• Animal Bioscience
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• v.35 no.12
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• pp.1850-1859
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• 2022

Objective: The quantitative reverse transcription polymerase chain reaction (qPCR) is the most accurate and reliable technique for analysis of gene expression. Endogenous reference genes (RGs) have been used to normalize qPCR data, although their expression may vary in different tissues and experimental conditions. Verification of the stability of RGs in selected samples is a prerequisite for reliable results. Therefore, we attempted to identify the most stable RGs in the hypothalamic-pituitary-gonadal (HPG) axis in sows. Methods: The cycle threshold values of nine commonly used RGs (18S, HPRT1, GAPDH, RPL4, PPIA, B2M, YWHAZ, ACTB, and SDHA) from HPG axis-related tissues in the domestic sows in the different stages of estrus cycle were analyzed using two RG-finding programs, geNorm and Normfinder, to rank the stability of the pool of RGs. In addition, the effect of the most and least stable RGs was examined by normalization of the target gene, gonadotropin-releasing hormone (GnRH), in the hypothalamus. Results: PPIA, HPRT1, and YWHAZ were the most stable RGs in the HPG axis-related tissues in sows regardless of the stages of estrus cycle. In contrast, traditional RGs, including 18S and ACTB, were found to be the least stable under these experimental conditions. In particular, in the normalization of GnRH expression in the hypothalamus against several stable RGs, PPIA, HPRT1, and YWHAZ, could generate significant (p<0.05) elevation of GnRH in the preovulatory phase compared to the luteal phase, but the traditional RGs with the least stability (18S and ACTB) did not show a significant difference between groups. Conclusion: These results indicate the importance of verifying RG stability prior to commencing research and may contribute to experimental design in the field of animal reproductive physiology as reference data.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.42-47
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• 1988

This experiment was carried out to investigate the sex ratio of produced lamb after artificial insemination into the cervix with spermatozoa from the top and bottom portion of ram semen separated by diluting semen a column of protein, and after implantation into the uterus with normal morulae and blastocyst cultured in BMOG-3 and Ham F-10 medium containing H-Y anbibody and complement treated with spleen and testis, respectively. All embryos developed to morulae and blastocyst were cultrued in medium under gas phase of 5% CO2 in air at 37$^{\circ}C$, 24 hrs. Estrus of ewes induced by a MAP vaginal sponge and 750 IU PMSG during the non-breeding season. The result obtained in these experiments were summarized as follows: 1. The ratio of heating, lambing, and prolificacy after artificial inseminatin into the cervix of ewes induced by a MAP vaginal sponge and 750 IU PMSG during the non-breeding season were 100%, 40% and 2.74 heads, respectively. 2. Involving 44 ewes, spermatozoa from the top of the protein column produced 23.1% male and 76.9% female lambs, while spermatozoa from the bottom of the column produced 81.3% male and 18.8% female offspring, respectively. 3. The sex ratio (male-to-female) of progeny produced after implantation with normal morulae and blastocyst cultured in medium containing H-Y antibody and complement treated with spleen and testis were 21.0%, 79.0% and 17.4%, 82.6%, respectively.

• Korean Journal of Animal Reproduction
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• v.9 no.1
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• pp.9-30
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• 1985

The study was carried out to find out the changes of the sex hormone levels in the milk of Holstein cows during the reproductive stages such as the estrous cycle, pregnancy and periparturient period. The FSH, LH, estradiol-17$\beta$ and progesterone from the milk samples were assayed by radioimmunoassay methods. The results of this study were summarized as follows: 1. The levels of progesterone and estradiol-17$\beta$ were similar among inter-quarters, but they were higher in after milking than before milking times, with no statistical significance. 2. The milk progesterone levels during the estrous cycles reached a peak mean level of 3.55$\pm$0.26ng/$m\ell$ at 15 days after estrus and they did not show any differences among the length of estrous cycles. The estradiol-17$\beta$ levels during the estrous cycles showed a peak level of 36.40$\pm$2.38pg/$m\ell$ at estrus, and decreased(17.20$\pm$0.46 pg/$m\ell$ to 18.65$\pm$1.26pg/$m\ell$) at luteal phase. 3. The FSH levels during the estrous cycles ranged from 2.25$\pm$0.23mIU/$m\ell$ to 4.35$\pm$0.24mIU/$m\ell$ showing significant changes. The LH levels during the estrous cycles gradually increased and remained a peak level of 10.90$\pm$0.36mIU/$m\ell$ from 20 to 25 days after estrus. 4. The progesterone levels during the pregnancy were decreased from 30 to 60 days after artificial insemination, and therafter continuously increased until 240 days. The estradiol-17$\beta$ levels during the pregnancy were 24.56$\pm$1.19pg/$m\ell$ at day 30 after artificial inseminaton, and increased rapidly until 180 days. The levles were agagin decreased by 26.17$\pm$3.03pg/$m\ell$ until 210 days and markedly increased by 68.00$\pm$8.70pg/$m\ell$ until 240 days. 5. The prolactin levels during the pregnancy were 31.27$\pm$2.31ng/$m\ell$ and 42.60$\pm$2.37ng/$m\ell$ at day 150 and 240 after artificial insemination respectively. The LH levels during the pregnancy reached a peak of 27.47$\pm$7.90mIU/$m\ell$ at day 30 after artificial insemination, and thereafter gradually decreased. 6. The progesterone levels during the periparturient period reached a peak of 4.61$\pm$0.34ng/$m\ell$ at day 3 prepartum, and thereafter gradually decreased, and showed 2.05$\pm$0.60ng/$m\ell$ at day 7 postpartum. The estradiol-17$\beta$ levels during the periparturient period showed high level from 207.23$\pm$6.04pg/$m\ell$ at day 1 prepartum to 239.90$\pm$13.90pg/$m\ell$ at day 2 prepartum, and thereafter began to decline and reached 51.87$\pm$1.72pg/$m\ell$ at by 7 postpartum. 7. The prolactin levels during the periparturient period showed relatively higher level at the time of parturition. The LH levels during the periparturient period rnage from 6.32$\pm$0.32mIU/$m\ell$ to 13.90$\pm$1.37mIU/$m\ell$ showing significant changes. 8. The progesterone levels(4.6$\pm$0.8ng/$m\ell$) of the pregnant cows were significantly higher than those (1.84$\pm$1.4ng/$m\ell$) of nonpregnant cows. The cows of artificial insemination from 61 to 90 days after parturition showed higher progesterone levels. 9. During 20 to 25 days after artificial insemination, the accuracy of pregnancy diagnosis from milk progesterone levels were 94.4% for nonpregnant cows(<2.3ng/$m\ell$), and 75.0% for pregnant cows( 3.2ng/$m\ell$). The average overall accuracy of pregnancy prediction for nonpregnant and pregnant cows 83.3% 10. The results obtained this study suggest that the understanding of the endocrinological mechanisms by means of milk hormone analysis during the estrous cycle, pregnancy and parturition would give the basic information needed for increasing efficiency of reproduction. This study would not only provide an accurate method of the early pregnancy diagnosis by milk progesterone levels but also contribute to the research of providing the method of detecting of FSH levels in milk, which was difficult in blood serum.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.265-270
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• 2012

In canine, oocytes are ovulated at the GV (germinal vesicle) stage and they have to fulfill maturation phase before reaching metaphase II stage. The efficiency of in vitro maturation is still very low. Therefore, the aim of this study was to investigate the effect of in vitro maturation on nuclear changes of immature canine oocytes recovered from different reproductive stages ovaries and different culture conditions. The oocytes were cultured in TCM-199 with supplement at 5% $CO_2$ and $38.5^{\circ}C$ for 72 h. The nuclear maturation of canine oocytes was evaluated with Hoechst 33342 stain under fluorescence microscope (Fig. 1). The results of this study detected differences in in vitro maturation rate between oocytes recovered from follicle status and non-follicle status ovaries. However, these differences were not significant as indicated in Table 1 and Fig. 2. In regard to the effect of culture condition with supplements, we did not found significant differences compared with control group (Table 2, Table 3). One of the reasons for this data could be the conditions that ovaries were exposed during slaughtering process or the long distant transportation of the ovaries. Although these data have not shown clearly significant differences results compared with control, furthermore the different reproductive status ovaries was beneficial for maturation of oocytes in vitro and can be a basic part of knowledge to improve in vitro maturation of canine oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.2
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• pp.192-200
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• 2019

Objective: Leukemia inhibitory factor (LIF) binds to a heterodimeric receptor composed of LIF receptor (LIFR) and glycoprotein 130 (GP130) to transmit signals into the cell. LIF plays an important role in reproduction by regulating immune response, decidualization, and implantation in several species. However, the expression of LIF and LIFR in the endometrium throughout the estrous cycle and pregnancy in pigs is not fully understood. Methods: We analyzed the expression of LIF and LIFR in the endometrium on days 0 (estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle, and days 12, 15, 30, 60, 90, and 114 of pregnancy, in conceptuses on days 12 and 15, and in chorioallantoic tissues on days 30, 60, 90, and 114 of pregnancy in pigs. We also determined the effects of estrogen and progesterone on the expression of LIF and LIFR in endometrial tissues. Results: The expression of LIF increased in the endometrium during the late diestrus phase of the estrous cycle and during mid- to late- pregnancy, while the expression of LIFR increased during early pregnancy. The expression of LIF was induced by increasing doses of estrogen, whereas the expression of LIFR was induced by increasing doses of progesterone. Conclusion: These results indicate that the expression of LIF and its receptor LIFR in the endometrium is regulated in a stage-specific manner during the estrous cycle and pregnancy, suggesting that LIF and its receptor signaling system may play critical roles in regulating endometrial function in pigs.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.165-171
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• 1993

While ornithine decarboxylase (ODC) is considered a key enzyme in the biosynthesis of polyamines, difluoromethylornithine(DFMO) acts as an inhibitor of polyamine synthesis. Cycling crossbred gilts were randomly assigned to one of two (treatment and control) groups (6/group). An indwelling silicone catheter was surgically implanted in the jugular vein of each animal. DFMO was dissolved in saline(200 mg/ml) and adminstered by i. m. injection at a dose of 80 mg/kg/day. The control group received an equivalent volume saline injection. DFMO was injected 3 times daily(08:00. 16:00. 24:00h) from day 16 of estrous cycle to 21 or until estrus. Once daily blood samples (10ml) were taken from day 14 until two days after the last DFMO treatment. Window blood samples were collected every 15 min for 8 h (from 08:00 to 16:00h) starting on day 16 and continuing until day 21 from one gilt per day. Serum progesterone (P$_4$), estradiol (E$_2$), LH and FSH were measured. Typical concentration profiles for P$_4$ and E$_2$ were seen during the follicular phase regardless of DFMO treatment. Injection of DFMO suppressed the preovulatory LH concentration in the serum(p<0.01) while having no effect on FSH profile. The present results indicate that DFMO had an inhibitory effect on LH secretion in the pig, but did not affect PI, E2 or FSH release.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.4
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• pp.460-466
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• 2004

This study was undertaken with the aim to establish a reliable radioimmunoassay (RIA) system for urinary pregnanediol glucuronide (PdG) and to employ it for monitoring the reproductive status of dairy cows. Urine and blood samples were collected from the Holstein cows both pregnant and non-pregnant. The samples were then investigated for evaluating the relationship between progesterone ($P_{4}$) in blood and PdG in urine adjusted with or without urinary creatinine basis. Biweekly urine collection was employed for three cows in estrous and those artificially inseminated, while urine from pregnant cows was collected on a monthly basis. P_{4}$ and PdG levels were measured by enzymeimmunoassay (EIA) and RIA techniques, respectively. Our results indicated the sensitivity of PdG for RIA being 35 pg/tube and the recovery rate of 100%. Urinary creatinine concentrations also fluctuated within a day, but change at midday was not noteworthy. Regardless of the time of urination the change in concentrations of PdG was relatively smaller and did not vary significantly. The urinary PdG concentration showed periodic changes as that with serum P_{4}$ levels during the cow's estrus cycle. The correlation coefficient rose when creatinine level in urine was adjusted but the change was also not significant. The concentrations of PdG during the luteal phase were detected between 8.2 and 17.4 ng/ml, three to five times higher than that in the follicular phase. The concentration of PdG from pregnant cows (21 days after conception) was three to four times higher than in the nonpregnant cows. Our finding suggests that the determination of urinary PdG could be reliably employed for early pregnancy detection. The urinary PdG level continued to raise until 30 days pre-partum while the concentration reached its peak at 30 ng/ml, after which it started to fall 18 to 30 days before parturition and finally fell to its nadir value one week after parturition. As the correlation coefficient between the urinary PdG and serum P_{4}$ was higher than that corrected by urinary creatinine it can be suggested that the adjustment is not needed. The concentrations of urinary PdG could be maintained stably for 2 days in urine samples stored at room temperature and extended to 8 days when the samples were pretreated by boiling for 30 minutes. In conclusion urinary PdG concentration even without the need for creatinine basis adjustment can be used directly for monitoring the reproductive status of dairy cows.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.175-180
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• 1999

Blood chemical values and sex steroid hormone concentration of repeat breeder and reproductive disorder in Hanwoo, which were bred in Chunchon areas, were examine to establish the basic physiological maker. The concentrations of albumin, blood urea nitrogen(BUN), calcium(Ca), cholesterol, creatinine, glucose, phosphorous, total protein and triglycerides in repeat breeder and reproductive disorder were 3.28$\pm$0.05 and 3.27$\pm$0.04 g/㎗, 11.07$\pm$0.61 and 12.69$\pm$0.88 mg/㎗, 9.98$\pm$0.10 and 9.56$\pm$0.11mg/㎗, 105.75$\pm$3.57 and 126.78$\pm$5.66mg/㎗, 1.62$\pm$0.05 and 1.65$\pm$0.09 mg/㎗, 67.46$\pm$9.07 and 76.97$\pm$3.76mg/㎗, 6.13$\pm$0.36 and 6.11$\pm$0.26mg/㎗, 5.82$\pm$0.08 and 6.18$\pm$0.08 g/㎗ , 12.82$\pm$1.46 and 15.19$\pm$1.61mg/㎗, respectively. Among the blood chemical values, Ca, cholesterol and inorganic phosphorous levels in repeat breeder and reproductive disorder were slightly higher than those in normal cows. The progesterone levels of pregnancy(0.18~6.56 ng/$m\ell$) and non-pregnancy(0.15~5.46ng/$m\ell$) cows at estrus cycle were showed similar trends from 0 day to 18 days. Those of pregnancy cows were increased gradually from 18 days to 180 days(5.90~7.51 ng/$m\ell$), while those of non-pregnancy cows were dramatically drop at 21 days (0.18$\pm$0.03mg/㎗). The concentrations of estrogen and progesterone in repeat breeder and reproductive disorder were 57.42$\pm$5.03, 56.76$\pm$8.73 pg/$m\ell$ and 6.26$\pm$0.83, 9.41$\pm$1.85 ng/$m\ell$ . respectively. These results indicate that blood chemical values in repeat breeder and reproductive disorder were not greatly difference in normal cows except for Ca, cholesterol, BUN and inorganic phosphorous. Since the progesterone levels in repeat breeder and reproductive disorder was maintained to the luteal phase levels of normal cows, it may cause of failing of conception and next estrus cycle.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.11
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• pp.1650-1655
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• 2003

Crossbred gilts (n=54) of about $26.38{\pm}0.85kg$ body weight and 25 weeks of age were randomly divided into 18 groups of 3 animals each. Three dietary treatments viz., $T_1$, $T_2$ and $T_3$ were formulated. The treatment $T_1$ containing maize grain (35%) and wheat bran (47%) along with soybean meal and fish meals served as control diet. Rice bran (RB) was selected as a single source of fat (13.02%) and fibre (17.12%), which was gradually increased in diets $T_2$ (41%) and $T_3$ (82%) replacing maize grain and wheat bran of $T_1$ at 50% and 100% in the diets $T_2$ and $T_3$, respectively. The fat and fibre levels of the diets were thus 3.46 and 5.24, 9.31and 9.69 and 11.61and 13.26% in $T_1$, $T_2$ and $T_3$, respectively. All the diets, however, contained almost similar concentration of CP (18.35${\pm}0.29%$). Each dietary treatment was offered to six replicated groups of 3 piglets in following completely randomized design and feeding was continued for 112 days during the growing phase. Growth, feed utilization, reproductive performance, nutrient utilization and different blood biochemical parameters were studied. Growth rate, feed intake and feed conversion were lower (p<0.01) in $T_3$ in comparison to $T_1$ or $T_2$. Digestibility of all the nutrients except EE was reduced significantly (p<0.01) in $T_3$. Serum glucose level decreased (p<0.01), whereas the urea and cholesterol concentration in the blood increased (p<0.01) in $T_3$. The duration of estrus of pigs fed diet containing 82% RB ($T_3$) was highly variable within the group, but all the groups showed statistically similar duration of estrous. Feed cost per unit gain was found to be comparable between control ($T_1$) and $T_2$ group. The results indicated that RB can be included up to 41% in swine diets replacing 50% of maize and wheat bran. Higher concentrations of ether extract and fibre beyond 9.31 and 9.69% in diet had detrimental effect on growth, nutrient utilization and reproductive performance.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.257-261
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• 2008

The objective of present study was to investigate the effect of seasons on reproductive performance of Hanwoo and Holstein heifers. Heat stress in summer or cold stress in winter stress to Hanwoo and Holstein heifers may bring reproduction failure, which would pose an important economic loss, even around Daegwallyeong region located in high mountainous area. Seasonal differences in the serum levels of LH, FSH and progesterone ($P_4$) in response to environmental factors (hot and cold) out of 20 pubertal Hanwoo heifers in Daegwallyeong, Gangwon Province and 20 non-lactating Holstein heifers in Chonan city of Republic of Korea at 2-3 years of age were compared. Blood samples for hormonal analysis were from jugular vein after detection of estrus repeatedly over four seasons within four-week intervals (Spring: May to June, Summer: July to August, Autumn: October to November and Winter: January to February). In Hanwoo heifer population, averages of LH and FSH concentration in spring and in summer were greater compared to those in winter (p<0.05). LH or FSH levels tended to be greater (p=0.06) in spring and less (p=0.09) in winter compared to the levels in autumn. Only in summer, cattle seemed to show lower LH or FSH secretion (p<0.05). Similar to the results in Hanwoo heifers, the serum concentrations of LH and FSH in Holstein heifers decreased further by heat stress in summer when P 4 levels were high during luteal phase. The results demonstrate significant effect of summer heat on reproduction of Hanwoo or Holstein heifers. Although parameters indicating the extent of heat stress were not measured in this study, we suggest that serum hormone levels could be considered as successful indicators of summer heat stress condition for Hanwoo and Holstein heifers even under rather cool summer climate.