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.

Contraceptive effect of a gonadotropin-releasing hormone vaccine on captive male brown bears (Ursus arctos).

Fertility control has traditionally been applied in zoos to control captive populations, and reversible contraception is important. However, contraceptive methods for male bears have not been reported. We aimed to establish a reversible contraceptive for male brown bears by investigating the effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac®) that was developed for the immune castration of pigs. We vaccinated six bears with two sequential doses of 400 (n = 2) or 600 µg Improvac® (n = 4) with a 1-month interval during the pre-breeding season (February to April). We compared the reproductive parameters (testosterone levels and semen parameters) of the six vaccinated and four non-vaccinated (control) bears once during the breeding season (May or June). To investigate whether the reproductive performance could be restored in the following year of contraception, we also compared the reproductive parameters once during the breeding season in two bears between the year with GnRH vaccination and the following year without vaccination. Vaccination treatments suppressed reproductive parameters in 5 bears, although vaccination with 400 µg of Improvac® was not effective in one bear. Testosterone levels and the rate of progressive sperm motility were significantly lower, and total sperm count and testis size tended to be lower in vaccinated bears, compared with the controls. Blood biochemical findings and direct observations after Improvac® vaccination did not reveal side effects. Moreover, testosterone levels and spermatogenic scores of two bears were restored in the following year. We confirmed that the Improvac® vaccine elicited a reversible contraceptive effect in male brown bears.

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.

Deciphering two rounds of cell lineage segregations during bovine preimplantation development.

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.

Macrophage ubiquitin-specific protease 2 contributes to motility, hyperactivation, capacitation, and in vitro fertilization activity of mouse sperm.

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selective Usp2 knockout (msUsp2KO) mice and specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KO mice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen-thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen-thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.

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.

Generation of viable calves derived from developmentally mature blastocysts produced by on-gel culture.

Conventional culture systems for bovine embryos are unable to support sustained embryonic development until the developmentally mature blastocyst stage. Although we have previously developed an on-gel culture system that enables bovine blastocysts to complete cell segregation events at day (D) 10 following in vitro culture, the development of D10 blastocysts to term has yet to be achieved. In this study, we attained full-term development of D10 mature blastocysts produced using an on-gel culture system. Two calves derived from on-gel-cultured embryos were vaginally born, showing normal birth and placental weights and no obvious morphological abnormalities. Moreover, we detected no abnormalities in blood metabolic profile analyses. Our findings indicate that on-gel culturing can be used to facilitate the development of developmentally mature blastocysts to term, and produce healthy viable calves. This culture system could make a valuable contribution to cattle production and would enable a range of analyses for characterizing bovine-specific pre-implantation development.

Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst.

Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to a posteriori externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.

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.

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.

Evolution of the sperm methylome of primates is associated with retrotransposon insertions and genome instability.

Changes in gene expression resulting from epigenetic and/or genetic changes play an important role in the evolutionary divergence of phenotypes. To explore how epigenetic and genetic changes are linked during primate evolution, we have compared the genome-wide DNA methylation profiles (methylomes) of humans and chimpanzees, which have a 1.2% DNA sequence divergence, of sperm, the frontal cortices, B cells, and neutrophils. We revealed that species-specific differentially methylated regions (S-DMRs), ranging from several hundred base pairs (bp) to several kilo base pairs (kb), were frequently associated with sequence changes in transcription factor-binding sites and insertions of Alu and SVA retrotransposons. We then generated a reference macaque sperm methylome map and revealed, in sperm, that both human and chimpanzee S-DMRs arose more frequently owing to methylation loss rather than gain. Moreover, we observed that the sperm methylomes contained many more hypomethylated domains (HMDs), ranging from 20 to 500 kb, than did the somatic methylomes. Interestingly, the sperm HMDs changed rapidly during primate evolution; hundreds of sperm HMDs were specific to humans, whereas most somatic HMDs were highly conserved between humans and chimpanzees. Notably, these human-specific sperm HMDs frequently occurred in regions exhibiting copy number variations. Our findings indicate that primate evolution, particularly in the germline, is significantly impacted by reciprocal changes in the genome and epigenome.

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.

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.

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.

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.