Ultrastructure of in-vivo fertilization in superovulated cattle

Heifers were induced to superovulate by treatment with PMSG or FSH. Subsequently, oestrus was induced with prostaglandins and artificial insemination was performed. Ova were collected from the oviducts and their ultrastructural features were related to an estimated time of ovulation based on the time of the LH peak. With the insemination schedule used, the estimated time of ovulation defined the time at which fertilization was expected to occur. The ova were characterized as unfertilized, fertilized or possibly fertilized, and a sequence of nuclear and cytoplasmic changes associated with fertilization was revealed. Within 4 h after the estimated time of ovulation formation of the female and male pronucleus was initiated, and at 5-7 h swelling of the pronuclei occurred. At 19 h the pronuclei were closely apposed and synkaryosis was seen, and at 23 h the first two-cell stage was obtained. Within 2-3 h after the estimated time of ovulation cortical granule release, development of conspicuous Golgi complexes, and transformation of the smooth endoplasmic reticulum occurred. At approximately 7 h parallel arrays of annulate lamellae appeared. In one third of the unfertilized ova deviant oocyte maturation was noticed.     

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.     

DNA Synthesis and pronucleus development in pig zygotes obtained in vivo: An autoradiographic and ultrastructural study

Porcine zygotes flushed from oviducts 48,52,56,60, or 64 hr after hCG were incubated 30 min in ³H-thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with ¹⁴C-thymidine. After this procedure, ova were prepared (i.e., at 51,55,59,63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively. The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56–56.5 hr after hCG, while the latest labelling was seen at 60–60.5 hr. At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucieolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron-dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electrondense material was found. It is concluded that (1) the S-phase in porcine zygotes is initiated around 56 hr post-hCG injection and is of a duration of 4.5–7.5 hr and (2) the progress of the S-phase is paralleled by the appearance of and complex interaction between different granules in the nucleoplasm. © 1995 Wiley-Liss, Inc.     

Character of preovulatory follicles and oocytes after different superovulatory treatments in heifers

The relationship between the opaqueness of the surface of bovine preovulatory follicles, degree of expansion of oocyte-cumulus investment, presence of perivitelline space, and abstriction of the first polar body was examined in heifers treated with PMSG (Group I), FSH-P (Group II), and FSH-P/GnRH (Group III). Follicles greater than 8 mm in diameter were inspected by laparoscopy 65 hours after treatment with cloprostenol in all groups and were classified as clear or opaque based on their surface appearance. Subsequently, oocytes were recovered from the follicles and characterized. The proportion of clear exceeded that of opaque follicles in all groups. The oocyte recovery rate was highest for clear follicles in Group I and II, while in Group III the rates were identical for clear and opaque follicles. The proportion of oocytes with expanded cumulus investment was highest in Group III. In Group II and I decreased proportions of expansion was seen especially among oocytes from opaque follicles. The proportion of oocytes with perivitelline space was highest in Group II and III. Again, this effect was most pronounced among oocytes from "opaque" follicles. The proportion of oocytes with a polar body was highest in Group III followed by Group II while only few oocytes in Group I displayed a polar body. It is concluded that treatment with FSH-P and especially in combination with GnRH reduced the incidence of follicular atresia as measured by opaqueness and improved oocyte quality as indicated by cumulus expansion, formation of the perivitelline space, and abstriction of the first polar body.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.