Narrowing the agronomic yield gap with improved nitrogen use efficiency: a modeling approach.

Improving nitrogen use efficiency (NUE) in the major cereals is critical for more sustainable nitrogen use in high-input agriculture, but our understanding of the potential for NUE improvement is limited by a paucity of reliable on-farm measurements. Limited on-farm data suggest that agronomic NUE (AE(N)) is lower and more variable than data from trials conducted at research stations, on which much of our understanding of AE(N) has been built. The purpose of this study was to determine the magnitude and causes of variability in AE(N) across an agricultural region, which we refer to as the achievement distribution of AE(N). The distribution of simulated AE(N) in 80 farmers' fields in an irrigated wheat system in the Yaqui Valley, Mexico, was compared with trials at a local research center (International Wheat and Maize Improvement Center; CIMMYT). An agroecosystem simulation model WNMM was used to understand factors controlling yield, AE(N), gaseous N emissions, and nitrate leaching in the region. Simulated AE(N) in the Yaqui Valley was highly variable, and mean on-farm AE(N) was 44% lower than trials with similar fertilization rates at CIMMYT. Variability in residual N supply was the most important factor determining simulated AE(N). Better split applications of N fertilizer led to almost a doubling of AE(N), increased profit, and reduced N pollution, and even larger improvements were possible with technologies that allow for direct measurement of soil N supply and plant N demand, such as site-specific nitrogen management.

Reproductive physiology of the female greater bilby (Macrotis lagotis Thylacomyidae): evidence for a male-induced luteal phase.

Endocrinology of the oestrous cycle, pregnancy and early lactation was investigated in captive Western Australian greater bilbies (Macrotis lagotis). Initially, six females were monitored for changes in urogenital cytology, plasma progestogen, pericloacal and pouch morphology in the absence of a male. This was followed by the introduction of a male and a reproductive assessment through mating, gestation and early lactation. In the absence of a male, there was no cyclical pattern of urogenital cytology, pericloacal or pouch development, and progestogen concentrations remained basal. Within 5 days of the introduction of a male, all females had a karyopycnotic index of 100%. Spermatozoa were present in the urogenital smear within 3 days of male introduction in all five females that gave birth. Five to 9 days after the introduction of a male, there was an increase in plasma progestogen concentration that remained elevated for 14-19 days. Six of the seven females gave birth approximately 3 days after reaching peak plasma progestogen concentrations. Gestation length ranged between 14 and 17 days. Plasma progestogen concentrations of the postpartum and early lactation period were lower (P < 0.0001) than during gestation, but greater (P < 0.0001) than those recorded before the introduction of a male. One female that gave birth early in the study that was examined until weaning of the pouch young showed a cyclical pattern of plasma progestogen secretion that ended at weaning. This study provides evidence that the luteal phase in the greater bilby is induced by the presence of a male. Similar to female reproductive physiology in the Peramelidae, elevated progestogen concentration in the greater bilby was extended into lactation.

Screening standards in assisted reproduction technologies: the screening of semen donors--where do we draw the line?

The most recent British Andrology Society guidelines have certainly stirred up much debate about the exclusion of donors that are seropositive for cytomegalovirus (CMV). Arguments will inevitably go back and forth regarding the statistical risk of donors passing on infection and the point at which that risk becomes unacceptable. As with many of these debates, a number of different opinions will be offered but a consensus will be almost impossible. However, the case of CMV highlights difficulties common to the screening of donors for many infectious and genetic disorders.

The ratio of X- and Y-bearing sperm in ejaculates of men with three or more children of the same sex.

PURPOSE: The present study evaluated the proportions of X-bearing and Y-bearing sperm within the semen of donors who were the declared fathers of three or more sons or daughters. METHODS: The proportions of sperm were determined using dual-color fluorescence in situ hybridization to identify the X and Y chromosomes. RESULTS: The only difference observed was in semen volume. There was no increase in the proportion of Y-bearing sperm for men with only sons (49.7 +/- 1.3%) or of X-bearing sperm for men with only daughters (44.8 +/- 2.6%). CONCLUSIONS: A preponderance of either sons or daughters in a family cannot be explained simply by an altered ratio of X-bearing and Y-bearing sperm in the father's semen.

Delay in the application of the cover glass is a potential source of error with the Makler Counting Chamber.

OBJECTIVE: To determine the effect of delaying the application of the cover glass of a Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel) on the apparent concentration of sperm or Dynabeads (Dynal Pty Ltd, Carlton, Victoria, Australia). DESIGN: Systematic alteration of the time taken to apply the cover glass. SETTING: Comprehensive infertility service. PATIENT(S): Fresh semen from a patient or washed sperm from a donor. INTERVENTION(S): For the Makler Counting Chamber, a 5-microL drop of the sperm or bead suspension was placed in the center of the lower platform and the cover glass was applied either immediately (0 seconds) or after a delay of 5, 10, or 30 seconds. MAIN OUTCOME MEASURE(S): The concentration of beads or sperm counted using an Improved Neubauer Hemocytometer or a Makler Counting Chamber. RESULT(S): There was a progressive increase in sperm concentration with a longer delay in applying the cover glass when sperm was suspended in either seminal plasma or culture medium. Repeating the experiments with Dynabeads resuspended in phosphate-buffered saline, serum, or seminal plasma showed the same trend, although the suspension of beads in serum seemed to settle the least over the first 10 seconds. CONCLUSION(S): Delay in the application of the cover glass is a potential source of error with the Makler Counting Chamber and should be avoided.

The fate of cryopreserved human embryos approaching their legal limit of storage within a West Australian in-vitro fertilization clinic.

Human embryos can only be stored in the first instance for 3 years in Western Australia, according to the West Australian Reproductive Technology Act. Thereafter, an application must be made to the local regulatory body, the Reproductive Technology Council, for an extension. Of the 650 batches of embryos frozen between 8 April 1993 and 31 October 1995, 170 (26.2%) batches were still in storage after 2.5 years. A reminding letter was sent at this time to the couples to whom the embryos belonged, i.e. 6 months before the expiry of the initial storage period, asking for clarification of what was to be done with the embryos. A large proportion of patients (64.7%) chose to either extend the storage period or thaw and transfer the embryos. Curiously, more batches of embryos were discarded (18.8%) than donated to other couples (5.9%). Contact with the patients was lost in a small but significant proportion of cases; more of these had been unsuccessful in their treatment (20.4%) than had achieved a pregnancy (4.3%).

The transfer of two embryos instead of three to reduce the risk of multiple pregnancy: a retrospective analysis.

PURPOSE: Our purpose was to investigate whether a reduction in the number of embryos transferred from three to two would help reduce the incidence of multiple pregnancies and yet leave the pregnancy rate unaffected. METHODS: Women were treated in a routine clinical in vitro fertilization program and the results analyzed retrospectively. RESULTS: There was no reduction in the pregnancy rate when two embryos were transferred compared with three. Indeed, there was actually an increase in pregnancy rate after the transfer of two embryos in those cases with one or more embryos remaining after the transfer. CONCLUSIONS: The transfer of only two embryos compared to three in women younger than 40 years of age does not compromise the chance of pregnancy. Triplets were not seen in the limited series of patients when only two embryos were transferred, but the incidence of twins remained the same. Further consideration should be given to strategies that enable the transfer of single embryos without compromising the pregnancy rate.

Detection of herpes simplex virus DNA in semen of men with genital HSV-2 infection.

BACKGROUND AND OBJECTIVES: Previous studies, using viral culture, have suggested that herpes simplex virus (HSV) isolation from semen is rare. This study attempts to investigate further the role of semen in sexual transmission of HSV. GOALS OF THIS STUDY: To evaluate semen samples for HSV DNA with a sensitive polymerase chain reaction (PCR) test. STUDY DESIGN: Laboratory examination of 255 stored semen samples collected from 15 healthy men with genital HSV-2 infection during a prospective clinical trial. RESULTS: Herpes simplex virus DNA was detected in 8 (3.1%) semen samples, 6 of which were collected during a herpes recurrence. Herpes simplex virus DNA was not detected in any of the 18 samples collected during acyclovir therapy. CONCLUSION: Herpes simplex DNA can be detected in semen, although it appears closely associated with clinical HSV reactivation. More detailed studies will be needed to assess the role HSV-2 in semen plays in transmission of infection.

Internal quality control and external quality assurance in the IVF laboratory.

The existence of internal quality control programmes and external quality assurance schemes is important in enabling the maintenance of good service to patients. All aspects of our work in laboratories involved in the diagnosis and treatment of human infertility can benefit from such programmes and schemes, moving the work from being a subjective art form to an objective science. Equally, many clinical procedures are amenable to such scrutiny. Acceptance and introduction of such schemes and programmes will rely initially on the self-motivation of the laboratories themselves and then pressure brought to bear by accrediting authorities.

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection.

This case report describes the birth of a baby following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) carried out on the second day after oocyte pick-up of in-vitro-matured metaphase I and germinal vesicle stage oocytes. The couple had a history of three failed intrauterine insemination attempts and reduced fertilization rates in two previous in-vitro fertilization (IVF) cycles. In the IVF-ICSI treatment cycle, 6/11 mature oocytes became fertilized following ICSI on the first day. However, the patient failed to conceive following the transfer of three embryos. Five oocytes were immature (two at metaphase I stage and three with a germinal vesicle) and these were cultured overnight. All had extruded a polar body by the following day and ICSI was therefore performed; four oocytes became fertilized, and were cryopreserved at the pronulear stage in propanediol. In the next treatment cycle, transfer of frozen embryos was planned. The pronuclear zygotes were thawed and cultured for 24 h prior to the transfer of two embryos in a cycle stimulated with low doses of follicle stimulating hormone. This resulted in a pregnancy and the delivery of a healthy baby boy. In-vitro maturation of metaphase I and germinal vesicle oocytes which are routinely collected in IVF-ICSI cycles, followed by second day ICSI fertilization, may provide a valuable source of embryos for infertile couples.

A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme.

A total of 364 consecutive patients requesting in-vitro fertilization (IVF) treatment were divided randomly into two groups. In the first group, two embryos in the original IVF cycle were allowed to divide prior to transfer, with any remaining embryos being cryopreserved at the pronucleate (PN) stage. In the second group, all the embryos were allowed to divide to the early cleavage (EC) stage, and the best two replaced; any suitable remaining embryos were frozen at the 2- to 4-cell stage. A total of 134 cycles (36.8%) fulfilled the study criteria for a fresh embryo replacement and supernumerary embryos cryopreserved. In the PN group, 72 out of 182 (39.6%) patients had a fresh embryo replacement accompanied by embryo cryopreservation, which was not significantly different from the EC group (62/182; 34.1%). The livebirth rate per fresh embryo transfer in the EC group (17/62; 27.4%) was significantly higher than that for the PN group (8/72; 11.1%; P < 0.05). Embryo survival following thawing was similar for the PN (96/129; 74.4%) and EC (79/102; 77.4%) stages. Although not significant, the livebirth rate following the transfer of thawed embryos was higher in the PN group (11/44; 25.0%) than in the EC group (4/38; 10.5%). Following one fresh and two freeze-thaw embryo replacements, the observed cumulative viable pregnancy rates were comparable for patients in both the PN (40.2%) and EC (41.1%) groups.

Clinical value of tests for assessing male infertility.

The laboratory assessment of the male partner of an infertile couple is an important aspect of the overall investigation of that couple. The laboratory tests are designed essentially to determine whether (a) the semen samples contain adequate numbers of normal motile sperm, and the sperm are able (b) to migrate to the site of fertilization and (c) to fertilize oocytes. Within this framework, tests can be viewed as being either descriptive, in terms of describing the ejaculate and sperm, or assessing functional qualities of the sperm. Irrespective of the nature of the test, it must satisfy simple criteria, namely being reproducible and able to discriminate between the fertile and infertile populations reliably. External quality assurance programmes now exist for semen analysis and allied techniques to help laboratories to standardize their reporting and to identify the source of possible errors.

Agricultural intensification and ecosystem properties.

Expansion and intensification of cultivation are among the predominant global changes of this century. Intensification of agriculture by use of high-yielding crop varieties, fertilization,irrigation, and pesticides has contributed substantially to the tremendous increases in food production over the past 50 years. Land conversion and intensification,however, also alter the biotic interactions and patterns of resource availability in ecosystems and can have serious local, regional, and global environmental consequences.The use of ecologically based management strategies can increase the sustainability of agricultural production while reducing off-site consequences.

Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection.

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.