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Estrus synchronization and artificial insemination in Korean black goat (Capra hircus coreanae) using frozen-thawed semen

  • Kim, Kwan-Woo;Lee, Jinwook;Kim, Keun Jung;Lee, Eun-Do;Kim, Sung Woo;Lee, Sung-Soo;Lee, Sang-Hoon
    • Journal of Animal Science and Technology
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    • v.63 no.1
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    • pp.36-45
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    • 2021
  • Presently, there is an increased demand for livestock products all over the world which has led to more devotion on improving livestock population. Although goats have been bred for a long time in Korea, but there is not much research conducted on traditional Korean black goat (Capra hircus coreanae) compared to other livestock populations. Mutton consumption has been dramatically changing from medicinal use to edible meat and this trend directs the black goat populations declining and also mutton import quantities are increasing consistently. The present study introduced a new estrus synchronizing technique with subsequent artificial insemination (AI) for Korean black goats to enable crossbreeding with non-native breeds for the small or subsistent farmers. Our data highlighted that, the percentage of motile sperm from the electro-ejaculated samples declined significantly after freezing and melting. In addition, the sperm motility significantly declined with regard to sperm incubation period (0, 5, 60, and 120 min at 37℃) and was negatively correlated (64.2 ± 7.9%, 63.3 ± 5.8%, 49.9 ± 6.3%, and 35.9 ± 7.6%, respectively) in frozen-thawed sperm samples. Moreover, the E2 levels were unchanged even 24 h after controlled internal drug releas (CIDR) withdrawal. But, 48 h and 72 h after CIDR removal, E2 levels increased significantly. These data helps us to consider the two time points for AI; CIDR removal after 24 h, at which E2 decreases, and after 48 h, as the time at which progesterone increases. Additionally, the AI after 48 h of CIDR removal group exhibited significantly higher pregnancy and parturition rates (42.9%) compared to AI after 24 h after CIDR removal 28.6% group. In conclusion, these studies will propose an optimal estrus synchronisation process with subsequent timing of AI and also will promote the Korean black goat breeding industry.

Prepartum and/or postpartum supplementation with monensin-molasses multinutrient blocks to optimize fertility and calf performance in primiparous beef cows

  • Catussi, Bruna Lima Chechin;da Silva, Laisa Garcia;Schalch, Fernando Jose Junior;Auder, Rafaela Maria Sutiro Angelieri;Gomez, Juan Fernando Morales;Mingoti, Rodolfo Daniel;Morgulis, Sergio Carlos Franco;Baruselli, Pietro Sampaio
    • Animal Bioscience
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    • v.35 no.11
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    • pp.1675-1688
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    • 2022
  • Objective: Pregnant Nelore heifers (n = 417) were used to evaluate the effects of supplementation with monensin-molasses multinutrient block (B) during pre and/or postpartum on reproductive and progeny performance. Methods: Heifers were allocated in four treatments: i) CC: heifers received control supplement (C) in loose meal form (0.06% of body weight [BW] offered daily before and after parturition; n = 108); ii) CB: received C before parturition and B (0.07% of BW offered weekly after parturition; n = 117); iii) BC: received B before and C after parturition (n = 103) and iv) BB: received B before and after parturition (n = 89). During pre and postpartum periods, concentration of metabolites/hormones and cow/calf performance was evaluated over time. Cows were synchronized twice for fixed timed artificial insemination (FTAI) using an estradiol/progesterone-based protocol. Data was analyzed by orthogonal contrasts (C). Results: B increased pregnancy at first FTAI (p = 0.04) and overall pregnancy rate (C1: CC vs BB+BC+CB; p = 0.05). Supplemented cows had greater body condition score (BCS) only at parturition (D0; p = 0.04) and at D40 (p = 0.02). B increased BW (p = 0.03), glucose concentrations (p = 0.01) and subcutaneous fat thickness (p = 0.03) only at D40. Concentrations of insulin were higher in supplemented cows (p = 0.008). Calves born by cows supplemented before and after parturition (C2: BB vs BC+CB) were heavier at 80 (p<0.001), 120 (p<0.001), 170 (p = 0.002) and 210 (p = 0.02) days old. Conclusion: Regardless of period of treatment, block supplementation increased pregnancy at first FTAI and overall pregnancy rate. Additionality, block supplementation during both pre and postpartum periods improved progeny weight until weaning. Block supplementation can be a tool to optimize fertility and calf performance in Nelore primiparous cows.

Improvement of Reproductive Efficiency of Artificial Insemination Following Estrus Induction in Dog II. Semen Freezing and Artificial Insemination in Dog

  • Lee, Y. R.;S. Y. Choe
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.92-92
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    • 2002
  • Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to evaluate semen quality after cryopreservation and to evaluate the Pregnancy rate after insemination (AI). Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifene (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. The ejaculated semen to freeze was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM: TES, 209 mM: citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 cm above the surface of liquid nitrogen (LN$_2$) for 23 min. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, the pregnancy rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only. In addition, frozen-thawed semen can be used successfully far artificial insemination in dog.

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Sexual Behaviors, Estrus Detection and Conception of Heifers Synchronized by Progesterone Intravaginal Device(Prid) and Synchromate-B

  • Im, K.S.;Kim, C.K.;Voss, H.J.;Allen, S.;Zheng, X.;Foote, R.H.
    • Korean Journal of Animal Reproduction
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    • v.9 no.2
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    • pp.140-147
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    • 1985
  • Seventy four Holstein heifers were randomly assigned over three trials to PRID-7+PG-6 and Synchromate B-9 regimens to synchronize estrus cycle for embryo transfer. Sexual behaviors; moounting, standing, orientation, chin-resting, sniffing, licking, rubbing and butting, vaginal swelling and mucus discharge were observed between 06-08, 12-14 and 18-20 h on 1st day and 00-02, 06-08, 12-14 and 18-20 h on 2nd day after removal of hormones. Synchromate-B treatment (81.6%) showed higher synchronized estrus rate than PRID treatment (77.8%) during observation period. Standing estrus was observed within 74 h after PG injection in PRID and within 52 h after removal of implant in Synchromate-B. About 68% of heifers in PRID and 74% of heifers in Synchromate-B showed standing estrus between 0-14 h on 2nd day after removal of the hormones. Synchromate-B resulted in a tighter synchrony of standing estrus than PRID. Incidence of average mounting and standing per head during observation period was 22.3 and 16.6 in PRID and 28.1 and 13.6 in Synchromate-B. The PRID showed peak in active mounting at 18-20 h on 1st day, however, the Synchromate-B showed at 0-2 h on 2nd day after removal of hormone. Active standing was shown between 18 h on 1st day to 20h on 2nd day in PRID, however, between 0-14 h on 2nd day after removal of hormone is Synchromate-B. There was slight difference in pattern of active mounting and standing during estrus between PRID and Synchromate-B. Conception rate of synchronized heifers transferred with fresh and frozen embryos by non-surgical and surgical methods was higher in synchromate-B (62.5%) than in PRID (38.5%). Chin-resting showed highest incidence among 6 sexual behavioral components in the both treatments. Synchromate-B showed higher incidence of chin-resting (16.6) than PRID (10.7). Synchromate-B group showed also higher incidence of orientation, sniffing and butting than PRID group. Synchromate-B resulted in more active sexual behaviors than PRID. The pattern of incidence of chin-resting, licking and butting was almost symmetrical in PRID with their peak values at 6-8 h on 2nd day, however in Synchromate-B chin-resting and sniffing was symmetrical with their peak values at 12-14 h on 2nd day after removal of hormone. There was tendency to increase vaginal swelling according to time passage of synchronized estrus in the both treatments. Incidence of mucus discharge in Synchromate-B was slightly higher than in PRID. Twenty to 40% was false negative in conception rate by tall painting before re-estrus day as judged by rectal palpation.

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Improved pregnancy rate and sex ratio in fresh/frozen in vivo derived embryo transfer of Hanwoo (Bos taurus coreanae) cows

  • Jihyun Park;Wonyou Lee;Islam M. Saadeldin;Seonggyu Bang;Sanghoon Lee;Junkoo Yi;Jongki Cho
    • Journal of Animal Science and Technology
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    • v.65 no.4
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    • pp.779-791
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    • 2023
  • This study aimed to assess the effects of embryonic developmental stage, quality grade, and fresh or frozen/thawed conditions on the pregnancy rate and sex ratio of live offspring in Hanwoo (Bos taurus coreanae) cows. The quality and developmental stage of in vivo-derived (IVD) transferred embryos were evaluated using the standard criteria of the International Embryo Technology Society. The recipient cows were synchronized using conventional (estradiol benzoate and progesterone) protocols before embryo transfer. Embryos were transferred to 297 cows, and pregnancy was monitored for 60-70 days after embryo transfer. The pregnancy rates of fresh and frozen/thawed embryos were 56.90% and 52.49%, respectively. Pregnancy rates varied according to embryo quality (56.18% for grade 1 vs. 36.67% for grade 2). Pregnancy rates also varied by developmental stage and cryopreservation (67.86% vs. 63.49% for stage 4-1, 64.00% vs. 54.72% for 5-1, and 50.00% vs. 47.83% for 6-1, in fresh embryos vs. frozen/thawed embryos, respectively). For stage 7-1, the pregnancy rates were 72.73% for fresh embryos and 20.00% for frozen/thawed embryos. In 66 fresh embryos, the sex ratio of live offspring was 5:5, whereas it was 4(female):6(male) for frozen/thawed embryos among the 95 frozen/thawed embryos. The miscarriage rate was approximately 3% higher for frozen/thawed embryos than for fresh embryos (18.1% for fresh vs. 21.1% for frozen). Seasonal fertility rates were 33.3% in spring, 55.67% in summer, 52.8% in autumn, 60.0% in winter. The following male-to-female ratios were observed in different seasons: 6.7:3.3 in spring, 4.0:6.0 in summer, 5.5:4.5 in autumn, and 3.3:6.7 in winter. The current data revealed no significant differences in pregnancy rates between fresh and frozen/thawed IVD embryos. However, there was a lower pregnancy rate with advanced-stage frozen/thawed embryos (stage 7-1). The current study provides comprehensive results for the better optimization of embryo transfer in Hanwoo cattle to obtain the desired fertility rate, pregnancy rate, and sex ratio of calves. These results provide important insights into the factors that influence the viability and success of IVD embryo transfer in Hanwoo cows and may have practical applications for improving breeding programs and reducing production costs.

Effects of hCG Administration on AI or Cloned Embryo Transfer of Korean Native Heifers (한우 미경산우에 있어서 인공수정 및 복제수정란 이식시 hCG 투여에 의한 수태율 향상)

  • 최선호;성환후;장유민;최재혁;임기순;양병철;연성흠;이장희;류일선
    • Journal of Embryo Transfer
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    • v.17 no.3
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    • pp.227-234
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    • 2002
  • This study was performed to investigate the improvement of the pregnancy rate of Al or clone embryo transfer on hCG administration in Korean Native heifer. A total of 42 heifers were treated with control, CIDR(withou E2 capsule). hCG after 7 day of hi, the pregnancy rate were spewed 53, 46, 71%. These results were significant different among the treatments(P<0.05). When the hCG were adminstrated at cloned embryo transfer, pregnanacy rate were control, hCG 5.8%, 10.4% respectively and there was no significant different between treatments. Plasma P4 concentration of hCG treatment in heifers were 3 times higher than control on 13~16 day after heat. After this, plasma P4 concentration of CIDR and hCG treated heifers were kept the 2~3 times levels. IGF-I concentration were showed no differences between pregnancy and non-pregnancy. hOG and CIDR. IGF-II concentration were revealed the differences between pregnancy and non-pregnancy in CIDR group but there was no differences in hCG administration group. Plasma cortisol were high at heat and 16 day after heat and CIDR treated group was higher than the other group. These results suggest that hCG administration was improve the pregnancy rates on Al and cloned ET, accompanying the incline of P4 concentration.

Study on the Propagation of Acanthopanax Plants II. Characteristics of Seed and Growth of Embryo in Stratifying Treatment (오갈피나무속(屬)(Acanthopanax) 식물의 번식(繁殖)에 관한 연구(硏究) II. 종자형질(種子形質)과 배(胚) 발육(發育)특성)

  • Ahn, Sang-Deuk
    • Korean Journal of Medicinal Crop Science
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    • v.1 no.1
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    • pp.16-23
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    • 1993
  • Seeds of Acanthopanx plants need about 2 years to germinate after sowing even if full matured fruits were harvested. I leave a room for doubt that this phenomenon could be brought by the immatured embryo in seed. If it is true, stratification method for the seed propagation of Acanthopanax plants would be used more effectively to promote the growth of embryo in relatively short time. Before stratification, seeds were devided into two parts. One of them was treated for 24hrs in the concentration of $GA_3$ 100 ppm. After stratification of non-treated and treated seeds, seeds for microtechnique were taken on interval of one month for three months, and fixed in Farmer's solution. The seed sizes of Acanthopanax plants were in biggest order A. sessiliflorus, A. seoulense, A. chiisanensis, A. koreanum and A. sieboldianum. The dehiscence phenomenon of seed coat didn't show in most of the seeds that stratified, but A. koreanum only dehisced in seeds treated in $GA_3$ 100ppm. The embryos of the stratified seeds that treated in $GA_3$ showed nomal growth, complete cotyledons and procambium in hypocotyl in any species, but the non-treated seeds could not expect the germination after sowing due to poor growing of the embryo in all species. In view of the results so far achieved, it has taken long times from sowing to germinate because the seed of Acanthopanax plants has immatured embryo.

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Roles of Theca and Granulosa Cells in Follicular Steroidogenesis in Rana dybowskii (북방산 개구리 여포의 스테로이드생성과정에 협막세포와 난구세포의 역할)

  • 안련섭;소재목;권혁방
    • The Korean Journal of Zoology
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    • v.39 no.3
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    • pp.273-281
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    • 1996
  • Previously, we have proposed a two-cell type model for follicular steroidogenesis inamphibians with Rana nigromacu lota. Present experiments were carried out to ascertain whether the model Is applicable to R. dybowskii. The role of theca layer were also reassessed by using granulosa cell-free pure theca layer (P-THEP). Theca/epithelium (THEP) layers, P-THEP layers, and granulosa cell enclosed-oocytes () were obtained from ovarian follicles of R. dybowskii by microdissection. Intact follicles (IFs) and different types of tissues were cultured for 6 hour in amphibian Ringer's m the presence or absence of FPII (0.05 gland/mi) or various steroid precursor (100 ng/ml). The amounts of product steroids converted by the components were measured by RIA. Exogenously added pregnenolone (P5) resulted in a marked increase in progesterone (P$_4$) by GCEOs (2143 pg/follicle) and IFs (2346 pg/follicle) but a smaller increase in P4 by THEP layer (495 pg/follicle). Addition of P$_4$ increased 17 a-hydroxyprogesterone (17 $\alpha$-OHP$_4$) levels by GCEOs (1118 pg/follicle) and IFs (1333 pg/follicle) but less by THEP layer (290 pg/follicle). However, much less amounts of P$_4$ or 17 $\alpha$-OHP$_4$ were producad by P-THEP layers than THEP in the presence of P5. Exogenous 1 7$\alpha$-OIIP$_4$ increased androstenedione (AD) levels by GCEOs (1415 pg/follicle) and IFs (561 pg/follicle) but not by THEP layers. In contrast, addition of AD resulted m a marked increase in testosterone (T) levels by TIIEP (2594 pg/follicle) and IFs (2223 pg/follide) but much less by GCEOs (339 pg/follicle). Exogenous T increased estradiol (E$_2$) levels by GCEOs (551pg/follicle) and IFs (887 pg/follicle), but not by THEP layer (<10 pg/follicle). Without addition of FPH or steroid precursors, very low or nondetectable levels of steroids were produced (< 20 pg/follicle) by all the types of follicular components examined. The data presented here indicate that the two-cell type model based on the study with R. nigromacu Iota is applicable to R. dybowskii and also suggest that the minor pathway, which convert P5 to 17$\alpha$-OHP$_4$, is not present in theca layer.

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Experimental Studies on Uterine Catecholamines (Catecholamines에 관(關)하여 -제5편(第五編) : 자궁(子宮) catecholamines에 관한 실험적(實驗的) 연구(硏究)-)

  • Lee, Woo-Choo
    • The Korean Journal of Pharmacology
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    • v.19 no.1
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    • pp.37-60
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    • 1983
  • The uterus receives adrenergic terminals from the mesenteric ganglia and considerably large amount of catecholamines have been shown to be contained in this organ. On the other hand, the activities of epinephrine, norepinephrine or adrenergic nerve on uterine motility is so complicated that many controversial results have been reporter. Recently, a large number of reports concerning the changes of uterine catecholamines content have appeared, but little is known about the role of uterine catecholamines in their activities on uterine motility. The present experiments were undertaken to determine the significance of the intrinsic uterine catecholamines in the physiology of uterus. Female albino rabbits weighing approximately 2 kg were employed in this experiment. uterine strip3 were prepared and suspended in a constant temperature $bath(38^{\circ}C)$ containing 100 ml of Locke's solution aerated with 95% oxygen and 5% carbon dioxide. Spontaneous motility was recorded on a smoked drum with an isotonic lever. The catecholamines concentration of the uterus was determined according to the Procedure described of Shore and Olin (1958). Human uterus obtained from patients was also used to determine the catecholam ines content of myometrium. Followings are summarized results. 1) On the non-pregnant rabbit uterine strips, epinephrine and norepinephrine significantly elevated the tonus and stimulated the spontaneous motility. Pretreatment with dichloroisoproterenol(DCI), an adrenergic beta-receptor blocker, enhanced the stimulatory activity of epinephrine or norepinephrine. On the other hand, pretreatment with dibenamine, an adrenergic alpha-receptor blocker, rendered the uterine muscle to exhibit inhibition after the administration of epinephrine or norepinephrine. Following the treatment with both DCI and dibenamine, epinephrine or norepinephrine produced no appreciable effects on the spontaneous motility of the uterus. These results suggest there exist both alpha and beta-adrenergic receptors in the uterine muscle and the response to epinephrine of the former is predominant over that of latter in the non-pregnant uterus of rabbits. The total catecholamines concentration of the non-pregnant uterus was $351\;m{\mu}g/g$ and the fractional concentrations of epinephrine and norepinephrine were $125\;m{\mu}g/g(35.7%)$ and $226\;m{\mu}g/g$ respectively. It is interesting to note that the catecholamines content of uterus was characterized by a high fractional corcentration of epinephrine relative to norepinephrine. 2) On the pregnant rabbit uterine strips, the effects of epinephrine and norepinephrine varied according to the period of pregnancy. The response to epinephrine of adrenergic beta receptor of uterus increased during pregnancy, and the effect of catecholamine was inhibitory in the early pregnancy but became stimulatory as the pregnancy progressed. This stimulating action on the uterine motility was found to occur through the action of norepinephrine. The uterine catecholamines concentration was markedly reduced during pregnancy. The catecholamines concentration was started to decrease in the early pregnancy, reached the lowest level in the mid-pregnancy and then started to increaae again in the late pregnancy when the total catecholamines content became the highest level of all. This increase of catefholamines in late pregnancy was chiefly due to the increase of norepinephrine. These results suggest that the uterine motility may be related to the catecholamines content, especially norepinephrine content in the uterus. 3) Bilateral oophorectomy of rabbits results in a marked shrink of the uterus in size. The spontaneous motility of the uterine segment of these animals was very weak and irregular. Norepinephrine produced inhibitory effect, whereas epinephrine was stimulatory or inhibitory effect on the uterine segment. The total catecholamines tontent in whole uterus was markedly reduced. The injection of estrogen into the oophorectornized rabbit increased the weight of uterus to approximately three times of that of oophorectornized animal. The apontaneous motility and the response to epinephrine and norepinephrine of the uterine segment were greatly enhanced. Both epinephrine and norepinephrine produced a marked stimulatory effects of the uterine motility. The uterine content of catecholamines, particularly epinephrine, was markedly increased. The injection of progesterone into the oophorectornized rabbit increaeed the weight of uterus to approximately 2.5 times of that of eophorectornized animal. The spontaneous motility of the uterine segment was weak and irregular. Epinephrine produced stimulatory effect at high concentrations but norepinephrine always prcdnced inhibitory effect on the uterine segment. The uterine content of catecholamines, particularly of norepinephrine, was markedly reduced. These results suggested that ovarian hormones play an important role not only on the growth and spontaneous norepinephrine of uterus but also on the catecholamines content and responee to epinephrine and norepinephrine of the uterus. 4) The intraperitoneal injection of reserpine(3 mg/kg) into the non-pregnant, pregnant and oophorectornieed rabbits markedly decreased the uterine content of catecholamines, particularly of the norepinephrine. The stimulatory response to epinephrine and. norepinephrine of the uterine segment of these reserpinized ratbits was markedly reduced whereas the inhibitory response to these catecholamines was enhanced. This finding further support the close relationship between the uterine catecholamines content and uterine response to epineptrire and norepinephrine. 5) In the human uterus, the concentration of epinephrine was actrally greater than that of norepinephrine and it was significantly greater during the proliferative phase of the menstrtal cycle. In the human pregnant uterus, the concentrations of toth epinephrine and ncrefinephrine were markedly reduced and showed about 45 percent rednction after 6-8 weeks of ectopic Pregnancy. At full term ana during labor, the concentrations of epinephrine and norepinephrine at placental sites were less than those found in the non-pregnant group. Of interest was the finding that the norepinephrine concentration of uterus from toxemic patients was two and half times higher than that of lower uterine segment of the nontoxemic pregnant individuals. Also the epinephrine concentraticn was slightly increaeed.

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Effects of Steroid Hormone in Avian Follicles

  • Caicedo Rivas, R.E.;Nieto, M. Paz-Calderon;Kamiyoshi, M.
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.4
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    • pp.487-499
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    • 2016
  • The aim of the present study was to examine the effects of testosterone (T) and estradiol-$17{\beta}$ ($E_2$) on the production of progesterone ($P_4$) by granulosa cells, and of the $E_2$ on the production of $P_4$ and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest ($F_1$) and third largest ($F_3$) preovulatory follicle were incubated for 4 h in short-term culture system, $P_4$ production by granulosa cells of both $F_1$ and $F_3$ was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or $E_2$. In the second experiment, $F_1$ and $F_3$ granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and $E_2$. Basal $P_4$ production for 48 h during 48 to 96 h of the cultured was about nine fold greater by $F_1$ granulosa cells than by $F_3$ granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not $E_2$, stimulated in a dose-dependent manner $P_4$ production in both $F_1$ and $F_3$ granulosa cells. In addition, when the time course of $P_4$ production by $F_1$ granulosa cells in response to oLH, cVIP, T and $E_2$ was examined for 48 h during 48 to 96 h of culture, although $E_2$ had no effect on $P_4$ production by granulosa cells of $F_1$ during the period from 48 to 96 h of culture, $P_4$ production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, $P_4$ production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when $F_1$ granulosa cells were precultured with $E_2$ for various times before 4 h culture with oLH at 96 h of culture, the increase in $P_4$ production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of $F_1$, $F_2$ and the largest third to fifth preovulatory follicles ($F_{3-5}$) were incubated for 4 h in short-term culture system with increasing doses of $E_2$. The production of $P_4$ and T by theca internal cells were increased with the addition of $E_2$ of $10^{-6}M$. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on $P_4$ production, and on $E_2$ in long-term action which may enhance the sensitivity to LH for $P_4$ production, and thus, in theca internal cells, $E_2$ in short term action may stimulate the production of $P_4$ and T.