Follicular cysts in cattle result from excessive growth of the dominant follicle without ovulation and still constitute a major reproductive disorder in this species. One key hormonal characteristic of cows with follicular cysts is the lack of an LH surge, although they have increased plasma estradiol concentrations. Another is a relatively high level of pulsatile secretion of LH that promotes continued growth of the dominant follicle. These LH characteristics seem to result from a functional abnormality in the feedback regulation of LH secretion by estradiol. Treatment with controlled internal drug release devices that increase circulating progesterone levels is effective in resolving follicular cystic conditions by 1) lowering pulsatile LH secretion and 2) restoring the ability of the hypothalamo-pituitary axis to generate an LH surge in response to an increase in circulating estradiol.

View full abstract

This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.

View full abstract

Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.

View full abstract

The aim of this study was to examine whether active metabolites of phytoestrogens (equol and para-ethyl-phenol) inhibit sensitivity of bovine corpus luteum (CL) to luteinizing hormone (LH) and to auto/paracrine luteotropic factors (prostaglandin E₂-PGE₂ and prostaglandin F_2α-PGF_2α), and whether they influence pulsatile progesterone (P₄) secretion by the bovine CL. In in vivo experiments, high levels of equol and para-ethyl-phenol were found in plasma and in the CL tissue of heifers and cows fed a soy bean diet (2.5 kg/animal/day), along with lower concentrations of P₄ (P<0.05). Both Prostaglandins (PG) and LH strongly stimulated P₄ secretion in cultured pieces of CL that were collected from cows fed a standard diet (P<0.01). There was no effect of PGs and LH on P₄ stimulation in CLs obtained from cows fed a diet rich in soy bean. Finally, we examined whether active metabolites of phytoestrogens participated in regulation of pulsatile P₄ secretion and LH-stimulated P₄ secretion in vitro using a microdialysis system. Equol and para-ethyl-phenol had no effect on basic and pulsatile P₄ secretion in CLs during 240 min of perfusion when compared to the control (P<0.05). However, they inhibited LH-stimulated P₄ secretion (P<0.05). Phytoestrogens and their metabolites may disrupt CL function by inhibiting PG- and LH-stimulated P₄ secretion.

View full abstract

The present study demonstrates the postnatal developmental changes in immunohistochemical localization of α-smooth muscle actin (SMA) and vimentin in the bovine testis. In the peritubular myoid cells of seminiferous tubules and the sub-epithelial and stromal cells of straight tubules and the rete testis, α-SMA starts appearing at around 4 months of age. Peritubular α-SMA attains the continuous mature pattern at around 5 months of age whereas sub-epithelial and stromal α-SMA increases with advancing age. Vimentin is localized in the perinuclear zone of Sertoli cells, peritubular and vascular wall cells, a few interstitial cells, and in the basal part of the epithelia of straight and rete tubules. Developmental changes are only evident in the Sertoli cell vimentin, which is basal and weak at birth and increases moderately until 4 months of age. From around 5 to 8 months of age when the Sertoli cells are under morphological transformation, vimentin intensity is considerably increased and the characteristic vimentin extensions connect the Sertoli nuclei to the basal membrane. These extensions get shorter at around 9 month of age as the Sertoli nuclei are positioned basally. The mature Sertoli cell perinuclear vimentin is strong and stable without infranuclear extension. In conclusion, the age of appearance of α-SMA coincides with the onset of postnatal division of spermatogonia, and vimentin may play a key role in stabilizing Sertoli cell nuclei during their transformation in bovine.

View full abstract

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean ± SEM, 66.9 ± 1.9 days; n=64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.

View full abstract

The relationship between basal body temperature and circulating progesterone levels were investigated in a female beluga. Body temperature and serum concentrations of progesterone were measured daily and at 2-4 week intervals respectively, in a female beluga that was in captivity for 7 years between 1996 and 2003. The beluga first ovulated in April, 2000 (13 years old). Thereafter, serum concentrations of progesterone showed cyclic changes, indicating that the ovulatory cycle had started. Serum concentrations of progesterone ranged from 0.1 ng/ml to 15.7 ng/ml. Body temperature also showed cyclic changes during the estrous cycle. Body temperature ranged from 34.9 to 35.9 C, and tended to reach the peak during the high progesterone phase. Mating behavior was observed during the low body temperature phase. The changes in body temperature positively correlated with the circulating progesterone levels. The length of the estrous cycle was 36.7 ± 3.9 (mean ± SEM) days based on the intervals between the days of mating behavior. This is the first report demonstrating that body temperature clearly changes during the estrous cycle in a captive female beluga. The present finding suggests that measurement of body temperature is a useful method for detecting the ovarian cycle of the beluga in captivity.

View full abstract

Killer whales were used to evaluate peripartum changes and interactions between body temperature and circulating progesterone concentration. Daily body temperature was measured between 0800 h and 0830 h before activity. Body temperature clearly increased during the first phase of pregnancy, and this was followed by a gradual decline until full term. The initial increase was paralleled by circulating levels of progesterone. A decrease in body temperature was first detected on day 5 (p<0.01) before parturition, and body temperature decreased significantly every 2 days until delivery. Five days before delivery, body temperature was 0.3 C lower than the mean value during the pregnancy period, and the decrease was more marked on day 1 before delivery (0.8 C). Serum progesterone levels during pregnancy showed a general pattern of initial rapid elevation (increasing phase), followed by a gradual decline (decreasing phase) throughout the remainder of pregnancy. However, statistically significant correlations between body temperature and progesterone pattern were only found only during the increasing phase. These results suggest that monitoring body temperature variation can be considered valuable for predicting impending parturition in killer whales.

View full abstract

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA^val-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

View full abstract

We examined the relations between plasma insulin-like growth factor (IGF) -I concentrations during treatment with CIDR-based or Ovsynch protocol for timed AI and conception and plasma steroid concentrations in early postpartum Japanese Black beef cows. Cows in the control group (Ovsynch; n=21) underwent Ovsynch protocol (GnRH analogue on Day 0, PGF_2α analogue on Day 7, and GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the Ovsynch+CIDR group (n=22) received Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Cows in the further treatment group (EB+CIDR+GnRH; n=22) received 2 mg of estradiol benzoate (EB) on Day 0 in lieu of the first GnRH treatment, followed by the same treatment as in the Ovsynch+CIDR protocol. Plasma IGF-I concentrations were determined on Days -7, 0, 7, 9 and 17. Conception rates were improved in the CIDR-combined groups (both CIDR-treated groups were combined) relative to Ovsynch group (P<0.05) for cows with low IGF-I concentrations (<1,000 ng/ml) on Days -7, 0, and 7, but improved conception rate produced by the CIDR-based protocols did not occur in cows with a high IGF-I concentration (≥1,000 ng/ml). Plasma estradiol-17β concentrations increased from Day 0 to 7 (P<0.05) and were unchanged from Day 7 to 9 in the Ovsynch group with low IGF-I concentrations on Day 0, while they were unchanged from Day 0 to 7 and increased from Day 7 to 9 (P<0.05) in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group. Plasma progesterone concentrations in the Ovsynch group with low IGF-I concentrations on Day 0 were higher on Day 14 than in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group (P<0.05). In conclusion, CIDR-based protocols may improve conception relative to Ovsynch in early postpartum beef cows with lower plasma IGF-I concentrations at the start of the protocols. This improvement is probably due to prevention of premature increases of estradiol-17β and progesterone concentrations, which occurred in cows with low IGF-I concentrations treated with Ovsynch, by the CIDR treatment.

View full abstract

The aim of present study was to optimize culture conditions for pig embryos. Initially, we evaluated three different basic culture conditions. When embryos from electro-activation (parthenotes) or in vitro fertilization (IVF-embryos) were cultured in PZM supplemented with 3 mg/ml bovine serum albumin (PZM-3) in 4-well dishes, in medium covered with oil in 4-well dishes or in droplets under oil, 0%, 33% and 20% of the parthenotes, and 11%, 23% and 20% of the IVF-embryos developed to blastocysts. Subsequently, we examined the development of embryos when they were cultured in 4-well dishes in medium covered with oil continuously for 7 days or cultured under the same conditions but with a change to fresh medium on Days 2 and 4. In this experiment, 23% (no medium change) and 34% (change) of the parthenotes developed to blastocysts, respectively. When IVF-embryos were cultured under similar conditions, 33% and 38% of the embryos developed to blastocysts. Further improvement was achieved when PZM was supplemented with FBS from Day 4. In this experiment, 47% of the parthenotes developed to blastocysts with an average cell number of 57 ± 7.7. In IVF-embryo group, 49% of the embryos developed to blastocysts with a mean cell number of 60 ± 6.1. These results indicate that a change to fresh medium and inclusion of FBS in the medium during the late stages of culture can generate a higher proportion of high-quality blastocysts.

View full abstract

Oogenesis is a critical event in the formation of gametes, which transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodeling, while the genomic information is stably maintained. Histone acetylation, the level of which is dramatically changed during oogenesis, has been implicated in the regulation of genome remodeling. In order to identify genes that are involved in the dynamic changes in histone acetylation levels during oogenesis, we performed suppressive subtraction hybridization (SSH) using unfertilized versus fertilized oocytes. Among the genes identified by SSH, we found histone deacetylase 4 (HDAC4), whose expression has been detected in only a few types of adult tissues. RT-PCR analysis revealed that the expression of HDAC4 was specific for full-grown oocytes. The HDAC4 transcript was barely detected in the growing oocytes, whereas it was readily detectable in fully grown oocytes. The expression was maintained at a high level until the MII stage, but decreased prominently after fertilization. Then, the expression level remained low until the morula stage. Immunocytochemistry localized the HDAC4 protein in the chromosome of fully grown oocytes. Although HDACs are most closely linked with transcriptional regulation owing to deacetylation of the core histones of the chromatin, previous studies have shown that all of the histone residues examined were highly acetylated in full-grown oocytes. Therefore, our results suggest that HDAC4 acts on a non-histone protein and plays a role in some other cellular functions of full-grown oocytes. To address this possibility, we examined the expression of p53BP1, which is associated with HDAC4 and plays a critical role in the DNA damage response. RT-PCR analysis revealed that p53BP1 was expressed specifically in fully grown oocytes, as was HDAC4. These results suggest that HDAC4 plays a role in stabilizing the genomes of fully grown oocytes.

View full abstract

The hypothesis of the present study is that active immunization of cows against inhibin would neutralize endogenous inhibin, increase circulating levels of follicle stimulating hormone, and subsequently affect follicular dynamics and the ovulation rate during the estrous cycle. Thirteen cows were immunized against inhibin α-subunit and, 6 cows were immunized with a placebo. Both groups were given 4 booster immunizations 7, 14, 21, and 34 weeks after the primary injection. Ovaries were examined daily after the 2nd, 3rd, and 4th booster immunizations by transrectal ultrasonography for 25 days. After the 4th booster immunization, blood samples were collected daily for one complete estrous cycle to measure FSH and LH. The results showed that the immunized cows generated antibodies against inhibin, and that they had higher FSH levels compared with the controls. The number of follicular waves during the estrous cycle was higher in the immunized cows (3 or 4 waves) than in the controls (2 or 3 waves). Moreover, the immunized cows had a greater number of follicles during the estrous cycle compared with the control cows. The maximum number of follicles was 14.8 ± 1.7 vs 5.4 ± 0.2 in inhibin-immunized and control cows, respectively, during the first follicular wave and 13.9 ± 1.9 vs 5.6 ± 0.7, respectively, during the ovulatory wave. Multiple ovulations were increased in the immunized cows. However, the ovulation rate varied greatly in the immunized animals. In conclusion, immunization against inhibin increased FSH secretions during the estrous cycle in the cows. Moreover, the immunized cows had a greater number of follicular waves during the estrous cycle and a greater number of follicles, and this could be used as a potential source of oocytes for use in IVF/embryo transfer programs.

View full abstract

The bed nucleus of the stria terminalis (BNST) is one of the brain areas densely innervated by noradrenergic neurons originating in the brain stem. The present study aims to determine the role of noradrenergic receptors in the BNST in regulating pulsatile luteinizing hormone (LH) secretion in female rats. Ovariectomized (OVX) or estrogen-primed OVX (OVX+E₂) rats received three 1-h-interval injections of 0.05 μmol of noradrenaline (NA), phenylephrine (α₁-adrenergic receptor agonist), clonidine (α₂-agonist), or isoproterenol (β-agonist) into the BNST. Injection of NA or α₁-adrenergic agonist into the BNST strongly suppressed pulsatile LH secretion in OVX+E₂ rats with a significant (P<0.05) decrease in the mean LH level for 3 h and LH pulse frequency, but α₂-and β-agonists did not affect any of the LH pulse parameters. In OVX animals, α₁- and α₂-adrenergic agonists caused a significant change in LH pulse frequency and amplitude, respectively, though the effect was not as apparent as the NA- or α₁-agonist-induced changes in OVX+E₂ animals. These results indicate that NA inputs to the BNST suppress pulsatile LH secretion via α₁-adrenergic receptors and that estrogen enhances this suppression.

View full abstract

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P<0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P<0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P<0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes

View full abstract

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF_2α injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF_2α; and Intact follicle, ovulation was induced by GnRH injection after PGF_2α. Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0=follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.

View full abstract

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 μl/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P<0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p<0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.

View full abstract

In response to changes in society and thus the marketplace, we need a vision for the future of our animal industries, including both on-farm and off-farm activities, that is "clean, green and ethical". Using small ruminants as a case study, we describe three "clean, green and ethical" strategies that farmers could use to improve reproductive performance. The first allows control of the timing of reproductive events by using socio-sexual signals (the "male effect") to induce synchronised ovulation in females. The second strategy, "focus feeding", is based on using short periods of nutritional supplements that are precisely timed and specifically designed for each event in the reproductive process (eg, gamete production, embryo survival, fetal programming, colostrum production). The third strategy aims to maximize offspring survival by a combination of management, nutrition and genetic selection for behaviour (temperament). All of these approaches involve non-pharmacological manipulation of the endogenous control systems of the animals and complement the detailed information from ultrasound that is now becoming available. Importantly, these approaches all have a solid foundation in reproductive biology. In several cases, they are currently used in commercial practice, but there is still room for improvement through both basic and applied research. Ultimately, these "clean, green and ethical" tools can be cost-effective, increase productivity and, at the same time, greatly improve the image of meat and milk industries in society and the marketplace.

View full abstract

Ultrasonography (US) has been applied to the ovary and the uterus of domestic animals from the late 1980s, and established in 1990s as a practical tool for animal production. US made it possible to detect pregnancy at a very early stage and, most importantly, to observe the real-time dynamics of follicular development and hence the discovery of follicular waves. This has greatly contributed to our understanding of ovarian physiology and helped us to develop several "pin-point" protocols for hormonal treatment. While US may not seem to fit preconceived ideas of a "green" technology, it does not contravene environmental priorities, and it is non-invasive ("ethical") and non-hormonal ("clean"). Using the US technology that is now commercially available at a reasonable price, we are able to estimate the best timing for AI and this allows us to plan either the use of precisely-timed nutritional supplements for fetal development or an immediate 2nd AI service to achieve a better economic efficiency. During the last few years, we have also begun to be able to observe in detail the local blood flow in individual ovarian follicles and CL using color Doppler ultrasonography in the cow. From the series of observations, we have found that: 1) the change of blood supply to an individual follicle closely relates to the dynamics of follicular growth and atresia; 2) the local blood flow detected in the theca externa of mature follicles rapidly increases around the onset of LH surge and is most active before ovulation; 3) the blood supply to the developing CL increases in parallel with CL volume and plasma progesterone concentrations; and 4) the local blood flow surrounding the mature CL acutely increases prior to the onset of luteolysis in response to uterine as well as exogenous PGF_2α. It is now clear that color Doppler ultrasound is very useful for observing echogenicity with local blood flow thereby providing an easily obtained estimation of the physiological status of follicles, CLs and early conceptus. Widespread commercial application of color US will depend on further technological developments that reduce the cost and improve performance and ease-of-use. Overall, US is now a most effective non-invasive tool for managing reproduction, at the level of both the individual animal and the herd system. In particular, US can help us to clarify potential problems in high-producing dairy cattle during the postpartum period.

View full abstract

For lactating dairy cows, we need management tools, that are "clean, green and ethical", cost-effective and easy to use. Specific tools are needed for artificial insemination (AI) after oestrus detection within a few months of calving, and for managing the complex nutritional requirements of cows between successive calvings. Assessment of energy deficit by measurement of body condition score (BCS) has been useful in the past but we now need more sophisticated ways to measure the relationship between adipose tissue and fertility. For this reason, we have focused our attention on the cells of the adipose tissue, the adipocytes, and the role of the hormone that they produce, leptin. This hormone affects pulsatile LH release and, in dairy cows, it seems to be linked to the first postpartum ovulation. Adipocytes are always sensing energy status and they control leptin secretion dynamically, so blood leptin concentrations can change acutely, even when there is no detectable change in BCS. Leptin secretion seems to be determined by the secretory activity of each adipocyte as well as the total mass of adipocytes in the body of the animal (as measured by BCS). The strong relationship between BCS, leptin concentration and reproductive function in dairy cows suggests that we should reconsider the interval of the recovery from prepartum and postpartum damages, the need for high milk yields at the last lactation causing the dry-off stress and the subsequent troubles. We should also re-assess the current drive to reduce calving interval because milk yields during the early stages of lactation are economically very important but high yields seem to cause several metabolic and reproductive disorders in modern dairy cows. In general, the thinking has been that calving interval must be short because short intervals are more profitable. However, if we remember that main product from dairy cows is milk and that a short calving interval is very difficult without reproductive problems, then a longer calving interval might be more sensible and also more profitable. We have example of an extended calving interval in Japan, Supercows which are very rare cows yielding remarkable high milk. Finally, we probably need to improve dairy cows genetically if we are to achieve the goal of "clean, green and ethical" dairy farming. This paper reviews data relevant to these strategies and we conclude that more basic and applied research will be required if we are to find ways to reach that goal.

View full abstract

Cumulus-oocyte complexes (COCs) and early embryos rely on a histotrophic nutrition source for energy production and the synthesis of macromolecules. There is accumulating evidence suggesting that the balance of supply and demand for energy and other anabolic substrates during oocyte maturation and very early stages of development programmes subsequent developmental potential, and this may include subsequent fetal growth trajectory. One example is the role of glucose (Glc) during cumulus-oocyte complex maturation. Glucose is an essential nutrient for maturation, especially its role during cumulus expansion. Our laboratory has shown that during in vitro culture, too little glucose during cumulus-oocyte complex maturation affects meiotic competence. We have focussed on glucose (Glc) metabolism through the hexosamine biosynthesis pathway (HBP) during COC maturation in vitro. The HBP in somatic cells is regarded as a "fuel-sensing" pathway and its interaction with cell signalling systems and transcriptional regulation is increasingly apparent. Up-regulation of the HBP during oocyte maturation in vitro has negative consequences for subsequent development. Another example is the role of hypoxia (low O₂) during peri-compaction development. My laboratory believes that ruminant embryos during compaction, blastulation and subsequent development in the uterine cavity lack a key hypoxia responsive element. Because of this, hypoxia is important for normal development in ruminants but perturbs further development in rodents. The implication of these examples to the fundamental concept of peri-conception nutritional programming of development are discussed.

View full abstract

Reproductive efficiency and performance in dairy cattle has been declining over the last 20 years in Japan. Recently, this has resulted in serious problems in dairy herds. There have been changes in the animals themselves and in the animal's environment, e.g. the genetic potential for milk yield has improved, the herd size of dairy farms has been expanding, the housing system of dairy cattle has been changing from tie stall to free stall, and total mixed ratio (TMR) feeding systems have become widespread. It is not clear whether the animals have adapted adequately to these environmental changes. Up till now, reproductive management has been undertaken by monitoring animal performance and/or hormonal treatments, and this has resulted in acceptable reproductive performance. However, conception and pregnancy rates have continued to decline, and the calving to calving interval has continued to extend. Consequently, the average parity and the average age at culling in dairy cattle have been declining. The most common causes of culling in dairy cattle are reproductive disorders in Japan. Therefore, in future, prevention of reproductive disorders will be required for both reproductive efficiency and animal welfare. During late pregnancy and the transition from the pregnant, non-lactating state to the non-pregnant, lactating state, dramatic changes are occurring in the dam including an increase in the fetal-placental mass and calving. Thus, the demand for energy, protein and minerals by the conceptus increases, the space within the abdominal cavity and the rumen capacity decreases due to the increased volume of the conceptus, the local immunological relationship between the fetal and maternal placenta dramatically changes, and physiological homeostasis is activated to adapt to these changes. If cattle can't maintain homeostasis before calving, the incidence of reproductive diseases will be greatly increased. We have been examining reproductive, nutritional and immunological factors to predict the occurrence of reproductive disorders as early as possible and to develop strategies to prevent them. It is unlikely that all dairy farms in Japan will be able to improve reproductive performance with general advice, due to the wide variation in feeding systems and management in Japan. Therefore, specific advice needs to be tailored to each herd, based on management systems employed within each herd. We have proposed that it is important to identify common risk factors across herds, to use these factors as an index for disease control and reproductive management and finally to demonstrate the relationship between these factors and fundamental improvement of the reproductive efficiency. We propose this approach with the aim of providing a breakthrough in improving reproductive performance in dairy cattle in Japan. The focus of this review will be that such an approach is important for improvement of the reproductive efficiency in Japanese dairy cows.

View full abstract

Internationally, reproductive performance of dairy cattle is declining. Under the low production, pasture-based and seasonal calving system used in New Zealand, reproductive performance is also declining, but remains higher than for many other industries. The seasonal calving system requires a high level of reproductive performance to ensure a 365 day inter-calving interval. About 80% of cows are detected in oestrus and inseminated in the first 3 weeks of the seasonal breeding programme and have a conception rate of 55% to 65% to this first insemination. This results in ∼75% and ∼90% of cows being pregnant by 6 weeks and by the end of the breeding programme, respectively. However, reproductive performance is declining due to multiple factors including increasing herd size, reduced oestrus detection sensitivity and specificity, declining body condition score at calving and increased rate of body condition score loss postpartum. Continuing selection for increased milk production is also having negative effects on reproduction. The mechanisms by which high production and negative energy balance are reducing reproductive performance are becoming better understood. However, practical solutions for the problem are challenging for veterinarians and consultants, especially given the consumer preference for less interventions.

View full abstract