Development and ultrastructure of first-generation meronts of Sarcocystis cruzi in calves fed sporocysts from coyote feces

The development of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) meronts was studied in seven 7- to 10-day-old calves filled 4, 7, 11, 15, 22, 25 and 28 days postinoculation (DPI) with 5 x 10(7) sporocysts from feces of coyotes. No meronts were found 4 and 7 DPI. Young and intermediate meronts with 1-16 nuclei were found in endothelial cells of arteries in mesenteric lymph nodes, but not in kidneys 11 DPI. Mature meronts were noted in endothelial cells of arteries, arterioles, or capillaries of many organs of calves killed 15 to 25 DPI. No first-generation meronts were found 28 DPI. By electron microscopy, all stages of the first-generation merogony were found free within the host cell cytoplasm and not within a parasitophorous vacuole. The appearance of intranuclear spindles preceded the formation of merozoites by endopolygeny. Mature meronts measured 41.0 x 17.5 (34-50 x 15-24) microgram, contained approximately 100-350 merozoites, and had 2 to 4 relatively small residual bodies, 2.8 microgram in diameter. Merozoites measured 6.3 x 1.5 (5.5-7 x 1 microgram) and contained most of the organelles characteristically found in coccidian merozoites. Micropores were observed in merozoites, but not in young and intermediate meronts. Merozoites were seen free in the lumen of blood vessels, in intracellular areas, and free within the host cell cytoplasm.     

Development and Ultrastructure of First-Generation Meronts of Sarcocystis cruzi in Calves Fed Sporocysts from Coyote Feces*

SYNOPSIS. The development of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) meronts was studied in seven 7- to 10-day-old calves killed 4, 7, 11, 15, 22, 25 and 28 days postinoculation (DPI) with 5 × 10⁷ sporocysts from feces of coyotes. No meronts were found 4 and 7 DPI. Young and intermediate meronts with 1–16 nuclei were found in endothelial cells of arteries in mesenteric lymph nodes, but not in kidneys 11 DPI. Mature meronts were noted in endothelial cells of arteries, arterioles, or capillaries of many organs of calves killed 15 to 25 DPI. No first-generation meronts were found 28 DPI. By electron microscopy, all stages of the first-generation merogony were found free within the host cell cytoplasm and not within a parasitophorous vacuole. The appearance of intranuclear spindles preceded the formation of merozoites by endopolygeny. Mature meronts measured 41.0 × 17.5 (34–50 × 15–24) μm, contained ～ 100–350 merozoites, and had 2 to 4 relatively small residual bodies, 2.8 μm in diameter. Merozoites measured 6.3 × 1.5 (5.5–7 × 1 μm) and contained most of the organelles characteristically found in coccidian merozoites. Micropores were observed in merozoites, but not in young and intermediate meronts. Merozoites were seen free in the lumen of blood vessels, in intracellular areas, and free within the host cell cytoplasm.     

Biology and pathogenicity of Eimeria spinosa Henry, 1931 in experimentally infected pigs.

A single-species isolate of E. spinosa from a diarrheic weaned pig was used to determine the endogenous development and pathogenicity of this swine coccidium. Seven out of 14 inoculated pigs developed endogenous stages or passed oocysts of E. spinosa in their feces. Immunosuppressive treatment with cyclophosphamide had no effect on the susceptibility to infection with E. spinosa in young pigs. The endogenous stages developed within the apical cytoplasm of the enterocytes lining the distal part of the villi in the posterior jejunum. The asexual development comprised three generations of meronts, which were seen at 5, 7 and 9 days post-infection (DPI). Meronts of the first generation measured 6-8 microns and produced 10-14 merozoites 4-6 microns in length. The second generation of meronts measured 6-8 microns and contained 10-20 merozoites 4-6 microns in length. Third generation mature meronts (8-10 microns) on DPI 9 contained 12-20 merozoites measuring 5-7 microns, which were more crescent-shaped and less blunt than the merozoites at 5 and 7 DPI. Merogony continued after formation of the gametes and the first fully developed macrogametes (10-14 microns), microgametes (9-12 microns), and oocysts were also seen at 9 DPI. The prepatent period was 8 or 9 days, but the patent period was not determined. In the present study E. spinosa infection did not produce overt clinical signs. Pathological changes consisted of an inflammatory infiltration in the lamina propria of the posterior jejunum, Peyer's patches activation and sporadic erosions scattered at the villous tips. No villous atrophy in association with a large number of endogenous stages was observed.     

An Ultrastructural Study of First- and Second-Generation Merogony in the Coccidian Sarcocystis tenella1

ABSTRACT. Sporocysts of the coccidian Sarcocystis tenella were originally isolated in the feces of a coyote. Sporocysts used for inoculation of lambs were obtained from experimentally infected dogs. At 14, 16, and 19 days postinoculation (DPI) of lambs with the sporocysts, various developmental stages of first-generation meronts were found within cells located between the endothelium and internal elastic membrane of mesenteric arteries. At 19, 21, and 25 DPI, second-generation merogony occurred in cells associated with capillaries and arterioles of kidney glomeruli and convoluted tubules. Meronts of both generations were bounded by a double pellicular membrane and were situated free in the host cell cytoplasm. Merozoites formed by endopolygeny that involved multiple intranuclear spindles of a single, large irregular nucleus. First-generation meronts measured 22.6 × 17.1 μm (19–28.7 × 7.5–24 μm) and contained 120–240 merozoites, which measured 7.1 × 1.6 μm (4.8–7.5 × 1.3–1.8 μm). Corresponding values for second-generation meronts were 13.2 × 9.2 μm (8.3–15 × 7–13.5 μ), 32–80, and 5.8 × 1.7 μm (5.6–6.2 × 1.4–2.2 μm).     

New observations on first-generation merogony of Eimeria tuskegeensis in Sigmodon hispidus

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Complete Development of Caryospora bigenetica (Apicomplexa: Eimeriidae) In Vitro1

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocysts sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

New Observations on First-Generation Merogony of Eimeria tuskegeensis in Sigmodon hispidus1

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

The effect of Lactobacillus buchneri on the fermentation, aerobic stability and ruminal degradability of maize silage

AIMS: To evaluate the effect of Lactobacillus buchneri, heterofermentative lactic acid bacteria (LAB), on the fermentation, aerobic stability and ruminal degradability of whole-crop maize silages under laboratory conditions. Two homofermentative LAB were tested for the purpose of comparison. METHODS AND RESULTS: Maize was harvested at early dent [290 g kg(-1) dry matter (DM)] and one-half milk line (355 g kg(-1) DM) stages. Both homofermentative LAB were applied at 1 x 10(5) CFU g(-1) of fresh forage. Lactobacillus buchneri was applied at 1 x 10(5), 5 x 10(5) and 1 x 10(6) CFU g(-1) of fresh forage. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-l anaerobic jars. Three jars per treatment were sampled on day 60. After 60 days of storage, silages were subjected to an aerobic stability test lasting for 5 days, in which CO(2) production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. Both homofermentative LAB increased the concentration of lactic acid and the numbers of yeasts, and decreased the concentration of acetic acid and impaired the aerobic stability of silages. In contrast, applying L. buchneri decreased the concentration of lactic acid and increased the concentration of acetic acid of the silages. Under aerobic conditions, silages treated with 5 x 10(5) and 1 x 10(6) CFU g(-1) of L. buchneri, had lower pH, CO(2) production and the numbers of yeasts than the silages treated with 1 x 10(5) CFU g(-1) of L. buchneri (P < 0.05). However, all doses of L. buchneri and both homofermentative LAB did not affect in situ rumen DM, organic matter and neutral detergent fibre degradability of the silages. CONCLUSIONS: Lactobacillus buchneri was very effective in protecting maize silages exposed to air under laboratory conditions. All doses of L. buchneri, especially 5 x 10(5) CFU g(-1) or more, markedly decreased the numbers of yeasts and improved the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of L. buchneri, as a silage inoculant, can improve the aerobic stability of maize silages by inhibition of yeast activity.     

Development of Ox-Coyote Cycle of Sarcocystis cruzi1

The ox-coyote cycle of Sarcocystis cruzi was studied by killing 38 calves between 4 and 153 days postinoculation (DPI) with 55 × 10³-5 × 10⁸ sporocysts from the intestines of coyotes. At 4 DPI, a zoite was found within the lumen of a mesenteric lymph node artery. At 7 DPI, zoites were found in mononuclear cells and in endothelial cells in mesenteric arteries. First generation meronts (41.0 × 17.5 μm in diameter) occurred 7–26 DPI in mesenteric lymph nodes. At 19–46 DPI, second generation meronts occurred in kidneys, muscles, and other tissues: renal meronts were 19.6 × 11.0 μm, and intramuscular meronts were 25.0 × 11.1 μm. Merozoites were found in the peripheral blood 17 DPI and later at 24–46 DPI. They divided by endodyogeny in mononuclear cells. Sarcocysts were seen first in the heart at 45 DPI and contained one or two metrocytes. At 55 DPI, sarcocysts containing only metrocytes were found in striated muscles, heart, and in smooth muscles of the urinary bladder, rumen, omasum, abomasum, and small intestine. At 67, 87, 112, and 153 DPI, sarcocysts were found only in striated muscles and in the heart. At 67 DPI, sarcocysts were up to 360 μm long. They contained only metrocytes and were not infective to the dog. At 86 DPI, sarcocysts contained mostly bradyzoites, a few metrocytes, and were infective to a coyote. The thin-walled sarcocysts grew to a maximum length of 800 μm and contained bradyzoites that were 10.9 × 3.0 μm. At 90 DPI, two mature sarcocysts were found in 2 of 73 sections of brain and spinal cord; hundreds of sarcocysts were present in sections of tongue and heart of this calf. Gametogony occurred in the small intestine of the coyote. Macro-and microgamonts were found in goblet cells of the small intestines of coyotes 6 h after the ingestion of infected meat. Microgamonts were few and contained 3–11 slender gametes. Oocysts were seen at 12 h and sporulation was completed 9 DPI. The prepatent period in the coyote was 8 days. The ox-coyote cycle is compared with ox-dog cycle.     

Development of the Swine Coccidium Eimeria debliecki Douwes, 1921 in Mammalian Cell Cultures1

Sporozoites of Eimeria debliecki entered human fetal lung and porcine kidney cells grown in cultures and underwent one merogenous cycle, terminating in the production of second-generation trophozoites. Sporozoites were intracellular 1 h post-inoculation (PI) and developed into sporozoite-shaped meronts at 40 h PI. These meronts, one of which was motile, had from two to ten nuclei. Sporozoite-shaped meronts then developed into elongate or spheroidal meronts with 10 to 24 nuclei by two days PI. Ten to 26 first-generation merozoites were formed by budding from the meront surface. Mature first-generation merozoites were most numerous three days PI. Most meronts had ruptured and released nonmotile merozoites into the culture medium by four days PI. Merozoites that were not released became rounded and developed into second-generation trophozoites. Refractile bodies were present in all developmental stages. No further development was observed five through eight days PI.     

Penetration of Eimeria tenella sporozoites under different oxygen concentrations in vitro.

Sporozoites of Eimeria tenella (Wisconsin strain) were inoculated onto monolayers of normal chicken kidney fibroblasts and cultured in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and gentamicin under either aerobic, 5% CO2/95% air, or anaerobic conditions. Penetration of fibroblasts by sporozoites under CO2 or anaerobic conditions at 2 and 24 hr postinoculation was 3-4 times greater than that in the aerobic atmosphere. Effect of reduced oxygen concentrations, i.e., 20.0, 12.5, and 5.0% oxygen, was also investigated in an N2-O2-CO2 incubator. Under 5.0 and 12.5% oxygen at 2 and 24 hr postinoculation, the number of sporozoites that penetrated was about 4 and 2 times greater, respectively, than under 20.0% O2. These results indicate that lower oxygen concentrations provide for greater penetration by E. tenella sporozoites in cultured cells.     

Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures

The efficiency of aerobic incubation was compared with incubation under various oxygen and carbon dioxide conditions for the isolation and subcultivation of three strains of Mycoplasma hyorhinis from VERO-cell cultures and subcultivation of three laboratory strains. Under anaerobic conditions with a low oxidation-reduction potential (at or below -115 mV) as obtained in jars, with catalysts, containing mixtures of 5%-10% CO2 in H2, very poor or no growth of any of the six M. hyorhinis strains was observed. When traces of oxygen were present (that is, under conditions with higher oxidation-reduction potentials, e.g. when omitting the catalyst in the above gas mixtures or in 5% CO2 + 95% N2) isolation from cell cultures was successful in most tests, but subcultivation of these primary isolates was seldom possible under these semi-anaerobic conditions. However, in most cases these primary isolates could be subcultivated aerobically, although aerobic conditions were unsatisfactory for isolation in about half of the experiments. Isolation of M. hyorhinis was optimal in 5% O2 + 95% N2, under which condition the isolates could also always be subcultivated. Isolation failed occasionally when 5% O2 + 5% CO2 + 90% N2 was used, thus indicating that 5% CO2 was slightly inhibitory. 5% CO2 in air and 10% CO2 either in air, H2 or N2 were also inadequate for isolation from cell cultures. In contrast to the findings with these cell culture-adapted M. hyorhinis strains, the laboratory strains could be subcultivated easily under all conditions tested except those with an oxidation-reduction potential at or below -115 mV; 100% CO2 was inhibitory for all 6 strains. Our findings may partly explain why M. hyorhinis is often considered "non-cultivable" on artificial media once adapted to cell cultures. The findings emphasize the need to employ also a micro-aerophilic condition (5% O2 in 95% N2) in the examination of cell cultures for mycoplasma.     

Development of the swine coccidium Eimeria debliecki Douwes, 1921 in mammalian cell cultures

Sporozoites of Eimeria debliecki entered human fetal lung and porcine kidney cells grown in cultures and underwent one merogenous cycle, terminating in the production of second-generation trophozoites. Sporozoites were intracellular 1 h post-inoculation (PI) and developed into sporozoite-shaped meronts at 40 h PI. These meronts, one of which was motile, had from two to ten nuclei. Sporozoite-shaped meronts then developed into elongate or spheroidal meronts with 10 to 24 nuclei by two days PI. Ten to 26 first-generation merozoites were formed by budding from the meront surface. Mature first-generation merozoites were most numerous three days PI. Most meronts had ruptured and released nonmotile merozoites into the culture medium by four days PI. Merozoites that were not released became rounded and developed into second-generation trophozoites. Refractile bodies were present in all developmental stages. No further development was observed five through eight days PI.     

In vitro cultivation of the vascular phase of Sarcocystis capracanis and Sarcocystis tenella

Sporozoites of Sarcocystis capracanis and S. tenella (Apicomplexa) penetrated all four cell types tested (bovine monocytes, BM; bovine pulmonary artery endothelial cells, CPA; Madin-Darby bovine kidney; and ovine monocytes). Sporozoites of S. tenella developed to meronts in BM and CPA; those of S. capracanis developed to meronts in BM only. Both species of Sarcocystis developed to large first-generation meronts followed by small meronts. At 40 to 50 days after inoculation (DAI) of sporozoites, considerably more merozoites of S. tenella were harvested from CPA (24.9 X 10(6) merozoites/75-cm2 flask; n = 4) than from BM (1.9 X 10(6) merozoites/75-cm2 flask; n = 4). Merozoites of S. capracanis were most numerous in BM at 88 to 100 DAI during which time 2.1 X 10(6) merozoites/75-cm2 flask (n = 4) were harvested.     

Suppression of Nippostrongylus brasiliensis (Nematoda)-induced lysophospholipase activity and peripheral eosinophilia by Eimeria nieschulzi (Apicomplexa)

Interspecific interactions between Nippostrongylus brasiliensis and Eimeria nieschulzi were studied by measuring fecal lysophospholipase (LYPH) activity and relative numbers of peripheral eosinophils in rats singly or concurrently infected with one or both parasite species. Three groups of 10 rats each were inoculated with 2 X 10(3) N. brasiliensis L3 larvae and/or 5 X 10(5) E. nieschulzi sporulated oocysts. Groups 1 and 2 were infected with E. nieschulzi or N. brasiliensis, respectively. Group 3 rats were infected first with N. brasiliensis, followed on day 8 postinoculation (PI) with E. nieschulzi. Each rat served as its own control. Results revealed LYPH levels rose steadily in Group 2 rats, reaching significant peaks on days 10 and 12 PI before decreasing to control levels. Lysophospholipase activity in Groups 1 and 3, however, did not differ from control values. Group 2 rats also demonstrated peripheral eosinophilia, with peak values occurring on days 10, 12, 14, and 16 PI, while rats in Groups 1 and 3 exhibited no eosinophilia. These results demonstrate that E. nieschulzi suppressed intestinal LYPH activity and relative peripheral eosinophilia and demonstrate that a host's immune response to a single parasite may be significantly altered when a second parasite species is present.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

Endogenous development of Eimeria minasensis in experimentally infected goats

The endogenous development of Eimeria minasensis was studied in 9 coccidia-free goat kids inoculated with 10(5) sporulated oocysts/kg body weight. Kids were killed 4, 7 (2 animals), 10, 13, 16, 18, 19, and 22 days after inoculation (DAI). In tissue sections of the intestines stained with hematoxylin and eosin and examined by light microscopy, 2 generations of meronts, gamonts, gametes, and oocysts were found. The first generation of meronts developed in cells deep in the lamina propria of the jejunum and ileum. Mature giant meronts (299.4x243.8 microm) found 16 DAI were visible to the naked eye and contained a large number of crescent-shaped merozoites. The second generation of meronts developed in the epithelial cells of crypts of the ileum and above the host cell nuclei. Mature meronts (11.5x10.1 microm) with 18-28 comma-shaped merozoites were first seen 16 DAI. Gametogenesis took place in epithelial cells of the crypts and villi of the terminal part of the ileum, cecum, and colon. Macrogametes (27.8x17.6 microm), mature microgamonts (21.3x17.0 microm), microgametes, and oocysts (30.5x19.4 microm) were found 19 DAI. Sexual stages were below the host cell nucleus.     

In vitro cultivation of meronts of Sarcocystis cruzi

Several established cell lines were tested for their ability to support in vitro development of meronts of Sarcocystis cruzi. Sporozoites penetrated bovine monocytes (BM), bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney cells and mouse macrophages, but developed to meronts in BM and CPA only. Sporozoites developed to large meronts that contained approximately 180-350 merozoites, whereas merozoites formed small meronts with 50-100 merozoites. Mature large meronts were present at 18-86 days after inoculation (DAI) in BM and at 16-72 DAI in CPA. Small meronts were present at 23-115 and 23-91 DAI in BM and CPA. Considerably more merozoites developed in CPA than in BM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that merozoites harvested at 36 and 48 DAI each had 1 unique protein as well as numerous common proteins.     

The effect of applying lactic acid bacteria at ensiling on the aerobic stability of silages

The effect of applying commercial lactic acid bacteria inoculants at ensiling on the aerobic stability of silages was studied under laboratory conditions. The silages used were wheat, hedysarum, corn and various sorghum cultivars at various stages of maturity. Three inoculants were used, two containing Lactobacillus plantarum, Enterococcus faecium and Pediococcus acidilactici (H/M F, Medipharm, USA and Sil-All, Alltech, UK) and one containing Ent. faecium (Lacticil, M74, Medipharm, Sweden). The inoculants were applied at 0.5 times 10⁶ cfu g^-1. Silages with no additives served as controls. After treatment, the chopped forages were ensiled in 1.5 1 anaerobic jars; there were six jars per treatment. After ensiling for 45 d, the silages were tested for aerobic stability in a test in which CO₂ production was measured along with chemical and microbiological parameters.
The inoculated silages that spoiled upon aerobic exposure faster than the controls were those of wheat and of the sorghum cultivar FS5 at the milk stage of maturity. This was evident from intensive CO₂ production and development of yeasts and moulds. Regression analysis indicated that aerobic deterioration of inoculated silages was associated with high levels of residual water-soluble carbohydrates and lactic acid and lack of volatile fatty acids. Aerobic spoilage of inoculated silages was attributed mainly to yeast activity.