본 연구는 우리나라 Holstein의 번식형질에 대한 유전모수를 추정하기 위하여, 2009년부터 2013년까지 분만한 농협 젖소개량사업소의 검정자료 경산우 144,793두의 211,899개의 번식기록을 이용하였다. 7가지 형질의 유전 및 오차 (공)분산을 WOMBAT 패키지를 이용하여 분석하였다. CTFS, GL, NRR, NS, FSTC, DO 및 CI의 유전력은 각각 0.064, 0.060, 0.005, 0.014, 0.012, 0.021 및 0.021로 추정되었다. 형질들 간의 유전상관은 –0.910에서 0.995로 나타났다. NRR과 NS, NRR과 FSTC 간에는 부의 유전상관이 나타났으나, DC와 CI는 강한 양의 유전상관을 나타냈다. CTFS와 FSTC는 DO 및 CI와 유의하게 양의 유전상관관계를 나타냈다(0.479~0.862). CTFS와 FSTC 사이의 낮은 유전상관은 두 형질이 서로 다른 유전적 측면에 있다는 것을 의미한다. 본 연구는 번식능력 개량을 위한 기초 자료를 제공할 수 있을 것이라 판단한다.

A pregnancy diagnosis is an important standard for control of livestock’s reproduction in paricular dairy cattle. High reproductive performance in dairy animals is a essential condition to realize of high life-time production. Pregnancy diagnosis is crucial to shortening the calving interval by enabling the farmer to identify open animals so as to treat or re-breed them at the earliest opportunity. MicroRNAs are short RNA molecules which are critically involved in regulating gene expression during both health and disease. This study is sought to establish the feasible of circulating miRNAs as biomarkers of early pregnancy in cattle. We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from 12 non-pregnant cows (“open” cows: samples were collected before insemination (non-pregnant state) and after pregnancy check at the indicated time points) on weeks 0, 4, 8, 12 and 16. Using small RNA sequencing we identified a total of 115 miRNAs that were differentially expressed weeks 16 relative to non-pregnancy (“open” cows). Weeks 8, 12 and 16 of pregnancy commonly showed a distinct increase in circulating levels of miR-221 and miR-320a. Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with pregnancy in cattle. Their application in the field of reproductive biology has opened up opportunities for research communities to look for pregnancy biomarker molecules in dairy cattle.

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and ‘EOMES transcription factor mRNAs’ were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

An experiment was carried out to assess the effect of feed selenium-lysine (Se-Lys) supplementation on milk compositions and serum biochemical indices in Saanen dairy goats in Korea. A total of twelve 36 months old Saanen lactating dairy goats (47±6.21 kg) fed the similar dry matter intake twice a day at 2% of BW (DMI) (10.9% moisture of concentrate and 19% moisture of roughage), milk yield (2.5 kg/d) and parity (2) were randomly selected and subjected for the present study, divided into two groups with six goats in each group. The goats in the control group received rice hulls (10 g/ day) only, and did not receive Se-Lys; goats in the treatment group were fed 0.06 g of Se-Lys with 10 g of rice hulls every day before feeding roughage for six weeks. The milk sample was collected every week, and its compositions were analyzed. The results of the present study showed that there is no significantly increased milk production in Se-Lys treated group goats when compared with control group goats. But, Se-Lys treatment significantly increased the milk protein content (3.98±0.16%), fat (3.72±0.27%), lactose (4.07±0.14%), total solids (12.51±0.28%) and urea (14.42±1.45 mg/dl) content as compared to the control group goats (p<0.05). The somatic cell counts (207,740±28.81 cells/ml) were significantly lower in the Se-Lys treated group than in the control group (p<0.05). Also, the results of the current study showed that supplementation of Se-Lys were significantly decreased the blood biochemical indices of IL-6 (34.34±6.04 pg/ml), TNT-α (0.56±0.22 ng/ml), MDA (5.07±1.03 ng/ml), GPx-1 (9.07±5.17 ng/ml), sCD4 (2.64±1.02 ng/ml) and sCD8 (5.08±2.08 ng/ml) level when compared with without addition of Se-Lys group dairy goats (p<0.05). On the other hand, the selenoprotein P (1,580.18±127.62 ng/ml) level was significantly higher in Se-Lys supplemented group than in the control group (p<0.05). Based on the study results, it was concluded that feed Se-Lys supplementation may improve milk yield with positively improved protein, fat, lactose, total solids, urea content, and biochemical indices without negative effects on milk production traits.

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/ c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

Although some factors, including season, age, type of estrus (natural estrus vs. induced estrus) and semen type (conventional vs. sexed), affect the conception rate following artificial insemination (AI) in dairy cattle, there is little information about the influence of ovarian characteristics, such as preovulatory follicle (PF) location at estrus, on fertility in dairy cattle. In most breeds of cattle the right ovary appears to function more actively than the left and about sixty percent of pregnancies in dairy cattle occur in the right horn of uterus (Reece and Tuner, 1938). Our study aimed to compare conception rates in dairy cattle between PFs that developed in the left ovary and those that developed in the right ovary at estrus. In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy cattle. In total, 955 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and dairy cattle were divided into two groups on their PF locations: (i) the PF located in the left ovary (L-PF); and (ii) the PF located in the right ovary (R-PF). Pregnancy was diagnosed by rectal palpation or ultrasonographic examination 60 days after AI. The conception rate was 38.1% in all dairy cattle. Conception rate was higher in the R-PF (40.8%) than in the L-PF (33.2%). In summary, PF development in the right ovary was associated with increased conception rates in dairy cattle.

The objective of this study was to evaluate the effect of treating dairy cattle with exogenous human chorionic gonadotrophin (hCG), five (5) days post artificial insemination (AI) on serum progesterone (P4) concentration and pregnancy rate. In this experiment, five days after AI, cows were assigned randomly to two groups namely: a) treated group (67) which were administrered with 1500 IU hCG (Chorulon) and b) control group (61), which received no treatment. On day 5, 10, 15 and 20 after the artificial insemination, blood samples from a total of 8 cows (4 from each group) were collected and were analyzed for serum P4 concentration. Cows were detected for estrus according to standing heat by visual observation. Cows that were detected still in estrus after days 18-24 were re-inseminated and recorded as not pregnant (open). Pregnancy diagnosis was conducted by ultrasonographic examination and transrectal palpation of the uterus on approximately 60 days in cows that observed to be not in estrus. The conception rate in hCG treated and control groups were 52.5 and 36.1%, respectively. The results proved that there were no significant differences in conception rate between two groups (p=0.0568). However, pregnancy rates were reduced by hCG treatment. Average serum P4 concentrations did not differ between Hcg-treated and control groups on day 5 (0.377 versus 0.375 ng/ml). On day 20 serum P4 concentrations were greater in the treated group compared with the control group (3.085 versus 2.010 ng/ml). The treatment with hCG seemed to increase P4 level compared with the control. In conclusion, the results of this study showed that 1500 IU of hCG administered on 5 day post AI increased conception rate in dairy cows. This was supported by the results on serum P4 concentration which was greater in hCG treated group.

Ensuring timely ovulation concerning the service is valuable. A satisfactory conception rate can be achieved by making sure that ovulation occurs within 7-18 hours after artificial insemination (AI). Delayed ovulation is one of the disturbances commonly encountered in repeat breeding animals. Although demanding research, many studies have not been conducted. Therefore, we aimed to examine the relation between ovulation confirmation and conception rate in dairy cattle. The research findings showed that the signs of true estrus were bred 12 hours after the onset of estrus by AI in cattle. Also, the performance of AI on ovulation was confirmed by the presence of fluctuant Graafian follicles through rectal palpation. From the results, we confirmed that cow encountered delayed ovulation were bred again. The Conception rate in cows with confirmed ovulation was 51.9%, while for those without confirmed ovulation were 33.3%. In conclusion, the results indicate that ovulation confirmation will likely increase conception rate.

High reproductive efficiency is a prerequisite for dairy animals in order to optimize dairy production. An accurate and early pregnancy diagnosis is a crucial aspect for better reproductive management in livestock. The indigenous, visual and clinical methods have various limitations including accuracy, sensitivity, specificity, later stages of applicability and requires highly skilled manpower. This hurdles the researchers to ensue further research on developing novel early pregnancy diagnostics for dairy animals. However, the advantage of molecular techniques like proteomics has given a new hope to look for pregnancy biomarkers in animal research. In this study, we assayed the pregnancy-associated glycoprotein (PAG) levels using anti-bPAG antibody. Serum plasma samples were collected 30, 60, 90 and 120 days after insemination. Cattle diagnosed pregnant with singleton are used for this experiment in which blood samples were collected. The plasma PAG level is gradually increased after insemination to until 60 days and drastically decreased after that. The PAG level was approximately 2-fold greater during 30 days compared with 60 days. These results concluded to validate our other pregnancy diagnosis methods including circulating microRNA and cell free DNA concentration (data not shown). Despite this proteomic approach, validating all other molecular technique results may give the exact time for early pregnancy diagnosis.

Growth rate during rearing, which varies depending on provided nutrition, has been related with age at 1st calving (AFC). This study investigated the effect of upgrowth parameters during the rearing period on the reproduction of nulliparous Holstein heifers. The study comprised 77 successively born heifers from the same herd. Growth rate and fertility traits were measured during rearing and fertility parameters were recorded in lactations 1. Growth parameters (body weight, height, heart girth and body length) were measured at the approximate birth time, 270 and 450 d of age. Reproduction data collected included age at 1st breeding, number of services per conception (S/C), pregnancy rate to 1st artificial insemination, AFC. Animals were subsequently divided into 4 AFC groups for analysis: <23 mo, 23∼ 25 mo, 26∼30 mo and >30 mo. The AFC reflected both upgrowth rate and heifer reproduction, with later calving heifers smaller. Increased skeletal growth (at 270 and 450 d) was related with a reduced AFC (p<0.05). Early calving animals (<23 mo) had the best reproduction as nulliparous heifers, with most conceiving at first service (87.5%). Fertility in the first lactation was the worst in the oldest AFC group (>30 mo). In the 1st lactation period, a number of services per conception (3.1±0.3) increased with increasing AFC (>30 mo). Sub-optimal upgrowth related with an increased AFC could be mitigated by improved monitoring of replacement heifers during the rearing period.

genetic selection has resulted in modern dairy cow with very high milk yields but reduced fertility, due mainly to an increase in reproductive disorders. The study was conducted to estimate the proportion of reproductive disorders among dairy cattle. The factors analyzed were; milk yield, cow parity, periparturient disorders, and reproductive status. The result of this study showed the incidence of reproductive disorders in high yielding dairy cows was prevalent. Repeat breeding was the major postpartum reproductive problem comprising (42.3%) of the samples. Higher reproductive disorders were also associated with lower parity. These disorders subsequently reduced reproductive performance by prolonged intervals from calving to first artificial insemination and from calving to pregnancy. Abnormalities in the reproductive system such as repeat breeder, silent heat, anestrous and ovarian cysts, as well as lower fertility have been recorded during the summer months (June to August). In conclusion, the study revealed that incidence of repeat breeder is mostly prevalent in dairy cows. Moreover, the incidence of reproductive disorders was higher in summer season and hence appropriate preventive and or therapeutic measures as per the type of abnormalities need to be undertaken.

The objective of this study was to analyze the accuracy of estrus detection of heat detector and analysis of estrus behavior (mounting and mounted), and the evaluation of conditions required for improving reproductive efficiency in Holstein dairy cows fitted with a estrous detector. The heat detection system consists of estrous detector based on wireless sensor and an electric bulletin board displayed estrus behavior data. When cow mounting other cows, the accuracy of estrus behavior displayed an electric bulletin board were 87.5% (mounting other cows only), 100% (mounting other cows but not standing), 80.0% (mounting other cows with standing for 1∼4 seconds), 90.0% (mounting other cows but not standing for 1∼4 seconds), 80% (mounting other cows with standing for more than 5 seconds) and 90.0% (mounting other cows but not standing for more than 5 seconds). When cow mounted other cows, the accuracy of estrus behavior displayed an electric bulletin board were 100% (mounted other cows but not standing), 100% (mounted other cows with standing for 1∼4 seconds), 100% (mounted other cows but not standing for 1∼4 seconds) and 100% (mounted other cows with standing for more than 5 seconds). Circadian distribution of first observed in estrus were 59.1% (am 8∼pm 6) and 40.9% (pm 6∼am 8). Distribution for the number of estrus behavior were 40.9% (less than 3 times), 36.4% (4∼6 times) and 22.7% (more than 4 times). The conception rates relative to interval from first estrus behavior to insemination for estrus periods were 23.1% (less than 11 hours) and 55.6% (12∼20 hours).

Embryo transfer (ET) technology is of high importance in modern cattle breeding programs. ET is one step in the process of removing one or more embryos from the reproductive tract of an outstanding donor female and transferring them to one or more recipient females. Embryos also can be produced in the laboratory via techniques such as in vitro fertilization (IVF). But the actual transfer of an embryo is only one step in a series of processes that may include some or all of the following: superovulation and insemination of donors, collection of embryos, isolation, evaluation and short-term storage of embryos, micromanipulation and genetic testing of embryos, freezing of embryos and embryo transfer. Cryopreservation and direct transfer of frozen-thawed embryos is common-place with pregnancy rates near that of fresh embryos. Polymerase chain reaction (PCR) technology is currently being used for sexing embryos, and this technology will be used for “embryo diagnostics” and “embryo genomics” in the future. Although, many limitations and problems remain to overcome, these and other new technologies promise to change livestock breeding drastically in the next decade.

This study aimed to improve the reproductive efficiency of dairy herds by comparison and analyzing estrous appearance rate, conception and non-conception rate according to the stage of lactation using the lactation and reproductive records of average (less than 10,000 liters milk in 305 days) and high yielding (more than 10,000 liters milk in 305 days) Holstein cows (n=102). Milk production and reproduction data were collected between January 2010 and December 2012 from Holstein cows kept in the commercial dairy farms. Average (n=32) and high yielding (n=24) Holstein cows used to analyze the relationship between milk yield and reproductive performance. Our results showed that estrous appearance rate according to the stage of lactation was 25.0% (30∼59d), 40.6% (60∼ 89d), 25% (90∼110d) and 9.4% (>111d) in average yielding cows and 16.7% (30∼59d), 20.8% (60∼89d), 12.5% (90 ∼110d) and 50.0% (>111d) in high yielding cows, respectively. Conception rate according to the stage of lactation was 87.5% (30∼59d), 61.5% (60∼ 89d), 75.0% (90∼110d) and 66.7% (>111d) in average yielding cows and 25.0% (30∼59d), 0% (60∼89d), 33.3% (90∼ 110d) and 50.0% (>111d) in high yielding cows, respectively. Days between parturition and conception was 23.7% (<149d), 0% (150∼209d) and 0% (>210 d) in average yielding cows and 69.0% (<149 d), 77.8% (150∼209d) and 38.9% (>210d) in high yielding cows, respectively. Conception rate from 110 days postpartum in high yielding cows was 41.7% (110∼150d), 50.0% (151∼180d) and 50.0% (>181d). Body condition score (BCS) in 120 days postpartum was 2.64±0.1 in average yielding cows and 2.28±0.1 in high yielding cows, respectively.

The oriental tobacco budworm, Helicoverpa assulta, is an oligophagous insect feeding on a few species of Solanaceae plants, including Nicotina tabacum. This study investigated the relationship between H. assulta and plants, focusing on oviposition preference and larval performance of the specialist in host and non-host plants. (1) In choice experiments, N. tabacum and Phaseolus vulgaris were put in a cage with 5 females and 10 males. Most of eggs (ca. 98%) were layed in N. tabacum, whereas few eggs in P. vulgaris. (2) In non-choice experiments, N. tabacum, Datura stramonium, or P. vulgaris was individually put in a cage with 3 females and 6 males, resulting that N. tabacum was the most preferred host, followed by D. stramonium and P. vulgaris. Oviposition was delayed about 2 days in P. vulgaris (a non-host plant) compared to the two host plants. (3) Larval performances were also compared with the three plants. Larval growth was better in N. tabacum followed by D. stramonium and P. vulgaris, High mortality (ca. 100%) of larvae was observed in P. vulgaris, suggesting toxic component(s) derived from the non-host plant might cause the death. The ovipositional avoidance in P. vulgaris is current being investigated to search for the oviposition deterrent, which could be useful for the environment-friendly pest management of H. assulta.

The objective of this study was to investigate the effects of abnormal ovarian cycles after superovulation treatment of Holstein Donor Cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with PGF2α and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. The cows were considered to have resumed ovarian cyclicity on the day of ovulation if followed by regular ovarian cycles. Seventy two point seven percentage of the cows(16/22) had normal resumption of ovarian cyclicity(resumption within 40 days after superovulation), and 27.3%(6/22) had delayed resumption(resumption did not occur until＞40 days after superovulation). Delayed resumption Type Ⅱ(first ovulation did not occur until ≥40 days after superovulation, i.e. delayed first ovulation 13.6%) were the most common types of delayed resumptions. The mean numbers of total ova from < 10 and 10≤ of corpora lutea(CL) was 7.8±1.8 and 12.7±2.7, respectively. The number of transferable embryos differed between < 10 and 10≤ CL was 5.4±1.3 and 8.1±3.4, respectively. Four point five percentage of the cows(1/22) did not resumption their ovarian cyclicity until 60 days after superovulation treatment. Diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.

today's dairy production management systems, reproductive efficiency affects the income of any farm by influencing overall milk production, genetic gain, amount of replacement heifers, and wise cullling decisions. Nutrition, management, and genetics play a major role achieving this maximum herd-reproductive performance. Several reports using trace minerals on the diet of cattle have shown reproductive effects. The main justification for using blocks, to provide deficient nutrients is, therefore, their convenience for packaging, storage, transport and ease of feeding. Dairy cattle are annually exposed to prolonged periods of elevated humidity and heat which reduce feed intake and reproductive performance. The objectives of the present study were to evaluate the effects of mineral on reproductive performance of dairy cows during Summer. The experimental group of cows had access ad libitum to the mineral lick in the following composition : Zn, Fe, Mn, Cu, I, Co, Se. Environmental heat load was described using the THI (temperature humidity index) because thermoregulation in cows is affected by both Temp and RH. The THI was calculated: THI=(0.8×Temp)+[RH×(Temp— 14.4)]+ 46.4. All cows were monitored for estrus twice daily in the morning and late afternoon. And any animal found in estrus was artificially inseminated. Pregnancy diagnosis was performed via ultrasonography at 45 to 70 d after insemination. The visitation and intake of mineral were higher with the maximum THI. The mean intake of mineral block was 23.1 kg in Summer and 17.7 kg in other seasons. The reproductive performance was considerably improved by mineral supplementation. The factors influencing consumption of mineral mixtures include: soil fertility and forage type, available energy and protein, individual requirements, salt content of the water, palatability of mineral mixture, availability of fresh minerals and physical form of minerals. These results show that minerals have a great impact on animal's reproductive physiology.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.