Effect of pronuclear DNA microinjection on the development of porcine ova in utero

The objective of this study was to assess the effect of various aspects of pronuclear DNA microinjection on the early development of porcine ova in utero. Estrus was synchronized and superovulation was achieved in sexually mature gilts by the administration of allyl trenbolone, PMSG and hCG. Donor gilts were bred at 12 and 24 h after the onset of estrus. Ova were recovered between 60 and 62 h after the administration of hCG. One-cell ova that exhibited pronuclei after centrifugation were randomly allocated in equal numbers from each donor across one of two pairs of treatments: micro-DNA (ova were injected with two gene constructs that code for the human complement regulatory proteins decay accelerating factor and membrane cofactor protein) and control (ova were centrifuged only) or micro-buffer (ova were injected with buffer only) and pierced (a pipette was inserted into one pronucleus). Ova were transferred by treatment pairs to recipients. Treatments were segregated by oviduct. Ova were recovered after 120 h in utero, fixed and stained with 1% orcein. The proportion of ova that possessed > or = 80 nuclei, the mean number of nuclei present and proportion of ova that formed blastocysts were all significantly (P<0.05) greater for control and pierced ova than for micro-DNA and micro-buffer ova. No difference in these parameters was observed between micro-DNA and micro-buffer ova. These results demonstrate that pronuclear microinjection of a buffer alone can adversely affect the early development of porcine ova in utero.     

In vitro fertilization in the rabbit after delayed ovum recovery

Rabbit ovum donors were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ova were recovered 16-17 h post-hCG from oviducts immediately after killing and from excised oviducts held in saline 30 min at 33 degrees or 38 degrees C prior to ovum recovery. In vivo-capacitated spermatozoa were used to inseminate both groups of ova. Data revealed a decrease in fertilization rates following a 30-min delay at 38 degrees C in ovum recovery. Thus, 64% (44/69 ova) were fertilized with rapid recovery, whereas 43% (39/90 ova) were fertilized following a 30-min delay. The decrease in fertilization imposed by delay in ovum recovery was apparently overcome when oviduct storage was at 33 degrees C. Under these conditions, 69% of inseminated ova were fertilized. Ova inseminated with in vitro-capacitated sperm showed a similar response to delayed ovum recovery. Embryonic development in culture of ova obtained from mated does was not affected by delay in recovery at 33 degrees or 38 degrees C provided mated does had been injected only with hCG. Ova from mated does receiving both PMSG and hCG were adversely affected by a 38 degrees C delay. The data emphasize the importance of rapid ovum recovery from oviducts and suggest the possibility of altering conditions to overcome damaging effects of delayed recovery.     

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.     

Transport of fertilised and unfertilized ova in sows

Transport of fertilised and unfertilized ova was studied in 22 crossbred (Landrace x Yorkshire) multiparous sows. Sows in the inseminated group (I-group, n=11) were inseminated once with 100ml of BTS extended semen from two fertile boars with a total of 10 x 10 (9) spermatozoa during the second oestrus after weaning between 18 and 8h prior to estimated time of ovulation, as estimated from the first oestrus after weaning. All the sows were slaughtered between 36 and 48 h after ovulation in the second oestrus after weaning by stunning and bleeding. After slaughter, the reproductive tract was immediately recovered, the isthmus was divided into three equal segments, and the number of ova was determined in each segment and in the upper third of the uterine horn from the UTJ. There were no significant differences (P>0.05) either in the intervals from ovulation to slaughter (42.3+/-6.2h versus 43.2+/-5.4h) or in the numbers of corpora lutea (CL) (18.2+/-5.5 versus 15.9+/-3.5) between the non-inseminated (N-group) and the inseminated groups (I-group), respectively. Ova recovery rate was 92.5% in the N-group and 82.9% in the I-group (P>0.05). In the I-group, ova had passed 2.2+/-0.3 segments whereas in the N-group, ova had passed 2.6+/-0.3 segments (P=0.38). It can be concluded that there is no difference in the transportation of either fertilised or unfertilized ova in the reproductive tract of pigs.     

Use of purified porcine follicle-stimulating hormone for ovarian stimulation of macaque monkeys

At present, in nonhuman primates, ovarian stimulation with heterologous gonadotropin preparations is the only reliable way to produce substantial numbers of competent ova for in vitro fertilization and embryo development studies. Preparations such as equine chorionic gonadotropin (eCG) and human menopausal gonadotropins (hMG or hFSH) have been used successfully, but eCG is crude and contains variable amounts of LH activity, while hMG/hFSH is very expensive and the supply is not stable. This study examined the use of a purified porcine FSH preparation (Folltropin V) for ovarian stimulation in rhesus monkeys. Twice-daily intramuscular injections of this preparation resulted in good follicular development, and was followed by a single intramuscular injection of hCG. Ova were collected laparoscopically 30 h post hCG, fertilized in vitro and then cultured until development ceased. Stimulation of 9 monkeys with Folltropin V yielded a mean of 20 ova per animal, of which 71% reached metaphase II and were inseminated; of these, 92% were fertilized in vitro and 48% developed into blastocysts in vitro. These results are similar to those reported by us and by others using eCG, hMG or an hFSH/hMG combination for ovarian stimulation of macaque monkeys. We conclude that Folltropin V is a suitable alternative preparation for ovarian stimulation in nonhuman primates and one that also has the advantages of being readily available and much less expensive than human gonadotropin preparations.     

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.     

Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of >/=-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of >/=-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.     

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.     

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.     

Recovery and culture of ova from Brucella abortus infected cows

One hundred and ninety-two ova were collected from 12 mixed-breed beef and dairy cows that had been artificially infected with Brucella abortus Biotype 1 Strain 2308. Each donor cow was treated for superovulation, and nonsurgical collections were performed on one, two or three occasions. A total of 27 nonsurgical collections on Days 6 through 12 (estrus = Day 0) resulted in the recovery of an average of 7.1 ova (4.4 fertilized and 2.7 nonfertilized). Ova were washed ten times in groups of not more than 10 and cultured for the isolation of B. abortus. A portion of the recovery medium, including sedimented uterine debris from each collection, was also cultured for isolation of the organism. Brucella abortus was not detected in any sample of the recovery medium or in any group of ova.     

In vitro fertilization of Siberian hamster oocytes

Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18:544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1 hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 hr prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.     

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.     

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Improved cleavage of bovine ICSI ova cultured in heparin-containing medium

Although heparin plays an important role in bovine sperm capacitation, there is no direct evidence for a role in embryonic development. The present study was designed to examine the effect of heparin on early development of bovine zygotes obtained by intracytoplasmic sperm injection (ICSI). Spermatozoa were treated with or without heparin, and the ICSI ova were cultured in a chemically defined medium + BSA, with or without heparin. Treatment of spermatozoa with heparin before ICSI or heparin in post-ICSI culture medium for 18 or 24h had a beneficial effect on pronuclear formation, cleavage rate (63% versus 76-83%), and number of cells in blastocysts (68 cells versus 82-109 cells; P < 0.05).     

Conditions for in vitro maturation and artificial activation of ferret oocytes

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.     

SD大鼠胞质内单精注射技术方法的建立

目的建立SD大鼠胞质内单精注射操作程序。方法和结果实验1：用直径为7～10μm和2-4μm的注射针以及相应的注射方法进行大鼠ICSI,ICSI后卵母细胞存活率（30．5％vs．61．3％）和卵裂率（12．5％vs．51．1％）均差异显著（P〈0．05）;实验2：分别在hCG后14h、16h和18h取卵进行ICSI,三组存活率（77．4％、74．1％vs．69．6％）差异不显著（P〉0．05）;14h和16h组的卵裂率（60．8％、56．0％vs．31．3％）与18h组差异显著（P〈0．05）;实验3：用不同的显微操作液H-mR1CM和H-mKRB进行大鼠ICSI,结果卵存活率相近（79．2％vs．75．9％）,卵裂率（70．5％vs．74．7％）差异不显著（P〉0．05）,但8-细胞发育率（43．5％vs．61．9％）差异显著（P〈0．05）,囊胚发育率差异极显著（P〈0．01）。结论大鼠ICSI时在注射hCG后14～16h取卵最佳,采用2—4pan直径的注射针、H—mKRB作为操作液更有利于卵的发育。     

Involvement of gonadotropins in induction of luteolysis in pigs

In our previous study we have demonstrated that treatment of endometrial explants with LH increased 13,14-dihydro-15-ketoprostaglandin F(2alpha) (PGFM) accumulation in pigs. This was particularly visible on Days 14-16 of the estrous cycle. Action of gonadotropin in porcine endometrium appears to be mediated by LH/hCG receptors whose number is dependent on the day of the estrous cycle. In the current study i.v. infusion (1 hour) of hCG (200 IU) performed on Days 10 (n=4) and 12-14 (n=4) of the porcine estrous cycle did not affect plasma PGFM (ng/ml+/-SEM) concentrations. In contrast, administration of hCG on Days 15-17 produced, depending on plasma PGFM level before the infusion period, three different types of response: I. plasma PGFM surge of amplitude 0.62+/-0.15 was observed when the mean basal pre-infusion PGFM plasma level was 0.23+/-0.05 (n=6 gilts); II. the delayed PGFM surge of amplitude 0.62+/-0.15 was determined when basal pre-infusion PGFM level was 0.80+/-0.20 (n=6); and III. lack of PGFM response to hCG was found when basal pre-infusion PGFM level was 1.09+/-0.61 (n=6). Concentrations of plasma PGFM before and after saline infusion did not differ on Days 12-14 and 16 of the estrous cycle. In the next experiment blood samples were collected every 1 hour on Days 12-19 of the estrous cycle to determine concentrations of LH, PGFM and progesterone in four gilts. In particular gilts, plasma peaks of LH closely preceded surges of PGFM in 72.7, 84.6, 75.0 and 66.6 percent, respectively. The highest PGFM surges followed a decline in plasma progesterone concentration. We conclude that the increased PGF(2alpha) metabolite production after hCG infusion during the late luteal phase of the estrous cycle as well as the relationship between plasma LH and PGFM peaks suggest the LH involvement in the elevation of endometrial PGF(2alpha) secretion in pigs, and, in consequence, induction of luteolysis.     

Laparoscopy for intrauterine insemination and embryo recovery in superovulated ewes at a commercial embryo transfer unit

Of 111 variable age, pedigree ewes subjected to a range of superovulatory regimens and then submitted to embryo recovery by laparoscopy, nine had adhesions corresponding to a mid-line laparotomy (presumably from a previous attempt to recover embryos) and could not have their embryos recovered by the laparoscopic technique. Of the remainder, 27 ewes (26.5%) had less than three ovulations or had prematurely regressing corpora lutea at the selected time for embryo recovery (Days 5 to 6 following insemination), and no attempt was made to recover embryos from them. For the 75 ewes subjected to laparoscopic ovum recovery following laparoscopic intrauterine insemination, the average number of ovulations (+/- SEM) was 7.9 +/- 0.6; the average ovum recovery (mean of values for each ewe) was 51.7% +/- 3.5; and the percentage of recovered ova that were fertilized was 87.3%. For a further nine 3-yr-old crossbred ewes the mean values for ovulation and ovum recovery were 7.6 +/- 1.2 and 70.1 +/- 7.7, and were not significantly different for the two insemination methods used (laparoscopic intrauterine vs cervical). In general, ovulation rates for ewes given pregnant mare serum gonadotrophin (PMSG) tended to be lower (5.2 +/- 0.7) than for those given porcine follicle stimulating hormone (pFSH, 7.7 +/- 0.8) or human menopausal gonadotrophin (hMG, 7.7 +/- 2.3). Ova recovery rates were similar on Days 5 and 6 (Day 0 = insemination), and were not affected by method of insemination (laparoscopic intrauterine vs cervical).     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.