In vitro determination of generation times for Entodinium exiguum, Ophryoscolex purkynjei and Eudiplodinium maggii

ABSTRACT. Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum . This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24–48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3–4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum , 15.0 h for E. maggii and 12.8 h for O. purkynjei . In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

A cryopreservation procedure for the rumen protozoon Entodinium caudatum: estimation of its viability by fluorescence microscopy

AIMS: To study the viability of a culture of the rumen protozoon Entodinium caudatum after a cryopreservation procedure by a fluorescence microscopy staining method. METHODS AND RESULTS: Fluorescence method is based on the different colour of cells depending on their membrane integrity. When the temperature effect was studied either by fluorescence or motility, the techniques were correlated (r = 0.727) and their slopes and intercepts were not different (P > 0.05). However, motility showed a higher variation coefficient (0.40 vs 0.12). There were no differences between cooling rates at cryopreservation (1 and 4 degrees C min-1) at 38, 15 or 5 degrees C, nor after thawing. CONCLUSIONS: Fluorescence staining is more accurate than motility for assessing protozoal viability. Viability after thawing was 0.50, and the number of viable cells per 250 microl straw was 320 and 420 for 1 and 4 degrees C min-1. SIGNIFICANCE AND IMPACT OF THE STUDY: This cryopreservation procedure seems to ensure culture recovery for E. caudatum.     

Effect of the rumen ciliates Entodinium caudatum,Epidinium ecaudatum and Eudiplodinium maggii,and combinations thereof,on ruminal fermentation and total tract digestion in sheep

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 × 2 × 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH₄-to-CO₂ ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.     

Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate Entodinium caudatum culture

The influence of insulin (17.4 nmol l-1) on total gas and methane production, the concentration of total and individual fatty acids and dry matter degradability was investigated in the rumen ciliate culture of Entodinium caudatum. The experimental groups consisted of control group (without insulin) and two groups with insulin application--single shot and long-term application (over 30 days). Fermentation activity of each experimental group was observed on two subgroups: whole protozoan culture (protozoa plus bacteria) and bacterial fraction (bacteria without protozoa). Long-term application of insulin significantly increased methane production and DM degradability in the whole protozoan culture. Total VFA concentration was significantly increased by long-term as well as single-dose application of insulin (by 255% and 158%, respectively). The growth of the protozoa was not influenced by insulin treatments. It can be concluded that the fermentation activity of the community of the rumen ciliate Entodinium caudatum culture was marked stimulated by application of insulin.     

Preliminary study on the requirements of Entodinium exiguum and E. caudatum for live or dead bacteria when cultured in vitro

Whether live bacteria are required to culture the rumen protozoa Entodinium exiguum and E. caudatum in vitro was studied. Treatments were protozoa plus antibiotics (PA), PA plus autoclaved bacteria (PAB) or protozoa plus live bacteria (PLB). Generation times at 24 h were 22.8 and 31.0 h for E. exiguum and E. caudatum. Protozoal concentrations were unaffected by the absence of bacteria up to 48 h. After 72 h, E. exiguum, concentrations were higher in PLB than PA or PAB. With E. caudatum differences between PLB and PA were only observed at 96 h. Thus, a requirement for live bacteria appears to be manifested in culture periods longer than 48 (E. exiguum) and 72 (E. caudatum) h. Although differences between PLB and PAB indicate a metabolic dependence for bacteria or a long-term antibiotic effect, non-significant differences between PAB and PA suggest that the effect is also related to a nutritive bacterial contribution.     

Fourteen morphotypes of Entodinium ovumrajae (Ophryoscolecidae, Entodiniomorphida) found in the Dromedary camel of Egypt

During a survey of the ciliate protozoal composition of the stomach contents of nine dromedary camels of Egypt, fourteen morphotypes of Entodinium ovumrajae, which has been considered as a species peculiar to camels, were found in six camels. Except for five morphotypes including one originally described as an independent species and its forms, these were newly detected. These morphotypes, divided into three groups, can be identified mainly by the morphology of their ectoplasmic processes. Each camel had on average, about five morphotypes of this species.     

Effect of the rumen ciliates Entodinium caudatum, Epidinium ecaudatum and Eudiplodinium maggii, and combinations thereof, on ruminal fermentation and total tract digestion in sheep

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 x 2 x 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH4-to-CO2 ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.     

Effect of plant secondary compounds on in vitro methane,ammonia production and ruminal protozoa population

Aims

The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied.

Methods and Results

Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)‐6000 (400 mg DM) as a tannin binder during 24‐h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg^?1 DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4·0 g kg^?1 in only Alpinia galanga (7·50), Cinnamomum verum (4·58), Pelargonium graveolens (18·7) and Pimenta officinalis (23·2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin‐binding agent PEG‐6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131·8) leaves, followed by Indigofera tinctoria (16·8) and Withania somnifera (10·2) leaves. Total and differential protozoal counts increased with added PEG in twenty‐two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG‐6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N‐digestibility as evidenced by increased accumulation of NH₃‐N with added PEG.

Conclusions

Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation.

Significance and Impact of the Study

It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.     

Rumen Ciliate Protozoa of the Blue Duiker (Cephalophus monticola), with Observations on Morphological Variation Lines Within the Species Entodinium dubardi

ABSTRACT. Protozoal concentrations were determined in rumen and cecal contents of 20 blue duikers ( Cephalophus monticola ). Ten animals of each sex were fed either a high concentrate or high roughage diet. Rumen protozoa were present in 19 of the 20 animals and concentrations ranged from 4.5 to 33.7 × 10⁶ per g of rumen contents. At the higher concentrations, protozoal cells equaled between 30–40% of the total rumen contents volume. No protozoa were found in cecal contents. Weight of rumen contents was higher in females than in males ( P < 0.01), and rumen protozoa concentrations were higher in males ( P < 0.05) and in those animals fed the high concentrate diet ( P < 0.05). All the protozoa were identified as belonging to a single species, Entodinium dubardi . However, an average of about 30% of the E. dubardi cells varied from the typical morphology of this species. These cells appeared to be on variation lines leading toward 7–10 other non-caudate species of Entodinium . The present data were used to evaluate and discuss the concept of variation lines within E. dubardi .     

Effect of the cryopreservation conditions on the viability of the rumen ciliate Diploplastron (Metadinium) affine

AIMS: To study the viability of Diploplastron (Metadinium) affine after its cryopreservation at two cooling rates, and the effect of procedure conditions on viability. METHODS AND RESULTS: There were differences in viability between cooling rates (1 and 4 degrees C min(-1)) at 15 or 5 degrees C, but not after thawing. When the equilibrium temperature (25 or 5 degrees C), the cryopreservant (glycerol or dimethyl sulfoxide [DMSO]) and the use of membrane protector were tested, there were no differences caused by the cryopreservant or the membrane protector. However, the equilibrium at 25 degrees C increased the viability (P = 0.005) compared with 5 degrees C. CONCLUSIONS: Viability after thawing was 0.10-0.19. Adding the cryopreservant (either glycerol or DMSO) at 25 degrees C instead of 5 degrees C improves viability of D. affine after thawing. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions of cryopreservation are largely dependent on the species of rumen protozoa. Number of viable cells after thawing would indicate the possibility of culture recovery for D. affine.     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

Effects of inoculum source,pH, redox potential and headspace di-hydrogen on rumen in vitro fermentation yields

This in vitro study aimed at understanding how abiotic, that is chemical and electrochemical potentials, and biotic factors combine to impact the outputs of rumen volatile fatty acid (VFA). Using a 48-run design optimized by means of an exchange algorithm, the curvilinear effects of pH, Eh and partial pressure of dihydrogen (H₂) on fermentation yields were investigated in 6-h batch cultures of mixed rumen microbes, fed on glucose so as to bypass the enzymatic hydrolysis and conversion steps preceding the glycolytic pathway. The role played by rumen microbiota in the expression of these effects was explored by testing three inocula grown on feeds supplying a microflora adapted to fibre, slowly degradable or readily degradable starch as the dominant dietary polysaccharide. Data were fitted to 2nd-order polynomial models. In fibre-adapted cultures, the yields of major VFA were mainly influenced by pH and H₂ partial pressure, in opposite ways. In wheat grain-adapted cultures, the VFA yields underwent the opposite influences of pH, in a curvilinear way for propionate, and Eh since acetate production yield was not significantly modified by any factor. In maize grain-adapted cultures, acetate production yield was not modified by any factor but H₂ in a quadratic way when the production yields of higher VFA underwent opposite influences of pH and Eh. In conclusion, the effects of environmental factors were dependent on the nature of the inoculum, a major source of variation, and more particularly on its adaptation to high- or low-fibre diets. These effects were loosely interrelated, the pH being the most active factor before the Eh and H₂ partial pressure.     

A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of concentrate percentage on ruminal pH and time-budget in dairy goats

The aim of this study was to compare rumen pH and time-budget in eight mid-lactation goats receiving two diets in a cross-over design (low-concentrate diet (L): 30% and high-concentrate diet (H): 60% concentrate). Feeding H increased daily intake (4.3 ± 0.08% v. 4.7 ± 0.08% of body weight for L and H, respectively) and daily milk production (3.01 ± 0.130 v. 3.50 ± 0.130 kg/day of 3.5% fat-corrected milk for L and H, respectively). It decreased milk fat and inverted the fat-to-protein ratio (1.07 ± 0.054 v. 0.94 ± 0.054 for L and H, respectively). As suggested by the percentage of time spent with rumen pH below 6.0 (23.4 ± 6.60% v. 39.9 ± 5.88% for L and H, respectively), H was more acidogenic than L. When offered H instead of L, goats spent less time eating (298 ± 17.5 v. 265 ± 17.5 min for L and H, respectively) and ruminating (521 ± 21.0 v. 421 ± 21.0 min for L and H, respectively) but more time resting (352 ± 27.1 v. 459 ± 21.1 min for L and H, respectively) over a 24-h period. They also tended to spend more time drinking (20 ± 2.9 v. 25 ± 2.9 min for L and H, respectively; P = 0.08) when offered H rather than L. These differences in activities were mainly observed during the first hours following feeding. When offered H, goats adapted their feeding behaviour around the feedings, which allowed them to limit the physiological disturbances potentially inducible by H and to increase milk production, without experiencing too much acidosis.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.