• Journal of Embryo Transfer
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• v.11 no.2
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• pp.103-109
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• 1996

The aim of this study was to evaluate the effect of recombinant bovine somatotropin (bST; Boostin-S, LG Chem) treatment with FSH (Super OV) or PMSG on superovulatory response for transvaginal ultrasound-guided oocyte retrieval (TVR) in calves. Eight Korean Native Cattle(KNC) heifer calves; 150 to 240 days old; were randomly assigned to four treatment groups: 1) FSH(75 mg); 2) FSH (75 mg) + bST(500mg) 3) PMSG(1;000 IU); 4) PMSG(1, 000 IU) + bST(500 mg). Experimental calves in group 1 (n=2) and 2(n=2) were weekly superovulated for 4 consecutive weeks with daily injection of FSH for 3days and the next day subjected to TVR session. Animals in group 3 (n=2) and 4(n=2) were weekly stimulated for 4 consecutive weeks with a single dose of 1, 000 IU PMSG. TVR was performed on 72 hours after PMSG injection. Calves in group 2 and 4 was received injection of 500 mg of bST every 10 days. At each TVR session, follicle number and size were recorded; the oocytes collected and graded according to cumulus and cytoplasm investment. Collected oocyte were determined viable oocyte according to morphological quality with granulation of oocyte and number and status of cumlulus cells. IVM and IVF were performed and assessed cleavage rate on day 3 after fertilization. A Sonovet 600(Medison, Co., Ltd) realtime ultrasound scanner with a 6.5 MHz convex transducer, fixed at the tip of 500 mm estended handle equipped with a needle guide was used in collecting oocyte. Differences between groups were analysed by chi-square test. The population of large follicle ($\geq$5 nun) and aspiration rate were not significant different among the 4 groups. But, the number of small follicles (<5 mm) and aspirated oocyte in the KNC calves treated with bST were 1.3~1.6 times higher than in KNC calves treat with FSH or PMSG alone. In conclusion, the administration of bST with FSH or PMSG at superovulation for TVR in calves was increase the nurnber of small follicle which was influenced the number of aspiratable follicle.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.353-357
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• 2008

A series of studies has been conducted to establish a base infrastructure for an ovarian follicle culture system in the porcine and this study was designed to develop an effective retrieval protocol of preantral follicles. Five different methods using collagenase type I (A) or IV (B, C1, C2 and C3), which employed different treatment durations and/or conditions, were employed and sliced ovarian tissue of prepubertal gilts was provided for the retrieval. A significant increase in total number of follicles retrieved was detected when collagenase IV (methods B or C) was used. In total, more ovarian follicles were retrieved by method B undertaking agitation and method C2 without the agitation than method C1 and C3, while the number of preantral follicles collected was the largest in method B. Neither incubation in 5% $CO_2$ in air atmosphere instead of the agitation nor increased duration of enzymatic treatment up to 120 minutes improved the efficiency of follicle retrieval. There were no differences in the number of follicles retrieved from intact ovaries and from used ovaries for oocyte collection. These results demonstrate the collagenase IV treatment with agitation is effective for retrieving porcine preantral follicles from the ovaries.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1711-1715
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• 2006

To predict the number of preantral (primordial, primary and secondary) follicles retrieved from bovine ovaries, we examined the relationship between morphological parameters of ovaries and number of preantral follicles retrieved mechanically. The preantral follicles were retrieved mechanically by slicing ovarian tissue and the influences of size of the ovaries, number of antral follicles, and presence of cystic follicle and corpus luteum on the retrieval were evaluated. Total 77 ovaries were used and significant (p<0.05) relationship was detected between the number of antral follicles and the presence of cystic follicles, and the retrieval number. More preantral follicles were retrieved from the ovaries having more than 20 antral follicles than those having less than 20 antral follicles (17,760${\pm}$5,637 vs. 3,689${\pm}$537) in the ovarian cortex. The retrieval number was significantly reduced in cystic ovaries compared with non-cystic ovaries (5,167${\pm}$825 vs. 20,631${\pm}$6,507). However, neither ovary size (<3.5, 3.5 to 4.0, 4.0 to 4.5 and >4.5 cm) nor the presence of corpus luteum affected the follicle retrieval. In conclusion, the number of preantral follicles retrieved from the ovaries can simply be predicted by the number of antral follicles and the presence of cystic follicles in the ovarian cortex.

• Journal of Embryo Transfer
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• v.5 no.1
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• pp.1-20
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• 1990

Follicular atresia is a universal and characteristic phenomenon of both non-mammalian and mammalian vertebrates. Generally it is estimated that greater than 99% of follicles become atretic in higher domestic animals and human. The number of selected follicles developing to the preovulatory stage are thus fewer. Follicles can become atretic at any stage of development. The previous studies emphasized on descriptive and retrospect aspects of a limited population of the fully grown preovulatory follicle. The main efforts in ovarian physilogical researches are focused on follicular development culminating in ovulation but recent advances have resulted in a better understanding of atresia. Nowadays, recent studies are concentrated on the induction of atresia in a selected population of follicles and of the associated cellular, endocrine, biochemical and molecular changes. The factors initiating atresia and follicle selections are worthy of investigations. Another intriguing question is whether one can predict when a follicle will become atretic, i.e., what biochemical markers indicate that a follicle is destined for atresia. It is generally agreed that atretic process may vary even in antral follicles at different stages of their differentiations and among species. The dicisive factors are follicular responsiveness and the hormonal milieu. Some generalizations can be made on the basis of experimental induction of atresia. Alteration of the pattern of follicular steroid production is associated with the initiation stage of atretic process. Atresia appears to be a process unfolding gradually and affecting progressively in increasing number of functions and components of the follicle. The oocyte may be the latest to be afflicted in the atretic process. The high steroidogenic activity of atretic follicles lends support to the notion that atresia is not necessarily a degenerative process and that atretic follicles may play an essential role in ovarian physiology. The simultaneous occurence of growth and atretic processes may render the search for regulatory mechanisms involved in atresia difficult extremely. The questions such as how follicles are selected to undergo ovulation rather than atresia or what the mechanism of atresia is remain unanswered. However, the factors regulating or modifying ovarian hormonal milieu for the initiation of follicular growth and maturation or of atresia are being elucidated.

• Biomedical Science Letters
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• v.16 no.4
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• pp.349-357
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• 2010

We have examined the histological changes of skin during hair follicle growth after depilation in C57BL/6N mice. We first studied on histological changes of number of mast cells and thickness of skin during hair follicle growth periods (telogen, 1 day, 3 day, 5 day, 10 day, 14 day, 17 day and 21 day after depilation) by toluidine blue, Giemsa and H&E staining methods. We second studied immunoreactive density of cytokines and Brdu labeled cells in skin during hair follicle growth periods after depilation in C57BL/6N mice by immunohistochemical methods. The histological changes on skin thickness was increased from telogen to 14 day. The number of mast cells was decreased in 3,5 and 10 day and increased in 14, 17 and 20 day after depilation. Immunoreactive density of cytokines [protein kinase C-${\alpha}$ (PKC-${\alpha}$), c-kit, and vascular endothelial growth factor (VEGF)] in 1, 3, 5, 10, and 14 day after depilation was mildly stained in bulge and cutaneous trunci m., but immunoreactive density of cytokines in 17 and 21 day was heavily stained in epidermis, bulge, outer root sheath (ORS), inner root sheath (IRS) and cutaneous trunci m.. Immunoreactive density of Brdu labeled cells in skin in 1 and 3 day was heavily stained in bulge, epidermis and connective tissue under the cutaneous trunci m.. In all periods, immunoreactive density of Brdu labeled cells in skin was heavily stained in bulge, subcutaneous tissue, cutaneous trunci m, ORS and IRS. These experiments suggest that histological changes related to hair follicle growth elevated mast cell counts, skin thickness and epidermis thickness and heavily stained immunoreactive density of cytokines and Brdu labeled cutaneous trunci m. and connective tissue under the cutaneous trunci m. after depilation in C57BL/6N mice.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.61-69
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• 2007

This study was designed to evaluate effects of BSA, PVA, gonadotropins and follicle shell during IVM of porcine oocytes and subsequent development to the blastocyst stage after IVF. Cumulus oocyte complexes (COCs) were cultured in TCM-199 media containing 4 mg/ml BSA and 1 mg/ml PVA during IVM for 44 hr. To compare the effect of gonadotropins on oocyte maturation, COCs were cultured with FSH+LH, FSH, LH and FSH-LH-free media during IVM. respectively. Also, different number of follicle shells (0, 2, 4 and 6) was used to examine whether the presence of follicle shell in culture medium affects oocyte maturation. The percentages of fertilization and blastocyst formation, respectively, were higher in the medium containing the PVA (49.0 and 17.9%) than those containing the BSA (40.0 and 12.2%). Significantly higher rates of Mil oocytes were in the presence of FSH+LH and FSH (88.6 and 85.1 %) compared to other treatments (64.0 and 53.4% at LH and FSH-LH-free media). Co-culture with inverted follicle shells in 2 ml maturation medium enhanced the developmental competence of porcine oocytes. In conclusion, PVA could be used as a macromolecules instead of BSA, and FSH and follicle shell played important roles in maturation of porcine oocytes.

• Korean Journal of Veterinary Research
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• v.40 no.4
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• pp.769-775
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• 2000

This study was investigated the ovarian response following superovulation treatment at different day of diestrus. The criterion for the presence or absence of a dominant follicle based on their morphological examination. Dominant follicle was puntured 48 hrs before the oneset of superovulation treatment by ultrasonography guided aspiration needle. Superovulation was induced by subcutaneous administration of FSH twice a day for 4 day in a decreasing regimen. There was no significant different between presence of dominont follicle and progesterone concentration/diameter of corpus luteum in HanWoo. Number of corpus luteum of donor after superovulation treatment was not significantly different in FSH administration at day 9, 11 and day 13 of estrus($14.5{\pm}4.5$, $15.5{\pm}5.6$ and $11.0{\pm}5.5$, respectively). But, the diameter of CL was significantly correlate(R2 = 0.757) with progesterone levels on day of superovulatory induction. After 7 days of artificial insemination, the embryos at 7 days were collected by uterine flushing after dominant follicle aspiration and superovulation treatment, and evaluated their quality by morphological criteria. Fifty five embryos with excellent, good and fair grade were transferred into 24 recipient cows. Seventeen offsprings, 1 of triplet, 4 of twins and 6 of singlet, were yield from 10 recipient cow. In conclusion, the present study showed that 1) dominant follicle can be determined by ultrasonography with rectal palpation by morphological evaluations, 2) superovulation response after follicular aspiration was not differ at day 9, 11 and 13 of estrus, 3) dominant follicle did not affect to progesterone concentration and diameter of CL, and 4) diameter of CL was significantly correlate to the level of progesterone concentrations in HanWoo.

• Korean Journal of Animal Reproduction
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• v.9 no.2
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• pp.97-104
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• 1985

This study was conducted to investigate the effects of unilateral ovariectomy on the weight of the remaining ovary, the change of number of ovarian follicle, number of corpus luteum and serum progesterone level. Sixty Sprague-Dawley female rats, 23$\pm$2 days old, were divided into 2 groups (control and unilaterally ovariectomized goup) with 30 heads per groups. Each group was again subdivided into 6 groups according to 6 experimental periods; Day 4, 8, 12, 16, 20 and 24 after uniteral ovariectomy. Five arts at every 4 day intervals were sacrificed for the measuring of ovarian weight and for quantitative histologic examination of ovary and at the same time, blood samples were taken for the determination of serum progesterone level of radioimmunoassy. The results obtained were as follows: During the experimental periods, a significant hypertrophy occured in the remaining ovary of unilaterally ovariectomized group from day 16 after operation. The average ovarian weight of control group at day 16 was 21.0$\pm$1.7mg, which is samller than that of unilaterally ovariectomized group weighing 50.5$\pm$8.4mg(P<0.01). The ovarian weight of the unilaterally ovariectomized rats at day 20 and day 24 was 75.9$\pm$2.2 mg and 63.3$\pm$7.0 mg, which is heavier than those of control group; 29.1$\pm$2.3 and 26.3$\pm$1.7 mg(P<0.01 and P<0.01). 2. A same degree of ovarian follicle development was observed in the unilaterally ovariectomized group. Following unilateral ovariectomy and there was no change in total number of follicles larger than 130$\mu$ during the period from day 4 till day 24 after operation. 3. Although the size fo ovarian follicle did not significantly change between two groups from day 4 till day 16, the size of vesicular follicle in unilaterally ovariectomized group (406.3$\pm$26.2$\mu$) was significantly greater as compared to that of control group (323.8$\pm$19.3$\mu$)(P<0.05). 4. Corpus luteum in unilaterally ovariectomized and control group began to a, pp.ar from day 16 after operation and then the number of corpus luteum slightly increased. The number of corpus luteum in unilaterally ovariectomized group at day 24 ws remarkably increased (13.7$\pm$1.41) than that of control (5.2$\pm$2.01)(P<0.01). 5. Serum progesterone levels in unilaterally ovariectomized group were slightly higher than those of control but there were no significant difference between treatment groups.

• Clinical and Experimental Reproductive Medicine
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• v.15 no.2
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• pp.135-147
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• 1988

To compare the stimulation effect of the ratio in follicle stimulating hormone and luteinizing hormone in induction of multiple follicular growth, the serum $E_2$ level, the diameter of follicle, number of aspirated follicles and cleavage rate of in vitro fertilized preovulatory oocytes as well as the pregnancy rate were evaluated. Forty one patients with irreparable tubal disease were stimulated by hMG(n=24) or FSH/hMG(n=17) for the purpose of in vitro fertilization and embryo transfer. The following results were obtained. 1. Serum estradiol($E_2$) levels on the day of hCG administration were $921.0{\pm}353.3\;pg/ml$ in hMG group and $1272.9{\pm}1060.6\;pg/ml$ in FSH/hMG group. The serum $E_2$ value of hMG group was significantly lower than that of FSH/hMG group. 2. The diameter of leading follicle by ultrasonogram on the day of hCG administration were $16.2{\pm}2.0\;mm$ in hMG group and $16.2{\pm}2.6\;mm$ in FSH/hMG group. No significant difference of follicle diameter between two groups was demonstrated. 3. The number of follicles with diameter above 10 mm by sonogram on the day of hCG injection were $3.91{\pm}2.32$ in hMG group and $6.52{\pm}3.86$ in FSH/hMG group. There was significant difference of number of follicles between two groups, (p< 0.01). 4. The number of oocytes found per patient at aspiration were $2.59{\pm}1.00$ in hMG group and 3. $76{\pm}2.31$ in FSH/hMG group. There was significant difference of number of aspirated oocytes between two groups. (p< 0.05). 5. The detection rate of preovulatory oocyte at aspiration were 68.4%(39/57) in hMG group (n=22) and 77.6%(38/49) in FSH/hMG group (n=13). 6. The cleavage rate of preovulatory oocyte at 44 hours after insemination were 74.4%(29/39) in hMG group(n=22) and 81.6%(31/38) in FSH/hMG group (n=13). When only hMG was used, one pregnancy was established in 15 patients to whom 29 zygotes were transferred. And a full term normal female baby was delivered by elective cesarean section. In the FSH/hMG group, five pregnancies out of 9 transferred patients were confirmed by serum ${\beta}-hCG$. Two pregnancies were spontaneously aborted before the 6th week of pregnancy. One patient aborted her baby at the 18th week of pregnancy because of incompetent internal os of the cervix. Two patients delivered two full term babies by elective cesarean section. From the above findings, paralell with the increase in the ratio of exogenous follicle stimulating hormone to luteinizing hormone, an increase in oocyte recovery was observed as well as an improvements in pregnancy rate. It was concluded that FSH enrichment early in the follicular phase had a beneficial effect in the controlled ovarian hyperstimulation.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.255-264
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• 1997

The purpose of this study was attempted to investigate the number changes of the growing and mature follicles in ovary following repeats of pregnant mare serum gonadotropin(PMSG) treatments for superovulation in nulliparous rats. Thirty two rats(Sprague-Dawely, about 200~250 gm) were randomized into 4 groups. Control group rats were sacrified at estrus phase confirmed by vaginal smear. PMSG-treated group 1 rats, PMSG-treated group 2 rats and PMSG-treated group 3 rats were sacrified at 48 hrs after injection once with PMSG 25 IU, after 2 repeated injection by a week interval, and 3 repeated injection, respectively. The uteri and ovaries of rats were removed and weighed and then were observed grossly and serial sections of all ovaries and some sections of uteri by paraffin embedding were stained with H-E. Number of ovarian follicles about 3 grades of small, middle and large follicles from seondary and follicles were investigated by LM photographies of ovary preparations. The criteria of the small, middle, and large follicles were based as small follicle with preantral follicles with 2~4 layers of granulosa cells surrounding the oocyte, as secondary follicles with more than 5 layers of granulosa cells and early signs of antral cavity or with small clefts on either side of the oocytes, and as tirtiary follicles with a single medium sized antral cavity or large well-formed antral cavity, respectively. In gross findings, the wall of the uteri in control group were thin, and those in 3 PMS-treated group were markedly thickened and some uterine lumen of those filled with fluid. In histological findings, the walls of the uteri from 3 PMSG-treated groups were hypertrophied and their blood and lymph vessels were dilated than those of control group. The ovaries fo 3 PMSG-treated groups were more increased in size and the cortexes were more developed and increased in width but there are no difference of development and changes in 3 PMSG-treated groups. The weight of the uteri and ovaries per rat in PMSG -treated group 1, 2 and 3 were a, pp.ared to be significantly increased 171.4$\pm$47.6%, 162.3$\pm$43.9%, 206.9$\pm$30.4%, respectively than those of control groups. The mean number of follicle per ovary in control group were a, pp.ared to be 17.1$\pm$3.5, 46.2$\pm$14.5, and 74.3$\pm$22.7 at large, middle and small follicles, respectively and total number of these 3 grade follicles per ovary were a, pp.ared to be 137.7$\pm$31.7. The mean number of follicle per ovary in PMSG-treated group 1 were a, pp.ared to be 25.6$\pm$7.3, 78.1$\pm$29.9, and 83.2$\pm$34.0, at large, middle and small follicles, respectively and total number of these 3 grade follicles were a, pp.ared to be 187.5$\pm$58.8. The mean number of follicle per ovary in PMS-treated group 2 were a, pp.ared to be 21.9$\pm$5.2, 67.8$\pm$16.8, and 68.0$\pm$14.9 at large, middle and small follicles, respectively and total number of these 3 grade follicles were a, pp.ared to be 157.7$\pm$26.2. The mean number of follicle per ovary in PMS-treated group 3 were a, pp.ared to be 21.7$\pm$4.8, 61.5$\pm$17.0, and 59.7$\pm$16.2 at large, middle and small follicles, respectively and total number of these 3 grade follicles were a, pp.ared to be 143.5$\pm$29.6. The number of follicles in PMSG-treated group 1 a, pp.ared to be more number than other 2 PMSG-treated gruops and tended to be decreased by frequency of PMSG-treatment.