M.J. Ranilla

Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep.

This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) levels in the serum of Churra and Merino ewes throughout gestation and the first month post partum. The oPAG levels were determined with an heterologus RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG, sensitivity was 4.0 ng/ml. The P4 levels were measured with a radioimmunological procedure, including a specific extraction step with petroleum ether (bp 60-80 degrees C) with a sensitivity of less than 0.1 ng/ml. There were no differences (P<0.10) in the oPAG profile between breeds from Weeks 1 to 18. From Week 18 to lambing, oPAG concentrations increased rapidly in Churra ewes (on average, from 250 to 650 ng/ml) while remaining relatively constant in the Merino ewes (around 250 ng/ml). No significant differences (P>0.05) were observed for mean weekly P4 levels between the 2 breeds. In both breeds, P4 increased throughout the whole length of gestation, with the highest level measured at Weeks 19-20, then declined 2 wk before parturition. No correlation was found between P4 and oPAG concentrations during gestation in either of the breeds. After lambing, oPAG and P4 levels decreased rapidly in 4 wk to basal values. In both breeds the oPAG concentrations at Weeks 19, 20 and 21 of gestation in ewes carrying male fetuses were higher than in those carrying female fetuses. From the results, we conclude that the breed and sex of the fetus could influence the production of oPAG.

Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet

Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW(0.75). Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH(3)-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.

Influence of exogenous fibrolytic enzymes and fumarate on methane production, microbial growth and fermentation in Rusitec fermenters.

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34.3 endoglucanase, 0.57 exoglucanase, 24.7 xylanase and 5.51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P 0.05). Supplementing with FUM increased (P 0.05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage.

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square to evaluate the effects of 4 diets on microbial synthesis, microbial populations, and ruminal digestion. The experimental diets had forage to concentrate ratios (F:C; DM basis) of 70:30 (HF) or 30:70 (HC) with alfalfa hay (A) or grass hay (G) as forage and were designated as HFA, HCA, HFG, and HCG. The concentrate was based on barley, gluten feed, wheat middlings, soybean meal, palmkern meal, wheat, corn, and mineral-vitamin premix in the proportions of 22, 20, 20, 13, 12, 5, 5, and 3%, respectively (as-is basis). Sheep were fed the diets at a daily rate of 56 g/kg of BW(0.75) to minimize feed selection. High-concentrate diets resulted in greater (P < 0.001) total tract apparent OM digestibility compared with HF diets, but no differences were detected in NDF digestibility. Ruminal digestibility of OM, NDF, and ADF was decreased by increasing the proportion of concentrate, but no differences between forages were detected. Compared with sheep fed HF diets, sheep receiving HC diets had less ruminal pH values and acetate proportions, but greater butyrate proportions. No differences among diets were detected in numbers of cellulolytic bacteria, but protozoa numbers were less (P = 0.004) and total bacteria numbers tended (P = 0.08) to be less for HC diets. Carboxymethylcellulase, xylanase, and amylase activities were greater for HC compared with HF diets, with A diets showing greater (P = 0.008) carboxymethylcellulase activities than G diets. Retained N ranged from 28.7 to 37.9% of N intake and was not affected by F:C (P = 0.62) or the type of forage (P = 0.31). Microbial N synthesis and its efficiency was greater (P < 0.001) for HC diets compared with HF diets. The results indicate that concentrates with low cereal content can be included in the diet of sheep up to 70% of the diet without detrimental effects on ruminal activity, microbial synthesis efficiency, and N losses.

Plasma pregnancy-associated glycoprotein and progesterone concentrations in pregnant Assaf ewes carrying single and twin lambs

Ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) concentrations were monitored weekly during the last three months of gestation and the first month postpartum in plasma of twelve Assaf ewes. The oPAG levels were determined with a heterologous RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG. The P4 levels were measured with a radioimmunological procedure with a sensitivity to less than 0.1 ng ml−1. The oPAG profiles in ewes carrying a single lamb and in those carrying twins were very similar from week 9 of gestation to lambing. Ewes which had twins presented numerically higher oPAG concentrations from week 12 to lambing, but that difference was only significant (P 0.05) was found between oPAG concentrations and lamb birth-weight at any week of gestation considered in this study. However, the lamb birth-weight was positively correlated (P < 0.10) with the P4 concentrations from weeks 9 to 19 of gestation. Results indicate that number of fetuses could influence the oPAG production in ewes during the last three months of gestation.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. II. Protozoa population and diversity of bacterial communities

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of dietary characteristics on microbial populations and bacterial diversity. The purpose of the study was to assess how closely fermenters can mimic the differences between diets found in vivo. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 (high forage; HF) or 30:70 (high concentrate; HC) with either alfalfa hay (A) or grass hay (G) as the forage. Total bacterial numbers were greater in the rumen of sheep fed HF diets compared with those fed HC diets, whereas the opposite was found in fermenters. The numbers of cellulolytic bacteria were not affected by F:C ratio in any fermentation system, but cellulolytic numbers were 2.7 and 1.8 times greater in sheep than in fermenters for HF and HC diets, respectively. Neither total bacterial nor cellulolytic numbers were affected by the type of forage in sheep or fermenters. Decreasing F:C ratio increased total protozoa and Entodiniae numbers in sheep by about 29 and 25%, respectively, but it had no effect in fermenters. Isotrichidae and Ophryoscolecinae numbers in sheep were not affected by changing F:C ratio, but both disappeared completely from fermenters fed HC diets. Total protozoa and Entodiniae numbers were greater in sheep fed A diets than in those fed G diets, whereas the opposite was found in fermenters. Results indicate that under the conditions of the present study, protozoa population in Rusitec fermenters was not representative of that in the rumen of sheep fed the same diets. In addition, protozoa numbers in fermenters were 121 and 226 times lower than those in the sheep rumen for HF and HC diets, respectively. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the diversity of liquid- and solid-associated bacteria in both systems. A total of 170 peaks were detected in the automated ribosomal intergenic spacer analysis electropherograms of bacterial pellets across the full set of 64 samples, from which 160 were detected in at least 1 individual from each system (sheep or fermenter). Diversity of liquid-associated bacterial pellets was greater with G diets in fermenters but seemed to be unaffected by diet in sheep. Bacterial diversity in solid-associated bacteria pellets was greater for G diets compared with A diets in sheep and fermenters. Different conditions in the fermenters compared with sheep rumen might have caused a selection of some bacterial strains.

The influence of diet type (dairy versus intensive fattening) on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production

The objective of this study was to evaluate the effects of increasing doses [0 (control: CON), 20, 60, 180 and 540 mg/L incubation medium] of garlic oil (GO) and cinnamaldehyde (CIN) on in vitro ruminal fermentation of two diets. Batch cultures of mixed ruminal microorganisms were inoculated with ruminal fluid from four sheep fed a medium-concentrate diet (MC; 50 : 50 alfalfa hay : concentrate) or four sheep fed a high-concentrate diet (HC; 15 : 85 barley straw : concentrate). Diets MC and HC were representative of those fed to dairy and fattening ruminants, respectively. Samples of each diet were used as incubation substrates for the corresponding inoculum, and the incubation was repeated on 4 different days (four replicates per experimental treatment). There were GO × diet-type and CIN × diet-type interactions (P 0.05) total volatile fatty acid (VFA) production at any dose. For MC diet, GO at 60, 180 and 540 mg/L decreased (P 0.05) on butyrate proportion were detected. Methane/VFA ratio was reduced (P < 0.05) by GO at 60, 180 and 540 mg/L for MC diet (0.23, 0.16 and 0.10 mol/mol, respectively), and by GO at 20, 60, 180 and 540 mg/L for HC diet (0.19, 0.19, 0.16 and 0.08 mol/mol, respectively), compared with CON (0.26 and 0.21 mol/mol for MC and HC diets, respectively). No effects (P = 0.16–0.85) of GO on final pH and concentrations of NH3-N and lactate were detected. For both diet types, the highest CIN dose decreased (P < 0.05) production of total VFA, gas and methane, which would indicate an inhibition of fermentation. Compared with CON, CIN at 180 mg/L increased (P < 0.05) acetate proportion for the MC (629 and 644 mmol/mol total VFA for CON and CIN, respectively) and HC (525 and 540 mmol/mol total VFA, respectively) diets, without affecting the proportions of any other VFA or total VFA production. Whereas for MC diet CIN at 60 and 180 mg/L decreased (P < 0.05) NH3-N concentrations compared with CON, only a trend (P < 0.10) was observed for CIN at 180 mg/L with the HC diet. Supplementation of CIN up to 180 mg/L did not affect (P = 0.18–0.99) lactate concentrations and production of gas and methane for any diet. The results show that effectiveness of GO and CIN to modify ruminal fermentation may depend on diet type, which would have practical implications if they are confirmed in vivo.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. I. Digestibility, fermentation parameters, and microbial growth

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of forage to concentrate (F:C) ratio and type of forage in the diet on ruminal fermentation and microbial protein synthesis. The purpose of the study was to assess how closely fermenters can mimic the dietary differences found in vivo. The 4 experimental diets contained F:C ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Microbial growth was determined in both systems using (15)N as a microbial marker. Rusitec fermenters detected differences between diets similar to those observed in sheep by changing F:C ratio on pH; neutral detergent fiber digestibility; total volatile fatty acid concentrations; molar proportions of acetate, propionate, butyrate, isovalerate, and caproate; and amylase activity. In contrast, Rusitec fermenters did not reproduce the dietary differences found in sheep for NH(3)-N and lactate concentrations, dry matter (DM) digestibility, proportions of isobutyrate and valerate, carboxymethylcellulase and xylanase activities, and microbial growth and its efficiency. Regarding the effect of the type of forage in the diet, Rusitec fermenters detected differences between diets similar to those found in sheep for most determined parameters, with the exception of pH, DM digestibility, butyrate proportion, and carboxymethylcellulase activity. Minimum pH and maximal volatile fatty acid concentrations were reached at 2h and at 6 to 8h postfeeding in sheep and fermenters, respectively, indicating that feed fermentation was slower in fermenters compared with that in sheep. There were differences between systems in the magnitude of most determined parameters. In general, fermenters showed lower lactate concentrations, neutral detergent fiber digestibility, acetate:propionate ratios, and enzymatic activities. On the contrary, fermenters showed greater NH(3)-N concentrations, DM digestibility, and proportions of propionate, butyrate, isovalerate, valerate, and caproate. Values of efficiency of microbial growth were greater in fermenters compared with sheep for 70:30 diets, but they were lower for 30:70 diets. Differences between fermentation in sheep and fermenters can be mainly attributed to the lack of absorption in fermenters, differences in solid retention time, and compartmentalization in the Rusitec system. In general, the Rusitec system simulated more closely the in vivo fermentation of high-forage diets compared with high-concentrate diets.

Influence of detachment procedure and diet on recovery of solid-associated bacteria from sheep ruminal digesta and representativeness of bacterial isolates as assessed by automated ribosomal intergenic spacer analysis-polymerase chain reaction

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square experiment designed to evaluate the efficiency of 3 detachment procedures (DP) to recover solid-associated bacteria (SAB) from ruminal digesta. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Bacterial biomass was labeled with 15NH4Cl. The DP were 1) MET: digesta was incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose under continuous shaking; 2) STO: digesta was mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 rpm; 3) FRE: digesta was immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of digesta 2 times in the treatment solutions. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the similarity between bacterial communities attached to the digesta and those in the pellet obtained after each DP. There were no significant F:C x DP or forage x DP interactions for any variable. On average, STO treatment detached 65.8% of SAB from ruminal digesta, about 1.2 and 1.5 times more than FRE and MET treatments, respectively. Total recovery of SAB in STO pellets (48.9%) was greater compared with FRE (31.7%) and MET (33.1%), values being greater for high-forage compared with high-concentrate diets. Similarity index between the bacteria attached to digesta and those in the pellets were lower for FRE (48.2%) compared with MET (54.1%) and STO (54.1%), which suggests that FRE could have destroyed cell integrity of some bacterial species, thus reducing the bacterial diversity present in the pellets. The STO method was the most effective removing SAB from digesta, but only a moderate similarity between the bacterial communities attached to digesta and those recovered in the bacterial pellets was obtained. Values of duodenal microbial flow estimated using SAB as reference bacteria were greater with FRE compared with STO and MET, but all DP detected similar differences between diets, and therefore did not influence the interpretation of results.

Effects of dilution rate and retention time of concentrate on efficiency of microbial growth, methane production, and ruminal fermentation in Rusitec fermenters

The objective of this study was to investigate the effects of 2 dilution rates (DL) and 2 concentrate retention times (RT) on microbial growth, methane production, and fermentation of a 30:70 alfalfa hay:concentrate diet in Rusitec fermenters maintained at similar pH. The DL were 3.78 (low DL, LDL) and 5.42%/h (high DL, HDL), and concentrate RT was either 24 h (T24) or 48 h (T48). Forage RT was 48 h in all fermenters. Apparent disappearance of diet DM and NDF was greater in HDL fermenters compared with LDL fermenters, but there was a significant DL x concentrate RT interaction, showing that the effect of DL was more pronounced in T48 compared with T24 fermenters. Methane production was not affected by DL, but was greater in T48 compared with T24 fermenters, which was consistent with the increased fiber degradation in T48 fermenters. Increasing DL augmented volatile fatty acid production and molar proportions of propionate, isovalerate, and valerate, and reduced those of caproate, but no effects were observed on acetate, butyrate, and isobutyrate proportions. Increasing concentrate RT resulted in greater volatile fatty acid production and proportions of acetate, butyrate, and caproate, but reduced those of propionate, valerate, and isovalerate. Ammonia-N production was not affected by concentrate RT, but was greater at HDL compared with LDL. Microbial growth was not affected by DL, but microbial growth efficiency was lower in HDL compared with LDL fermenters. Concentrate RT affected microbial growth and its efficiency, with both being greater in T48 compared with T24 fermenters. Carboxymetylcellulase and xylanase activities in ruminal fluid were greater in HDL compared with LDL fermenters, but were not affected by concentrate RT. There were DL x concentrate RT interactions for diet apparent disappearance, molar proportions of propionate, butyrate, isovalerate, and caproate, and acetate:propionate ratio, indicating that effects of DL on these variables were influenced by concentrate RT. The results would indicate that using higher DL and shorter concentrate RT than those typically used in Rusitec fermenters would contribute to improving the simulation of in vivo fermentation of high-concentrate diets.