Changes in interleukin-2, -4, -6 and -8 expression in the postovulatory sow endometrium after artificial insemination based on conceived or failed to conceive.

The establishment and maintenance of a pregnancy requires proper interaction between the endocrine and immune systems in the uterus. Therefore, it is crucial to understand how changes in endometrial cytokine levels facilitate reproduction. This study aimed to investigate how representative cytokines sequentially changed in the endometrium and whether conception could be attributed to these changes. In this study, artificial insemination was performed twice in 160 sows and ovulation was examined every 3 h using transrectal ultrasonography. Uterine endometrial tissues were obtained via repeated biopsies at 2, 4, 6, 8, 12, 16, and 20 h after ovulation and interleukin (IL)-2, IL-4, IL-6, and IL-8 expression was examined using real-time polymerase chain reaction. The conception rate was 91.9%. The IL-2 levels showed no differences in conception or time. The expression peaks of IL-4 and IL-6 were delayed in sows that failed to conceive within 4-6 h and 2 h, respectively, compared to those that did conceive. In sows that conceived, IL-8 was highest after 2 h, and no difference was observed at other time point, regardless of conception. In sows that failed to conceive, the increase in IL-8 levels might have been cancelled or terminated before the first sampling time. These results highlight the importance of timely increases and subsequent declines in the levels of some cytokines for the establishment of pregnancy. Differences in uterine capacity start just after ovulation; detection and correction of these deviations can improve the reproductive efficiency of sows.

Physiological high temperatures alter the amino acid metabolism of bovine early antral follicles.

Heat stress reduces the developmental competence of bovine oocytes during the growth phase; however, the detailed mechanisms remain unclear. Amino acids play various critical roles in follicular development, including protein synthesis and as energy sources. We performed in vitro growth (IVG) culture of oocyte-cumulus-granulosa complexes (OCGCs) to assess the amino acid metabolism of small follicles at high temperatures. We isolated OCGCs from early antral follicles (0.5-1.0 mm) and subjected them to IVG culture for 12 days. OCGCs in the heat shock group were cultured under a temperature cycle of (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, and 39.5°C: 9 h) to reproduce the body temperature of lactating cows under a hot environment. OCGCs in the control group were cultured at a constant temperature of 38.5°C for 24 h. Of the surviving OCGCs, those showing similar morphology and size between the groups were selected for amino acid analysis. We analyzed the free amino acids and their metabolites in the culture medium and calculated the depletion or appearance of molecular species. The depletion of three essential amino acids (isoleucine, leucine, and valine), two non-essential amino acids (aspartic acid and glycine), and ornithine was higher in the heat shock group (P < 0.05). Alanine depletion was lower in the heat shock group (P < 0.05). We concluded that heat exposure alters the amino acid metabolism of OCGCs isolated from early antral follicles, which might be involved with the diminished developmental potential of oocytes during summer.

Pre-maturational culture promotes the developmental competence of bovine oocytes derived from an 8-day in vitro growth culture system.

In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

Analysis of sequential ruminal temperature sensor data from dairy cows to identify cow subgroups by clustering and predict calving through supervised machine learning.

The present study investigated the applicability of a calving prediction model based on supervised machine learning of ruminal temperature (RT) data in dairy cows. The existence of cow subgroups for prepartum RT changes was also examined, and the predictive performance of the model was compared among these subgroups. RT data were collected from 24 Holstein cows at 10 min intervals using an RT sensor system. The average hourly RT was calculated and data were expressed as residual RTs (rRT = actual RT - mean RT for the same time on the previous three days). The mean rRT decreased beginning at approximately 48 h before calving to a low of -0.5°C at 5 h before calving. However, two cow subgroups were identified: cows with a late and small rRT decrease (Cluster 1, n = 9) and those with an early and large rRT decrease (Cluster 2, n = 15). A calving prediction model was developed using five features extracted from the sensor data (indicative of prepartum rRT changes) through a support vector machine. Cross-validation showed that calving within 24 h was predicted with a sensitivity of 87.5% (21/24) and precision of 77.8% (21/27). A significant difference in sensitivity was observed between Clusters 1 and 2 (66.7 vs. 100%, respectively), while none was observed for precision. Therefore, the model based on RT data with supervised machine learning has the potential to efficiently predict calving, although improvements for specific cow subgroups are required.

Effects of heat stress on the endometrial epidermal growth factor profile and fertility in dairy cows.

The endometrial epidermal growth factor (EGF) profile is an indicator of uterine function and fertility in cattle. The present study aimed to investigate the effects of heat stress on the endometrial EGF profile and fertility in lactating Holstein cows. The endometrial EGF profiles of 365 cows in the Hokkaido and Kyushu regions were examined between June and September (heat stress period, n = 211) and between October and January (control period, n = 154). EGF profiles were investigated using uterine endometrial tissues obtained by biopsy 3 days after estrus (Day 3). The proportion of cows with an altered EGF profile was higher between June and September than between October and January (41.2 vs. 16.2%, P < 0.05). The effects of rectal temperature on Days 0 and 3 on the endometrial EGF profile were also assessed in cows (n = 79) between June and September in the Kyushu region. A single embryo was transferred to cow on Day 7 to evaluate fertility (n = 67). Regardless of the rectal temperature on Day 3, the proportion of cows with an altered EGF profile was higher (64.1 vs. 30.0%, P < 0.05) and the pregnancy rate after embryo transfer (ET) was lower (26.7 vs. 51.4%, P < 0.05) in cows with a rectal temperature ≥ 39.5°C on Day 0 than in cows with a rectal temperature < 39.5°C on Day 0. The present results indicate that alterations in the endometrial EGF profile induced by an elevated body temperature on Day 0 contributed to reductions in fertility in lactating dairy cows during the heat stress period.

Relationship between the timing of insemination based on estrus detected by the automatic activity monitoring system and conception rates using sex-sorted semen in Holstein dairy cattle.

We investigated the optimal timing of artificial insemination (AI) for achieving pregnancy according to the onset/end of estrus detected by an accelerometer system in Holstein cattle. The conception rates of conventional semen were used as a reference. The conception rate from AI of sex-sorted semen was higher at -4 to 4 h (57.1%) from the end of estrus than those at -12 to -4 h (37.7%) and 12-20 h (30.3%), whereas AI at 4-12 h showed an intermediate conception rate (47.4%). Conversely, conception rates were similar in AI performed between 0 and 32 h from the onset of estrus. Regarding conventional semen, the interval from the onset and end of estrus did not affect conception rates. The present results suggest that the time of the end of estrus is the better indicator of optimal AI timing for sex-sorted semen than the onset of estrus.

Leptin receptor expression and its change in association with the normalization of EGF profile after seminal plasma treatment in repeat breeder dairy cows.

Factors associated with high milk production levels have been linked to alterations in the endometrial epidermal growth factor (EGF) profile, a cause of reduced fertility in dairy cows. Therefore, we examined the leptin system that connects nutritional status and reproduction in dairy cattle related to reduced fertility in repeat breeder cows. Plasma leptin concentrations were measured in 18 heifers, 20 high-yielding control cows, and 26 repeat breeder cows, showing an altered EGF profile. Then, all repeat breeder cows were infused with seminal plasma (SP) into the vagina at the next estrus to normalize the EGF profile, while heifers and control cows were infused with vehicle alone. All animals were examined for EGF profiles. Eighteen repeat breeder cows, nine heifers, and nine control cows were also determined for leptin receptor (Ob-R) expression levels in the estrous cycle before and after the infusion. SP normalized the EGF profile in 53.8% of the repeat breeder cows. Leptin concentrations were similar in all groups, regardless of the treatment results for the EGF profile. In contrast, Ob-R levels in repeat breeder and control cows were similar and higher than those in heifers before SP treatment. Ob-R in repeat breeders showing a normal EGF profile after treatment decreased to an intermediate level between heifers and control cows and may provide a clue to take measures against repeat breeding in dairy cows.

Effect of seminal plasma infusion into the vagina on the normalization of endometrial epidermal growth factor concentrations and fertility in repeat breeder dairy cows.

Epidermal growth factor (EGF) concentrations in the uterus show two peaks on days 2-4 and 13-14 during the estrous cycle in fertile cows. Loss of the two peaks has been linked to reduced fertility in repeat breeder cows. This study aimed to examine the effect of seminal plasma (SP) on normalizing endometrial EGF concentrations and restoring fertility in repeat breeder cows with low EGF concentrations on day 3. In study 1, we examined the effect of the deposition sites (the vagina and uterus) of SP on the endometrial EGF concentrations in repeat breeder cows. SP infusion into the vagina, but not uterus, on the first day of the estrus cycle (day 0) normalized the endometrial EGF concentrations (≥ 4.7 ng/g tissue weight) on day 3. In study 2, the effect of SP volume (0.5 and 10 ml of SP and 0.5 ml of SP diluted to 10 ml) on EGF concentrations was examined. All groups with SP infusion had increased EGF concentrations on day 3, and cows with 10 ml of SP and 0.5 ml of SP diluted to 10 ml showed the highest levels of EGF concentrations. In study 3, we examined the effect of SP infusion on fertility. SP infusion normalized two peaks of endometrial EGF concentrations in about 60% of repeat breeder cows and produced more pregnancies than the controls (44.4 vs. 19.4%). Therefore, we concluded that SP may contain an activity to normalize the EGF profile and restore fertility in repeat breeder cows with altered EGF profiles.

Monitoring follicular dynamics to determine estrus type and timing of ovulation induction in captive brown bears (Ursus arctos).

It is important to understand ovarian physiology when developing an artificial insemination (AI) protocol. Brown bears (Ursus arctos) have a breeding season from May to July, although the type of estrus (polyestrus or monoestrus) is still contested. The present study aimed to define the ovarian dynamics, including follicular waves and ovulatory follicle size, and estrus type in brown bears. Six brown bears were used for ovarian ultrasonography; four were observed between April and October (before the start and after the end of the breeding season) and two in June (breeding season). In addition, we attempted to induce ovulation by administering a gonadotropin releasing hormone (GnRH) agonist. We observed follicular development in April in four bears, but follicles did not develop to greater than 6.0 mm in diameter until May. Thereafter, a group of follicles developed to more than 6.0 mm and grew as dominant follicles, except in one bear. After ovulation and subsequent corpus luteum (CL) formation, the follicular waves disappeared. Furthermore, in three bears treated with GnRH, follicles between 8.2 to 11.2 mm in diameter at the time of treatment ovulated and formed CLs. In two bears, follicles between 5.8 to 8.8 mm ovulated spontaneously within the observation interval. Our results suggest that brown bears may be monoestrous animals. Therefore, AI can only be performed once during the breeding season. Our results also suggest that dominant follicles larger than 8.0 mm are a suitable size for inducing ovulation.

Relationships between the antral follicle count, steroidogenesis, and secretion of follicle-stimulating hormone and anti-Müllerian hormone during follicular growth in cattle.

BACKGROUND: The antral follicle count (AFC) in mammalian ovaries positively correlates with female fertility. To clarify the causes of differences in fertility between low and high AFC cows, we investigated follicular growth dynamics and hormone concentrations in plasma, follicular fluid, and in vitro growth (IVG) media at different stages of follicular growth. METHODS: Seven cows were divided into high AFC (n = 4, > 30 follicles) and low AFC (n = 3, < 30 follicles) groups based on the peak AFC detected by ultrasonography. These cows were subjected to estrous synchronization, daily ovarian ultrasonography, and blood collection. Their follicular fluid was collected from dominant follicles at different stages (selection, luteal, and ovulatory phases). In another experiment, we cultured oocyte-cumulus-granulosa cell complexes collected from early antral follicles (< 1 mm) for 12 days. Estradiol-17ß (E2), testosterone (T), progesterone (P4), and anti-Müllerian hormone (AMH) concentrations in follicular fluids and plasma were measured. Plasma follicle-stimulating hormone (FSH) concentrations were examined. E2, P4, and AMH concentrations were also measured in IVG media. RESULTS: The numbers of small (< 4 mm) and intermediate (4-8 mm) follicles were larger in the high AFC group than in the low AFC group (P < 0.05). The number of intermediate follicles was stable in the low AFC group, indicating consistent development. However, the number of these follicles fluctuated in the high AFC group. Plasma FSH concentrations were higher, whereas E2 and T concentrations were lower in the low AFC group (P < 0.05). E2 concentrations and the E2/P4 ratio in ovulatory follicles and IVG media on day 8 were higher in the high AFC group (P < 0.05). AMH concentrations in plasma and IVG media (P < 0.01) were higher in the high AFC group. CONCLUSIONS: The weaker response to FSH of granulosa cells caused low E2 production in the low AFC group, resulting in high FSH concentrations and the consistent development of intermediate follicles. Conversely, higher E2 concentrations suppressed FSH secretion in the high AFC group. Granulosa cells in the high AFC group had the ability to produce more AMH than those in the low AFC group throughout IVG culture.

Relationship between bovine endometrial thickness and plasma progesterone and estradiol concentrations in natural and induced estrus.

The objective of this study was to investigate cyclical changes in endometrial thickness in relation to progesterone (P4) and estradiol-17ß (E2) concentrations during natural and induced estrus in 15 cows. In the prostaglandin (PG) F2α-induced estrus group, ultrasonography (USG) at 6-h intervals was used to determine endometrial thickness 48-24 h before the PGF2α treatment until 24 h after ovulation (ovulation = Day 0). In the natural estrus group, USG was performed every 48 h from Day 3 to Days 15-18 after the first ovulation, and then every 6 h until 24 h after ovulation. Endometrial thickness was standardized using Day 13 as a reference day. Blood was collected during every USG examination and plasma P4 and E2 concentrations were determined. Endometrial thickness of the induced estrus group (n = 11) was greater than that of the natural estrus group (n = 9) between 60 and 12 h before ovulation (P < 0.05). In the natural estrus group, prior to an increase in endometrial thickness, a decrease in P4 and an increase in E2 were detected. In the induced estrus group, based on the time of ovulation, an increase in endometrial thickness was detected at the same time of a decrease in P4 before an increase in E2. These results suggest that decreases in P4 concentrations may be a cue to changes in endometrial thickness, while increases in E2 concentrations appear to sustain and/or enhance these changes.

Effects of pre-maturational culture duration on developmental competence of bovine small-sized oocytes.

We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

Effect of a single epidural administration of follicle-stimulating hormone via caudal vertebrae on superstimulation for in vivo and in vitro embryo production in Japanese black cows.

Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.7 ± 3.5 and 9.0 ± 6.0, respectively). Next, we compared in vitro embryo production by ovum pick-up and in vitro fertilization (OPU-IVF) between control (n = 12) and epidural groups (n = 12). The rate of development to transferable blastocysts was higher in the epidural group than in the control (23.3 vs. 10.5%, P < 0.001). In conclusion, a single epidural administration of FSH can induce follicular development comparable to that of the conventional superovulation protocol and may improve the productivity of OPU-IVF.

Relationship between the antral follicle count in bovine ovaries from a local abattoir and steroidogenesis of granulosa cells cultured as oocyte-cumulus-granulosa complexes.

The antral follicle count (AFC) is used as an indicator of cow fertility. We herein investigated the relationship between AFC and the steroidogenesis of granulosa cells and confirmed the developmental competence of oocytes derived from early antral follicles (0.5-1.0 mm) using in vitro growth culture. Slaughterhouse-derived ovaries were divided into high (≥ 25) and low (< 25) AFC groups based on AFC (≥ 2.0 mm). Oocyte-cumulus-granulosa complexes (OCGCs) collected from early antral follicles were cultured for 12 days. The total number, viability, and diameter of granulosa cells and estradiol-17ß and progesterone production during the culture were evaluated. Surviving oocytes on day 12 were subjected to in vitro maturation, and their volume and nuclear status were evaluated. Some oocytes were subjected to the evaluation of developmental competence to blastocysts. Although the total number and viability of granulosa cells did not differ between the groups, granulosa cell diameters were smaller in the high AFC group than in the low AFC group. The estradiol-17ß and progesterone ratio on day 8 was higher in the high AFC group than in the low AFC group. Oocyte volumes and nuclear maturation rates were greater in the high AFC group than in the low AFC group. The development rate to blastocysts was 9.1% in the high AFC group, while no oocytes developed to blastocysts in the low AFC group. Therefore, estradiol-17ß production by granulosa cells appears to be greater in high AFC cattle than in low AFC cattle, thereby promoting the acquisition of oocyte competence.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions.

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

A transvaginal endoscopy-based technique for performing ovarian examinations in sows.

For examining pig ovaries, which have complex structures, laparoscopy is a useful technique, but requires general anesthesia; therefore, it cannot be performed repeatedly within a short period of time. We report a transvaginal endoscopy-based technique for conducting ovarian examinations without general anesthesia. Sows were sedated in pig stalls. Using a colonoscope, the vaginal wall was punctured with a trocar. To avoid the trocar being caught in the broad ligament of the uterus or the connective tissue around the vagina, the trocar was inserted close to the external uterine os and between the 2:00 and 3:00 or the 9:00 and 10:00 positions (in a clockwise direction). Then, a urethroscope was inserted into the abdomen, and an examination was carried out after the ovaries had been moved towards the urethroscope camera via rectal palpation. This less invasive procedure may allow repeated examinations and will increase our understanding of ovarian dynamics in pigs.

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice.

The preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

Endometrial epidermal growth factor profile and its abnormalities in dairy cows.

Normal cows have 2 peaks in endometrial epidermal growth factor (EGF) concentrations on Days 2-4 and 13-14, and the absence of peaks has been linked to reduced fertility in repeat breeder (RB) cows. However, the timing of the peaks (i.e., day of examinations) was estimated for a few cows per cycle day. Therefore, the present study characterized EGF peaks and examined if the absence of peaks in RB cows indicate either peak loss or changed timing. In Study 1, 20 Holstein cows were examined for EGF concentrations between Days 1 and 6 using repeated biopsy of the uterine endometrial tissues. Sixteen cows exhibited increased EGF concentrations for 2-3 days between Days 2 and 5. All 16 cows exhibited increased EGF concentrations on Day 3. In Study 2, 10 cows were examined for EGF concentrations between Days 11 and 16. Increased EGF concentrations for 2-3 days were found in 7 cows between Days 12 and 15. All 7 cows exhibited increased EGF concentrations on Days 13 and 14. In Study 3, 12 RB cows were examined for endometrial EGF concentrations between Days 1 and 6. Four cows exhibited an increase of EGF concentrations on Days 3 and 4, whereas 8 cows (66.7%) exhibited low EGF concentrations throughout the study period. In conclusion, Days 3 and 13-14 are suitable days to examine a cyclic change of endometrial EGF concentrations. Further, low EGF concentrations on Day 3 in RB cows indicated an absence, but not altered timing, of the EGF peak.

Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations.

Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17ß and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17ß, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17ß after copulation.

Effects of ovarian storage condition on in vitro maturation of Hokkaido sika deer (Cervus nippon yesoensis) oocytes.

The effects of different preservation conditions (temperature 20-25 or 10-15 degrees C; duration 12 or 24 h) for ovaries of Cervus nippon yesoensis on in vitro maturation of oocytes were examined. When ovaries were kept for 12 h at 20-25 degrees C, maturation rate of oocytes was highest (71%); however, it declined when the preservation time was extended to 24 h (31%). When the preservation temperature decreased to 10-15 degrees C, the maturation rate after 12 h preservation decreased (51%) but it remained in same level even though preservation time is prolonged to 24 h (55%).