A practical method for the quantitative measurement of estrone conjugates (E₁C), pregnanediol-3-glucronide (PdG), follicle stimulating hormone (FSH), and monkey chorionic gonadotropin (mCG) in the excreta of non-human primates were described. In the series of studies, results suggest that 1) urinary and fecal steroid metabolites accurately reflected the same ovarian or testicular events as observed in plasma steroid profiles in captive Japanese macaques, time lags associated with fecal measurements were one day after appearance in urine; 2) these noninvasive methods were applicable to wild and free-ranging macaques for determining reproductive status; 3) hormonal changes during menstrual cycles and pregnancy could be analyzed by measurement of FSH, CG and steroid metabolites in the excreta in captive great apes and macaques; and 4) hormone-behavior relationships of macaques in their natural habitats and social setting could be analyzed. In macaques, between maternal rejection and excreted estrogen, but not excreted progesterone were associated, moreover, in male study, significantly higher levels of fecal cortisol were observed in high-ranking males. In addition, reliable noninstrumented enzyme-linked immunosorbent assay (NELISA) for detection of early pregnancy in macaques was established. These results suggest that the noninvasive characteristic of excreted hormone monitoring provide a stress-free approach to the accurate evaluation of reproductive status in primates.

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This paper describes several technical improvements in developmental engineering for livestock production, including their practical utility in the field. The artificial production of monozygotic twins via embryo splitting is shown to increase embryo productivity, while embryo sexing capability provides added value without compromising offspring productivity, with both techniques being adequate for practical field applications. It is also shown that: (1) the development of nuclear transfer utilizing oocytes collected from slaughtered ovaries and matured in vitro enables producing a large number of cloned embryos, (2) the intracytoplasmic injection of somatic cell improves the productivity of nuclear transplantation, and (3) the injection of sperm increases the rate of normal oocytes with male and female pronuclei allowing further preimplantation development. Finally, the removal of cytoplasmic lipid droplets from embryos following centrifugation alters an embryo's intrinsic sensitivity to low temperature allowing long-term preservation. Collectively, these techniques have clearly provided improvements in developmental engineering for livestock production.

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Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as β-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.

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When released into an appropriate environment, mammalian spermatozoa begin to capacitate and then continue until fully capacitated and able to fertilize. During capacitation in vitro, some cells `over-capacitate' and undergo spontaneous acrosome reactions; this would be highly undesirable in vivo since already acrosome-reacted spermatozoa are non-fertilizing. Recent studies have revealed that seminal plasma contains several small molecules that bind to specific receptors on the sperm plasma membrane and act as `first messengers', causing biologically important changes in availability of the `second messenger' cAMP. Fertilization promoting peptide (FPP), calcitonin and adenosine all regulate cAMP production, stimulating it in uncapacitated spermatozoa and then inhibiting it in capacitated cells; in contrast, angiotensin II stimulates cAMP throughout capacitation. The molecules that regulate cAMP appear to do so via G protein-modulated changes in membrane associated adenylyl cyclases (mACs). Both mouse and human spermatozoa have been shown to have Gα_s and Gα_i2, as well as several isoforms of mAC, located in the same regions as the specific receptors. Thus spermatozoa possess the required elements for several separate signal transduction pathways, many of which regulate mAC/cAMP and so maintain sperm fertilizing ability. In vivo, such responses could increase the chances of successful fertilization.

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We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin α5β1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin α5β1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of α5 and β1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin- mediated manner.

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Low birth weight (LBW) as a result of restricted fetal growth increases the risk for later metabolic diseases and adiposity. However the relationship between LBW and postnatal growth and adult body composition has not been fully investigated. We have used sheep to determine the effects of LBW on postnatal growth and body composition at maturity. LBW was induced by twinning and placental embolization. At birth, LBW lambs were 38% lighter than controls (2.8 ± 0.2 vs 4.4 ± 0.3 kg, P<0.05), but had caught up in bodyweight by 8 weeks after birth. At ~2.3 years, bodyweights were not different between groups, but there were reductions in absolute (-8%) and relative (-17%) brain weights of LBW sheep (P<0.05) compared to controls. X-ray absorptiometry showed that the mature LBW sheep, compared to controls, had greater amounts of lean muscle (38.1 ± 1.3 vs 35.3 ± 0.5 kg, P<0.05) and tended to have more body fat (12.2 ± 1.2 vs 9.6 ± 0.9 kg; P=0.1); at autopsy abdominal fat mass was greater in LBW sheep (3.06 ± 0.26 vs 2.20 ± 0.25 kg, P<0.05). Plasma leptin concentrations were not different between groups. We conclude that, in sheep, LBW is associated with early postnatal catch-up in body weight, but body composition is permanently altered such that, relative to controls, adiposity is increased and brain weight is decreased.

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The present study was conducted to investigate whether a portable CO₂ incubator was effective for in vitro maturation (IVM) of bovine, porcine and minke whale oocytes, and the effect of maturation media supplemented with different hormones; porcine follicle stimulating hormone (pFSH), estradiol-17β (E₂), or pregnant mare's serum gonadotropin (PMSG): human chorionic gonadotropin (hCG) for minke whale immature oocytes was also examined. In vitro maturation rates of bovine and porcine oocytes cultured in the portable CO₂ incubator were not significantly different from the standard CO₂ incubator. In minke whale IVM culture using the portable incubator, the maximum expansion of cumulus mass was observed by pFSH/E₂ and PMSG/hCG at the end of IVM culture. Moreover, the IVM culture period was shortened to 28-30 h from 96-120 h previously reported. The proportion of matured oocytes cultured in the medium supplemented with pFSH/E₂ (26.7%) was significantly higher (P<0.05) than that with PMSG/hCG (6.9%). The present study indicates that a portable CO₂ incubator is a useful device for minke whale IVM culture on a research base ship, and the addition of pFSH/E₂ into an IVM medium enhanced cumulus expansion and the proportion of minke whale matured oocytes.

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To establish a tool for the study of follicular growth and development, we xenotransplanted small pieces (approximately 1 mm³) of porcine ovarian cortical tissues containing only primordial follicles and small preantral follicles under the capsules of kidneys of severe combined immunodeficient (SCID) mice (8-10 weeks old). The changes in cell proliferation and cell death/apoptosis, and vascularization in xenotransplanted follicles during follicular growth and development were analyzed histochemically at 1-26 weeks after operation. Follicles in grafted ovarian tissues grew rapidly forming an antral cavity (a hallmark of tertiary follicles) at 1 week after grafting. The diameter of the follicles in transplanted tissues ranged from 0.5 to 1.5 mm, from 0.5 to 2.0 mm and from 0.5 to 3.0 mm at 1, 2 and 26 weeks after the operation, respectively. Histological observation of ovarian tissues at 26 weeks after grafting revealed that all grafts had abundant capillary vessels, which invaded from murine organs and surrounded the growing follicles. Grafted small preantral follicles developed to the antral stages at 1 week after grafting and growing antral follicles survived at 26 weeks after grafting. The oocytes in the growing follicles were easily recovered for evaluating the quality. Our simple xenografting sysytem is easy to use and a good experimental tool for the study of folliclular growth and development in porcine ovaries.

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The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (≥15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.

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Shortened and weakened estrous expressions could be one of the causes of poor heat detection rate. Non-specific acute stresses are assumed to depress expression of estrus by an increase of plasma progesterone which may originate from the adrenal cortex. The objective of the present study was to examine whether the adrenal cortex can secrete significant amounts of progesterone in response to exogenous adrenocorticotropic hormone (ACTH) in lactating cows. Four cows had estrus synchronized and were ovariectomized in the luteal phase. The cows were given 25 IU ACTH through an indwelling catheter 5 h after catheterization. Blood samples were collected at an interval of 30 min. In 3 of the 4 cows, plasma progesterone concentrations increased significantly 0.5-1.5 h after the first ACTH challenge with a mean peak value of 4.2 ± 0.4 (S.D.) ng/ml. A similar response was also observed after the second ACTH challenge. Peak plasma progesterone concentrations in the 3 cows after first ACTH challenge were comparable with the progesterone values in the luteal phase of each cow. The results suggest that lactating cows have the capability to secrete a significant amount of progesterone from the adrenal cortex.

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The aim of the present study was to assess follicular and oocyte development in the same gilts during three phases of their reproductive life [prepuberal gilts (PP; 6.0 months of age), puberal gilts (P; 9.5 months of age) and primiparous sows (S)]. Follicular development was stimulated by the injection of 1,000 IU of equine chorionic gonadotropin (eCG) followed by 500 IU of human chorionic gonadotropin (hCG) 72 h later. Cumulus-oocyte-complexes (COCs) were recovered by endoscopic ovum pick up/aspiration from preovulatory follicles of the left ovary, and the follicular fluid (FF) from the right ovary was collected 34 h after the hCG treatment by endoscopy. Altogether, 19 pigs were used in the PP and P trials and 12 in the S trial. From the left ovaries, 168, 190 and 82 follicles were aspirated and 106, 125 and 42 COCs, respectively, were recovered (recovery rate 61 ± 27, 63 ± 21 and 53 ± 22%, respectively). The mean number of follicles was greater in the P phase than in the PP phase (19.7 ± 6.8 vs. 15.7 ± 6.8; p=0.06) and S phases (14.2 ± 4.0; p<0.05). More uniform oocytes with an expanded cumulus were aspirated in the P and PP phases than in the S phase (90 and 78 vs. 46%; p<0.05). Furthermore, the meiotic configuration in oocytes (T I/M II stage) differed between the three phases (56 and 62 vs. 0%; p<0.05). Progesterone (P4) levels in FF decreased from 590.0 ± 333.6 (PP) to 249.1 ± 72.6 (P) and 161.4 ± 75.2 ng/ml (S) (p<0.05). Estradiol-17β (E2) levels differed between PP and P gilts and S sows (9.3 ± 2.9, 21.9 ± 10.6 and 94.0 ± 15.9 pg/ml, respectively; p< 0.05), and the P4/E2 ratio was 72, 15 and 5, respectively. These results indicate differences in follicular and oocyte development between the reproductive phases investigated. Puberal gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows must be taken into consideration in breeding. Any prediction of lifetime performance based on individual ovarian reactions of prepuberal gilts is unreliable.

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Gene expression of the porcine glycoprotein hormone α subunit (p-αGSU) was examined in LβT2 cells, which were established from the anterior pituitary lobe of the immortalized transgenic mouse and produce αGSU, and in CHO cells cloned from Chinese hamster ovaries. Expression of the reporter gene fused with p-αGSU gene upstream in LβT2 cells showed that the distal regions -540/-240 and -798/-541 are important for the activation of gene expression. In contrast, the transcriptional activity of the distal region of p-αGSU gene was repressed in CHO cells. The region -540/-240 contains an adequate enhancer, called pituitary glycoprotein hormone basal element, whereas the region -798/-541 has no distinguished element. Transfection of the expression vector containing cDNA of a pan-pituitary activator, Ptx1, whose putative binding sites are present scatted in the distal region of the p-αGSU gene, revealed unexpectedly that this factor significantly suppressed the expression of p-αGSU gene in LβT2 cells, indicating that Ptx1 is unrelated to the upregulation in the region -798/-541. Thus, this study demonstrated for the first time that the distal region -798/-541of the p-αGSU gene is indispensable for prominent expression of this gene in which an as yet unidentified factor may participate.

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This study aimed to evaluate the quality of porcine somatic cell nuclear transfer (SCNT) embryos by examining its gene transcription patterns. Embryos were produced by SCNT, intracytoplasmic sperm injection (ICSI) or under different conditions, and transcripts of genes for fibroblast growth factor receptor (FGFr) 2IIIc, FGFr72IIIb, X inactive-specific transcript (Xist), interleukin 6 (IL6), IL6 receptor (IL6r) α and c-kit ligand, were detected by real-time RT-PCR. The percentages of embryos in which these transcripts were detected were similar in SCNT and ICSI embryos. On the other hand, the transcriptional levels of the FGFr72IIIb and IL6rα genes were 0.5 times less and 2 times more, respectively, in SCNT blastocysts than those of ICSI blastocysts (p<0.05). When nuclear transfer was performed before or after activation of oocytes, embryos in the latter case showed significantly lower frequencies of having FGFr72IIIb (74% vs. 90%) and Xist (3% vs. 33%) transcripts compared to the former case embryos (p<0.05). When two lines of nuclear donor cells with different developmental potencies were used, the transcriptional profiles in the reconstructed embryos did not show any significant differences. Our study suggests that expression profiles of FGFr72IIIb, IL6rα, and Xist can be used as markers for the diagnosis of the developmental potency of porcine nuclear transfer embryos.

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Prenatal stress has been seen as a reason for reproductive failures in pig offspring mostly originated or mediated by changed maternal functions. Experiments were conducted in pregnant gilts (n=32) to characterize effects of elevated maternal glucocorticoids on the secretion of reproductive hormones (LH, progesterone) during the 1^st (EXP 1), 2^nd (EXP 2) and 3^rd (EXP 3) trimester of pregnancy (TP). Transiently elevated cortisol release was repeatedly achieved by application of 100 IU adenocorticotropic hormone (ACTH) (Synacthen Depot^®) six times every second day beginning either on day 28 (EXP 1), day 49 (EXP 2) or day 75 of pregnancy (EXP 3). Glucocorticoid concentrations were examined in umbilical blood vessels of fetuses which mothers were subjected to ACTH at 2^nd and 3^rd TP (EXP 4). Furthermore, the pituitary function of newborn piglets of EXP 2 was checked by a LH-RH challenge test. In sows, LH concentrations were at low basal level (0.1-0.2 ng/ml) but with pulsatory release pattern during each TP. The number of LH pulses/6 h (LSM ± SE) of saline treated Controls increased with ongoing pregnancy and decreased to the 3^rd TP (1.3 ± 0.2 in EXP 1 vs. 2.0 ± 0.1 in EXP 2 vs. 1.4 ± 0.1 in EXP 3, p<0.05). After ACTH treatment the number of LH pulses left unchanged in Experiments 1 and 2 (1.3 ± 0.2 and 1.5 ± 0.1) and decreased in EXP 3 (0.8 ± 0.2, p<0.05). Differences (p<0.05) were obtained comparing the LH pulse number of ACTH and saline treated sows at the 2^nd and 3^rd TP. Moreover, areas under the curve (AUC) of each LH pulse and of LH over baseline were significantly reduced by treatment. Levels of progesterone increased (p<0.05) for 150 to 170 min after each ACTH application both in EXP 1 and EXP 2, but not in EXP 3. The mean progesterone concentration was different between trimesters, and ACTH and Controls (1^st TP: 30.0 ± 0.9 and 24.4 ± 0.7 ng/ml; 2^nd TP: 35.5 ± 0.9 and 29.1 ± 1.0 ng/ml; 3^rd TP: 13.6 ± 0.2 and 13.1 ± 0.1 ng/ml; p<0.05). In fetuses (n=87) recovered 3 h after ACTH or saline (EXP 4), the plasma cortisol concentrations were significantly increased in umbilical vein (93.7 ± 5.5 vs. 47.0 ± 5.3 nmol/l) and artery (95.7 ± 5.4 vs. 66.4 ± 5.4 nmol/l), and in periphery (46.8 ± 5.3 vs. 27.1 ± 5.3 nmol/l) compared to controls. Plasma ACTH concentrations, however, did not differ in fetuses of both treatment groups. Postnatal LH-RH challenge tests (1^st and 28^th day post partum) induced LH surges in female piglets (n=67) both of ACTH and saline treated sows, but did not differ between groups (1^st day: 7.2 ± 0.8 vs. 8.1 ± 0.7 ng/ml; 28^th day: 10.5 ± 1.7 vs. 13.6 ± 2.2 ng/ml). However, basal LH of piglets whose mothers were submitted to ACTH during 2^nd TP was lower on 1^st day (1.7 ± 0.2 vs. 2.3 ± 0.2 ng/ml, p<0.05) but not on 28^th day (1.0 ± 0.2 vs. 1.1 ± 0.2 ng/ml). However in both groups, the basal LH was always higher on 1^st as on 28^th day (p<0.05). Thus, chronic intermittent ACTH administration is able to influence the release pattern of maternal reproductive hormones. However, these findings demonstrate that these effects are dependent on the stage of pregnancy. Furthermore, it was shown that maternal cortisol can cross the placenta during gestation and thus may affect maternal-fetal interactions and, as a result, reproductive function of offspring.

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The meiotic progression of Xenopus oocytes has been suggested to depend on the activity of cyclin-dependent kinase 2 (Cdk2). We examined whether Cdk2 is involved in the regulation of mammalian oocyte meiosis by injecting porcine oocytes with anti-Cdk2 antibody. At first, the cross-reactivity of the anti-Cdk2 antibody with Cdc2 kinase was evaluated by immunoprecipitation and immunoblotting experiments using porcine granulosa cell extract, and no cross-reactivity with Cdc2 kinase was observed in the antibody used. In the anti-Cdk2 antibody-injected group, 50.7% of the oocytes were arrested in the second metaphase after 50 h of culture and this rate was significantly lower than those in the non-injected intact oocytes or the oocytes injected with mouse IgG (84.5% and 86.7%, respectively). Most of the other oocytes in the antibody-injected group formed a pronucleus without polar bodies or with only one polar body. The cyclin B1 amount in the antibody-injected and activated oocytes was dramatically decreased compared with that in the intact or mouse IgG-injected oocytes after 50 h of culture. These results suggest that Cdk2 is involved in the meiotic maturation of mammalian oocytes, and that the block of Cdk2 activity results in the failure of cyclin B1 accumulation and second meiosis induction.

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This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (~53% vs ~68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (~47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (~20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (~15%) than did the non-delipated oocytes, whether centrifuged or not (~4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, ~39% were activated and male pronucleus formation was observed in ~40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.

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To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 μg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 μg/kg) and 164 (8.0 μg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.

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