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In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte-cumulus-granulosa complexes (OCGCs) derived from early antral follicles (0.5-1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

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.

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 degrees C on Day 0 than in cows with a rectal temperature < 39.5 degrees 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.

A severe negative energy balance and high circulating free fatty acids (FFA) in postpartum cows impair fertility. The lipotoxicity of FFA has been shown to decrease the quality of bovine oocytes in vitro. Therefore, excess FFA in cells is converted to triacylglycerol (TAG), a non-toxic form, to avoid lipotoxicity. We recently reported that the TAG content in oocytes was higher in postpartum lactating cows subjected to grazing management than in heifers (Theriogenology 176: 174-182, 2021). The present study inves-tigated the compositions of the energy metabolism-related lipids, FFA and TAG, in the plasma and oo-cytes of cows at different lactation stages under indoor intensive feeding management in order to obtain insights into lipotoxicity in oocytes, particularly those in early postpartum cows. Blood and oocytes were collected from 20 lactating cows categorized into the following lactation groups: 20-30 days in milk (DIM) (n = 5), 40-50 DIM (n = 5), 60-80 DIM (n = 5), and 130-160 DIM (n = 5). Daily energy balance data were obtained for 3 weeks prior to oocyte collection using the ovum pick up (OPU) method. The contents and compositions of FFA and TAG in plasma and oocytes were analyzed using liquid chromatography-mass spectrometry. As expected, plasma FFA was high at 20-30 DIM, decreased by 50 DIM, and was maintained at a low level for the remainder of the experimental period. Similar changes were observed in oocyte FFA and TAG with DIM as plasma FFA. Oocyte FFA positively correlated with plasma FFA (P < 0.05), but negatively correlated with the mean energy balance 1 and 21 days before OPU (P < 0.05). Relationships were noted between the composition and content of FFA in plasma and oocytes, with the FFA 16:1/16:0 and 18:1/18:0 ratios positively correlating with the total amount of FFA (P < 0.05). Elevated oocyte FFA in cows in the early postpartum period under intensive feeding management sug-gested that oocytes were at a high risk of FFA lipotoxicity. Fur

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.

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 degrees C on Day 0 than in cows with a rectal temperature < 39.5 degrees 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.

Impaired oocyte quality is one of the main causes of low fertility in modern high-yielding dairy cows. One of the potential factors of the impaired oocyte quality is the effects of free fatty acids (FFA). In fact, high FFA supplementation to culture media exacerbated oocyte developmental competence in vitro. Meanwhile, artificially induced high blood FFA levels in heifers did not affect the lipid composition of oocytes in vivo; however, the oocyte lipid profile of postpartum cows has not yet been investigated. Therefore, the profile of lipids involved in energy metabolism, including FFA and triacylglycerols (TAG), and their relationship between plasma and oocytes were compared among cows at different lactation stages. Heifers were used as a control group that was not affected by lactation. Plasma and oocytes were collected from heifers (n = 4) and 14 Holstein cows categorized to the early lactation stage: 25-47 days in milk (DIM) (n = 6), peak lactation stage: 61-65 DIM (n = 4), and middle lactation stage: 160-202 DIM (n = 4). The FFA and TAG profiles of plasma and oocytes were examined by liquid chromatography mass spectrometry. Plasma FFA positively correlated with oocyte TAG (P < 0.05). Plasma FFA and oocyte TAG were significantly higher in cows in the early lactation stage than in heifers (P < 0.05), while the peak and middle lactation stage groups had intermediate levels. The proportion of oleic acid in plasma increased concurrently with elevations in total FFA, while the compositions of oocyte FFA and TAG fatty acyls were constant regardless of plasma FFA concentration or oocyte TAG content. The present results suggest that high postpartum plasma FFA concentrations affect the quantity of oocyte TAG. Taken together with the adverse effects of high FFA concentrations on oocyte developmental competence in vitro, oocyte quality in postpartum cows may be impaired due to high circulating FFA concentrations. These results provide a more detailed understanding o

Impaired oocyte quality is one of the main causes of low fertility in modern high-yielding dairy cows. One of the potential factors of the impaired oocyte quality is the effects of free fatty acids (FFA). In fact, high FFA supplementation to culture media exacerbated oocyte developmental competence in vitro. Meanwhile, artificially induced high blood FFA levels in heifers did not affect the lipid composition of oocytes in vivo; however, the oocyte lipid profile of postpartum cows has not yet been investigated. Therefore, the profile of lipids involved in energy metabolism, including FFA and triacylglycerols (TAG), and their relationship between plasma and oocytes were compared among cows at different lactation stages. Heifers were used as a control group that was not affected by lactation. Plasma and oocytes were collected from heifers (n = 4) and 14 Holstein cows categorized to the early lactation stage: 25-47 days in milk (DIM) (n = 6), peak lactation stage: 61-65 DIM (n = 4), and middle lactation stage: 160-202 DIM (n = 4). The FFA and TAG profiles of plasma and oocytes were examined by liquid chromatography mass spectrometry. Plasma FFA positively correlated with oocyte TAG (P < 0.05). Plasma FFA and oocyte TAG were significantly higher in cows in the early lactation stage than in heifers (P < 0.05), while the peak and middle lactation stage groups had intermediate levels. The proportion of oleic acid in plasma increased concurrently with elevations in total FFA, while the compositions of oocyte FFA and TAG fatty acyls were constant regardless of plasma FFA concentration or oocyte TAG content. The present results suggest that high postpartum plasma FFA concentrations affect the quantity of oocyte TAG. Taken together with the adverse effects of high FFA concentrations on oocyte developmental competence in vitro, oocyte quality in postpartum cows may be impaired due to high circulating FFA concentrations. These results provide a more detailed understanding o

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.

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.

The objective of this study was to investigate cyclical changes in endometrial thickness in relation to progesterone (P-4) and estradiol-17 beta (E-2) concentrations during natural and induced estrus in 15 cows. In the prostaglandin (PG) F-2 alpha induced estrus group, ultrasonography (USG) at 6-h intervals was used to determine endometrial thickness 48-24 h before the PGF(2 alpha), 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 P-4 and E-2 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 P-4 and an increase in E-2 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 P-4 before an increase in E-2. These results suggest that decreases in P-4 concentrations may be a cue to changes in endometrial thickness, while increases in E-2 concentrations appear to sustain and/or enhance these changes.

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 (OCGC5) collected from early antral follicles were cultured for 12 days. The total number, viability, and diameter of granulosa cells and estradiol-17 beta 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-17 beta 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-17 beta production by granulosa cells appears to be greater in high AFC cattle than in low AFC cattle, thereby promoting the acquisition of oocyte competence.

To clarify the relationship between ovarian reserve and the developmental competence of bovine oocytes, oocyte-granulosa complexes (OGCs) collected from early antral follicles (≤1 mm) in ovaries with high (≥25) and low (<25) antral follicle counts (AFCs) were used. OGCs derived from different AFC groups were cultured for growth followed by maturation, fertilization and blastocyst formation. Viability of OGCs during growth culture was similar between groups; however, OGCs in the high-AFC group had a larger number of granulosa cells than the low-AFC group at 12 days of growth. The proportion of matured oocytes in the high-AFC group was higher than that in the low-AFC group. Mitochondrial activity of oocytes before maturation in the high-AFC group was higher than that in the low-AFC group; however, accumulation of reactive oxygen species was similar between groups. Cleavage rate in the high-AFC group tended to be higher than that in the low-AFC group, although blastocyst development was similar between groups. In conclusion, oocytes derived from ovaries with high AFC have higher maturational ability and fertilizability than those from low AFC. The difference may be caused by high proliferation of granulosa cells from ovaries with high AFC.

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.

In this study, to clarify the relationship between ovarian reserve and oocyte quality, cumulus-oocyte complexes (COCs) were collected repeatedly by ovum pick-up (OPU) from cows with high and low antral follicle counts (AFCs) at short (3–4 days) and long (7 days) intervals, and COC morphologies and oocyte fertilizability were examined. The relationship between AFC and follicular growth after OPU was also investigated. Cows showing AFC of ≥30 in at least one OPU session were grouped into the high-AFC group. At a short interval, follicular sizes and COC morphologies were similar between the different AFC groups. However, the normal fertilization rate was higher in the high-AFC group than in the low one, although total penetration rates were similar. At a long interval, the percentage of COCs with poor morphology in the high-AFC group was higher and the normal fertilization rate was lower than in the low one. In the low-AFC group, normal fertilization rates at short and long intervals were similar, and mean follicular size became larger at a long than at a short interval. However, mean follicular sizes at short- and long-interval OPU were similar in the high-AFC group. In conclusion, it is suggested that oocytes derived from cows with high AFC had higher fertilizability than those from cows with low AFC when OPUs were performed at a short (3–4 days) interval. However, oocyte quality in high-AFC cows was impaired by long-interval (7 days) OPU, possibly due to the degradation of follicles.

In sika deer, the normal method of estimating fetal age, based on fetal weight, is not applicable during the early pregnancy period. The objective of the present study was to describe the growth and development of sika deer fetuses and to establish a method for fetal age estimation during early pregnancy using ultrasonography. Five captive female Hokkaido sika deer (Cervus nippon yesoensis) were observed for estrus and mated (day 0) with an intact male. At two- or three-day intervals, fetuses were observed by rectal ultrasonographic scans until 59-61 days of gestation. The straight crown-rump length (SCRL), curved crown-rump length (CCRL), head length (HL), trunk depth (TD) and heart rate (HR) of the fetuses were measured. Linear regression equations were computed for each measurement together with fetal age. Analyses were conducted after transformation to a natural logarithm for SCRL and CCRL. All equations were significant (P<0.001), with SCRL becoming measurable earlier (day 20) than the others and yielding the best correlation (Days= -2.08+14.15 LnX: X=SCRL, Ln=natural logarithm). Therefore, we concluded that a precise estimation of fetal age in early gestation is best performed using SCRL measurements.

Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent.

We examined the relationship among morphological appearance (six groups) of bovine oocytes, ATP content and maturational/developmental ability. Oocytes with a brown ooplasm (with or without a dark region) had intermediate levels of ATP at the germinal vesicle (GV) stage and showed higher rates of first polar body (PB) extrusion than the other groups. Oocytes with a low level of ATP (oocytes with a pale ooplasm without dark clusters) and oocytes with a high level of ATP (oocytes with a black ooplasm) showed lower rates of PB extrusion. During in vitro maturation, ATP levels in oocytes decreased at around GV breakdown and increased toward metaphase II (MII). MII oocytes having a brown ooplasm with a dark region, which had good developmental capacity, had a relatively high level of ATP. MII oocytes with a brown or pale ooplasm without dark clusters, which had poor developmental capacity, had low ATP levels. MII oocytes with a black ooplasm, which had poor developmental capacity, had an unusually high level of ATP. These results suggest that the morphological appearance of bovine oocytes is closely related to their ATP levels and that cytoplasmic morphology will give an advantage for the selection of oocytes with a high maturational and developmental ability.