Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.     

CARBONIC ANHYDRASE AND CARBON FIXATION IN COCCOLITHOPHORIDS1

Rates of carbon fixation in coccolithophorids in culture, unlike many other algae, are carbon limited at ambient levels of dissolved inorganic carbon (DIC). Apparently, plants often rely on activity of carbonic anhydrase (CA) to raise the level of CO₂ in cells and achieve carbon saturation. However, CA activities in the coccolithophorids, Coccolithus (= Emiliania) huxleyi Lohmann and Hymenomonas (=Cricosphaera) carterae Braarud, were either not detectable or very low compared to activities in other systems, including other algae, higher plants, and representative animals. Furthermore, additions of CA to medium with 2 mM DIC at pH 8.1 resulted in nearly 30% enhancement of photosynthesis, but not coccolith formation. Although carbon fixation in coccolithophorids can be suppressed by the CA inhibitor acetazolamide, studies of CaCO₃ nucleation revealed a non-specific effect of the inhibitor. Using a 30 min assay based on pH decreases accompanying loss of dissolved. CO₃^2-, inhibition of crystal formation in the absence of CA at 1 mM acetazolamide was demonstrated for decalcified crab carapace, a tissue with which normal CaCo₃ deposition in vitro has been shown. The results suggest only a minor role for CA in coccolithophorids.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

CALCIFICATION AND CATION SORPTION OF CLADOPHOHA GLOMERATA (CHLOROPHYTA)1, 2

Clacium sorption by Cladophora glomerata (L.) Kutz grown in continuous-flow culture increased substantially as the alga aged (12.3–160 mg Calg dry wt). This reflected increased pectin layered in thickening cell walls followed by deposition of CaCO₃ around cells. The high levels of pectin (up to 23% of dry wt) may account for the plant's reported high affinity for cations. The onset of carbonate deposition coincided with the appearance of carbonabic anhydrase activity in cells. This suggests that carbonate deposition mey be a funtion of bicarbonate use as a source of CO₂ for photosynthesis. Calcium uptake appears to occur by active transport in that it exhibited saturation kinetics, occurred against a concentration gradient, depended on light, and was nearly abolished by treatments that allow diffusion. Although strontium competed for Ca for binding sites of pectin, it did not inhibit intermal transport of Ca. Consequently, the proposed carrier may be specific for Ca.