The effect of glucose in the medium used during in vitro culture on both cell death by apoptosis and the sex ratio of bovine blastocysts derived from in vitro-matured and in vitro-fertilized oocytes was evaluated. Oocytes were matured, inseminated, and cultured in vitro in mSOF medium with 10% FCS with or without glucose supplementation. Exposure to high concentrations of glucose (10, 20, and 30 mM) during bovine embryo development in vitro from zygote to blastocyst resulted in a decrease in the number of cells per embryo and an increase in the frequency of apoptotic cells. A significantly higher proportion of females was found among those embryos that developed under hyperglycemic conditions in vitro. Moreover, both murine and bovine blastocysts incubated for 6 hr in 20 mM glucose had a significantly higher number of apoptotic cells in comparison to control. In this study, we also determined whether blastocyst production of the X-linked inhibitor of apoptosis protein (XIAP) differs between the sexes. Our results show that female bovine blastocysts produce significantly higher amounts of XIAP mRNA than males and this could be crucial in explaining the higher proportion of female blastocysts observed following in vitro culture under hyperglycemic conditions which induce apoptosis. Moreover, a higher proportion of female murine blastocysts cultured under hyperglycemic conditions were implanted in the uterus (65.3 of implantations from embryos cultured with 20 mM of glucose are females vs. 49% in control). This mechanism provides an explanation for the significant reduction of male children born to diabetic mothers.

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.

No abstract

No abstract

Previously we have demonstrated in two monotocous species (bovine and sheep), a relationship between time of insemination, moment of ovulation, and embryo sex ratio. Here, we have analyzed in a polytocous specie (mice) if in addition to pre-conceptional mechanisms, also post-conceptional ones affect the offspring sex ratio. To verify this hypothesis we carried out two experiments. In the first experiment, we analyzed the effect of mating dynamics on the sex ratio of mice with synchronic male and female embryo development. Females were mated before and after ovulation and sacrificed 13 days later for sex determination of embryos and reabsorptions. A decreased litter size, and an increased offspring sex ratio in matings occurring later in oestrus, supported the view that a biased sex ratio may occur as the result of behavioral differences between the populations of X- or Y-bearing spermatozoa. In the second experiment, embryos developmentally synchronic and asynchronic with the recipient female endometrium were transferred, and again, 13 days later, females were sacrificed for sex determination of embryos and reabsorptions. The male proportion per litter found, indicated that our developmentally asynchronic transfers favored a sex ratio disbalance at birth. When combined, these results become the first experimental evidence supporting the view that both pre- and post-conceptional mechanisms of sex ratio distortion in polytocous species are not mutually exclusive and both may explain, under different conditions, sex ratio deviations at birth.

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.

Barbary sheep (Ammotragus lervia) are considered vulnerable species by the World Conservation Union (IUCN). The purpose of this study was to describe the basic characteristics of fresh semen, test the efficacy of commercial extender Triladyl, and collect necessary data that may help to create a frozen semen bank for the Barbary sheep in Spain. A total of 21 ejaculates were collected by rectal-probe electroejaculation from one dominant (D) and three minor (M) adult males housed in the Madrid Zoo. After ejaculation, semen volume, concentration, and mass motility were assessed. Remaining raw semen was diluted at 37°C with TRIS-based extender Triladyl (Minitüb, Tiefenbach, Germany) and 20% egg yolk to a final concentration of 200 × 106 sperm per mL. Diluted samples were kept at 5°C for 4 h and then loaded into 0.25-mL French straws, frozen at 5 cm above liquid nitrogen (LN2) for 10 min and then plunged into LN2. Samples were thawed in a water bath at 37°C for 30 s. Post-thaw semen survival was evaluated by sperm motility (%M), quality of movement (Q), normal acrosome status (%NAS), normal sperm morphology (%NOR), membrane integrity (hypo-osmotic test; %HOST), and sperm viability (eosin-nigrosin vital staining; %V), and were compared with the same parameters in the fresh semen. Data between D and M males were analyzed by one way ANOVA. Mean volume of ejaculates, total sperm concentration and mass motility of raw semen were respectively; 5.2 ± 1.56 mL, 2800.0 ± 1290.5 × 106 and 3.4 ± 0.4 for the D male, and 3.5 ± 3.2 mL, 251.2 ± 103.9 × 106, and 1.88 ± 1.4 for M males (P < 0.05). Remaining semen parameters evaluated in raw semen showed no differences between D and M males. However, post-thaw semen quality was significantly (P < 0.05) reduced in all analyzed parameters except %NAS and %NOR in M males groups as compared to the D male (Table 1). It can be concluded that Barbary sheep raw semen collected by electroejaculation is of sufficient quality to be used in an artificial insemination program and can be successfully frozen in commercially available Triladyl extender. However, the post-thaw viability of semen may considerably depend on the male reproductive status in the flock. Table 1. Characteristics of fresh and cryopreserved Barbary sheep semen This work was supported by CAM 07B/007/1999 (Analysis of ejaculate traits and development of methods of semen preservation in wild ungulates from the Madrid Zoo).

With no conclusive studies, a number of factors-mostly including thermal stress-have been suggested as the etiology for the sperm defect known as diadem/crater (Larsen nd Chenoweth, 1990;Vogler et al., 1993). A 1-year study conducted on 16 Bos taurus and Bos indicus bulls showed a consistently high percentage (up to 95.5%) of spermatozoa bearing a ''diadem/crater'' (D/C) defect in the semen of a 6-year-old Grey Brahman bull during the hotter months at an artificial insemination center in western Venezuela, an area representing the dry tropics. This sperm pattern reverted to essential normality during the cooler periods.In affected samples, phase contrast and transmission electron microscopy (TEM) both revealed typical diadem patterns of vacuoles in the sperm equatorial region (Fig. 1A,B) as well as vacuoles occurring in other regions of the sperm head. These were shown by TEM as deep invaginations within the nuclear chromatin (Fig. 1C,D). In affected samples, dead sperm ranged from 27.5% to 54% and sperm progressive motility varied between 45% to 60%. Associated sperm defects included deformed and detached heads, bent tails, proximal droplets, and damaged acrosomes. Semen with !15% D/C had depressed sperm mass activity (P 0.5%) compared with semen containing 15% D/C.Although a previous in vitro fertilization (IVF) study (Thundathil et al., 1998) determined defective zona binding and penetration only, we observed low fertilization rates. Here, IVF rates (fertilization, cleavage rates, and formation of both morulae and blastocysts on d7 and d8) were compared using semen samples containing high (95%), medium (52%), and negligible (0%) D/C levels. Both high and medium levels of sperm D/C were associated with low fertilization rates and poor development of morulae and blastocysts in comparison to the results of IVF using control semen.In conclusion, this condition evidently reflects an interaction between genetic predisposition and environmental effects, as this bull was the only 1 of 16 housed and monitored under identical circumstances to demonstrate elevated D/C levels during the hotter period of the year. To our knowledge, no previous report has shown the possibility of genetic  environmental interaction as a likely etiology for the D/C defect.