, respectively, n = 238 oocytes; P < 0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs--88.9% diplotene, expanded COCs--53.8% metaphase II (M-II), and denuded oocytes--69.2% degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG.     

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii)

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Premature elevation of progesterone shortens duration of ovulation in PMSG/hCG-treated prepuberal gilts

A surge of LH during the follicular phase triggers multiple pathways, including progesterone and prostaglandin synthesis before culminating in ovulation. Progesterone has been shown to be involved in the ovulatory process in many species. In prepuberal gilts treated with PMSG/hCG the follicular progesterone level has been shown to increase sharply before ovulation. This study was conducted to investigate whether premature elevation of progesterone can accelerate the ovulatory process in Large White PMSG/hCG-treated prepuberal gilts. Fifty-four Large White gilts were treated with 1000 IU, i.m. PMSG to stimulate follicular growth, followed 72 h later by 500 IU, i.m. hCG to induce ovulation. Gilts in the treatment group (n = 27) were given progesterone intermuscularly at 24 and 36 h after hCG. Ovaries were exteriorized to observe ovulation points during laparotomy under general anesthesia at 38 to 50 h after hCG. Ovulation in both groups commenced by 40.05 h after hCG and was completed by 47.71 h in the control group and by 42.87 h after hCG in the treated group. Progesterone shortened (P < 0.01) ovulation time by 4.84 h and the time required (P < 0.01) for the median proportion of follicles to ovulate (40.7 vs 43.5 h after hCG). Progesterone also increased (P < 0.01) the plasma progesterone concentration without altering follicular progesterone concentration.     

Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes

The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.     

The impact of dose of FSH (Folltropin) containing LH (Lutropin) on follicular development, estrus and ovulation responses in prepubertal gilts

FSH is favored over chorionic gonadotropins for induction of estrus in various species, yet little data are available for its effects on follicle development and fertility for use in pigs. For Experiment 1, prepubertal gilts (n = 36) received saline, 100 mg FSH, or FSH with 0.5 mg LH. Treatments were divided into six injections given every 8 h on Days 0 and 1. Proportions of gilts developing medium follicles were increased for FSH and FSH-LH (P < 0.05) compared to saline, but follicles were not sustained and fewer hormone-treated gilts developed large follicles (P < 0.05). No gilts expressed estrus and few ovulated. Experiment 2 tested FSH preparations with greater LH content. Prepubertal gilts (n = 56) received saline, FSH-hCG (100 mg FSH with 200 IU hCG), FSH-LH5 (FSH with 5 mg LH), FSH-LH10 (FSH with 10 mg LH), or FSH-LH20 (FSH with 20 mg LH). FSH-LH was administered as previously described, while 100 IU of hCG was given at 0 h and 24 h. Hormone treated gilts showed increased (P < 0.05) medium and large follicle development, estrus (>70%), ovulation (100%), and ovulation rate (>30 CL) compared to saline. There was an increase (P < 0.05) in the proportion of hormone-treated gilts with follicular cysts at Day 5, but these did not persist to Day 22. These gilts also showed an increase in poorly formed CL (P < 0.05). FSH alone or with small amounts of LH can induce medium follicle growth but greater amounts of LH at the same time is needed to sustain medium follicles, stimulate development of large follicles and induce estrus and ovulation in prepubertal gilts.     

Ovulation and follicular growth in gonadotropin-treated gilts followed by in vitro fertilization and development of their oocytes

We investigated whether porcine ovaries derived from FSH-pituitary (FSH-P) or hCG-treated animals can produce oocytes with better in vitro cytoplasmic maturation and in vitro embryonic development relative to those derived from saline-treated animals. The size of the follicle producing the oocyte was also studied. Each of 25 prepubertal gilts received 1 of 6 treatments by intramuscular injection: 1) saline (3 mL, once, n = 5); 2) FSH-P8-3 (8 mg, 3 times, with a 24-h interval, n = 4); 3) FSH-P16-3 (16 mg, 3 times, with a 24-h interval, n = 4); 4) FSH-P16-1-P4-2 (16 mg, once, 4 mg, twice, with a 24-h interval, n = 4); 5) FSH-P16-1 (16 mg, once, n = 4); or 6) hCG (100 IU, 3 times, with a 24-h interval, n = 4). The ovaries were removed by mid-ventral laparotomy 72 h after the first injection. The numbers of corpora hemorrhagica (CH) with each FSH-P treatment were similar (P > 0.05). However, compared with gilts treated with saline or hCG, those treated with FSH-P8-3 had a greater (P < 0.05) number of CH. Treatment with FSH-P8-3 or FSH-P16-3 induced significant growth of medium/large follicles (4 to 8 mm in diameter) compared with saline or FSH-P16-1. The same results were observed when FSH-P8-3 was compared with FSH-P16-P4-2 or hCG. After in vitro fertilization, the rates of male and female pronuclei in oocytes derived from medium/large follicles did not differ (P > 0.05) between treatments, but in oocytes derived from small follicles they were lower (P < 0.05) in saline-treated than in FSH-P16-1-P4-2-treated gilts. After 120 h in culture, the percentages of the inseminated oocytes from 1 to 3 mm or 4 to 8 mm follicles developing to > or = 2-cell did not differ (P > 0.05) between saline- and gonadotropin-treated gilts. However, a higher (P < 0.05) percentage of the inseminated oocytes from 4 to 8 mm follicles had developed to the morula stage or beyond, than those from the 1 to 3 mm follicles. In conclusion, administration of single or multiple doses of FSH-P induced ovulation, but only 8 or 16 mg FSH-P injected 3 times with 24-h intervals for 72 h induced growth of 4 to 8 mm follicles. The size of follicle from which the oocyte derived also had a significant effect on its development in vitro.     

猪卵母细胞体外成熟的发育潜力及核移植重构胚构建方法的研究

为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28 h、32 h、36 h、40 h、44 h、48 h、52 h和56 h的猪卵母细胞分别进行去核构建重构胚.研究结果表明,成熟44 h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48 h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44 h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40 h、36 h、32 h、28 h的卵母细胞的分裂率与囊胚率(P＜0.05).卵龄为48 h的卵母细胞融合率高于卵龄为52 h卵母细胞的融合率(P＜0.05).同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响.研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%.Hoechest染色法去核率显著高于盲吸法的去核率(P＞0.05),而与Spindle-view法去核率没有差异(P＞0.05).三种方法在融合率和囊胚率方面差异不显著(P＞0.05),但Hoechest染色法的分裂率较低,差异显著(P＜0.05).进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P＜0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考.     

Roles of MAPK and spindle assembly checkpoint in spontaneous activation and MIII arrest of rat oocytes

Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca(2+) rise, Sr(2+)-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.     

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.     

Cytogenetic analysis of Day-4 embryos from PMSG/hCG-treated prepuberal gilts

Prepuberal gilts were treated with pregnant mare serum gonadotropin (PMSG) to study the effects of its dosage on ovulation rate, fertilization rate after artificial insemination, embryo viability, and rate of development and incidence of chromosome abnormalities in Day-4 embryos. Gilts received 750 IU, 1250 IU or 1500 IU of PMSG, followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Gilts were inseminated 28 to 30 h following the hCG injection, and resulting embryos were collected on Day 4 post ovulation. Ovulation rate was higher in the 1250 IU group than in the 1500 IU group or the 750 IU group. The 1500 IU dose caused excessive stimulation of the ovary, resulting in the occurrence of large (>10mm diameter) unovulated follicles, reduced fertilization rate and low embryo recovery rate. There was no difference in the incidence of chromosome abnormalities among the three groups, although the 1500 IU group had higher embryonic mortality than the two lower dose groups. A dose of 1250 IU PMSG increased ovulation rate above that achieved by 750 IU and, therefore, increased the number of oocytes or embryos available for transfer or for other studies, without sacrificing embryo viability or increasing the incidence of chromosome abnormalities.     

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.     

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris)

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)