A. I. Martín-García

Effect of bromochloromethane on methane emission, rumen fermentation pattern, milk yield, and fatty acid profile in lactating dairy goats

Several technologies have been tested to reduce enteric methanogenesis, but very few have been successfully used in practical conditions for livestock. Furthermore, the consequences of reduced rumen methane production on animal performance and milk quality are poorly understood. The aim of this work was to investigate the effect of feeding bromochloromethane (BCM), a halogenated aliphatic hydrocarbon with potential antimethanogenic activity, to dairy goats on rumen methane production, fermentation pattern, the abundance of major microbial groups, and on animal performance and milk composition. Eighteen goats were allocated to 2 experimental groups of 9 animals each: treated (BCM+) or not (BCM-) with 0.30 g of BCM/100 kg of body weight per day. The BCM was administered per os in 2 equal doses per day from parturition to 2 wk postweaning (10 wk). After weaning, methane emissions were recorded over 2 consecutive days (d 57 and 58 on treatment) in polycarbonate chambers. On d 59, individual rumen fluid samples were collected for volatile fatty acid (VFA) analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. On d 69 and 70, daily milk production was recorded and samples were collected for determination of fat, protein, lactose, casein, and total solids concentration by infrared spectrophotometry, and fatty acid composition by gas chromatography. Treatment with BCM reduced methane production by 33% (21.6 vs. 14.4 L/kg of DMI) compared with nontreated animals, although it did not affect the abundance of rumen bacteria, protozoa, and total methanogenic archaea. The observed improvement in the efficiency of digestive processes was accompanied by a 36% increase in milk yield, probably due to the more propionic type of rumen fermentation and an increase in VFA production. The increase in milk yield was not accompanied by any changes in the concentrations or yields of fat, protein, or lactose. Despite the substantial decrease in methane production, only minor changes in milk fatty acid profile were observed, suggesting that ruminal biohydrogenation pathways were not affected. Compounds that influence terminal biochemical pathways for methane production deserve further development for future application in the dairy goat sector.

Effects of forage:concentrate ratio and forage type on apparent digestibility, ruminal fermentation, and microbial growth in goats.

The effects of forage type and forage:concentrate ratio (F:C) on apparent nutrient digestibility, ruminal fermentation, and microbial growth were investigated in goats. A comparison between liquid (LAB) and solid (SAB)-associated bacteria to estimate microbial N flow (MNF) from urinary purine derivative excretion was also examined. Treatments were a 2 x 2 factorial arrangement of forage type (grass hay vs. alfalfa hay) and high vs. low F:C (70:30 and 30:70, respectively). Four ruminally cannulated goats were fed, at maintenance intake, 4 experimental diets according to a 4 x 4 Latin square design. High-concentrate diets resulted in greater (P < 0.001) nutrient digestibility except for ADF. However, CP digestibility increased (P < 0.001) only for the high-concentrate diets including grass hay. Likewise, N retention, ruminal NH(3)-N concentration, and urinary excretion of purine derivatives increased (P < 0.05) with increasing concentrate in animals fed diets based on grass hay (0.23 vs. 0.13 g of retained N/g of digested N, 30.1 vs. 12.9 mg of NH(3)-N/100 mL, and 11.5 vs. 8.40 mmol/d, respectively), but not (P > 0.05) when diets included alfalfa hay. Total protozoa numbers and holotricha proportion were greater and less (P < 0.001), respectively, in high- than in low-concentrate diets. The F:C affected (P < 0.001) ruminal pH but not total VFA concentration (P = 0.12). Ammonia-N concentration was similar (P = 0.13) over time, whereas pH, VFA concentration, and protozoa numbers differed (P < 0.001) among diets. Estimated MNF was strongly influenced by using either the purine bases:N ratio obtained in our experimental conditions or values reported in the literature for small ruminants. There was a F:C effect (P = 0.006) on MNF estimated from LAB but not from SAB. The effect of F:C shifting from 70:30 to 30:70 in goat diets depends on the type of forage used. The MNF measured in goats fed different diets was influenced by the bacterial pellet (LAB or SAB). In addition, the purine bases:N ratio values found were different from those reported in the literature, which underlines the need for these variables to be analyzed directly in pellets isolated from specific animals and experimental conditions.

Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal fermentation, microbial abundance, and methane emissions in sheep

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3 NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3 NP (40 and 80 μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3 NP at one dose (100mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3 NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals.

Nutritional intervention in early life to manipulate rumen microbial colonization and methane output by kid goats postweaning.

The growing interest in reducing methane (CH4) emissions from ruminants by dietary means is constrained by the complexity of the microbial community in the rumen of the adult animal. The aim of this work was to study whether intervention in early life of goat kids has an impact on methane emissions and the microbial ecosystem in the rumen and whether the effects persist postweaning. Sixteen doe goats giving birth to 2 kids each were randomly split into 2 experimental groups: 8 does were treated (D+) with bromochloromethane (BCM) after giving birth and over 2 mo, and the other 8 does were not treated (D-). In both groups of does, 1 kid per doe was treated with BCM (k+) for 3 mo, and the other was untreated (k-), resulting in 4 experimental groups: D+k+, D+k-, D-k+, and D-k-. Methane emissions were recorded, and ruminal samples were collected from kids at 2 mo of age (weaning, W) and 1 (W+1) and 4 (W+4) mo later. At W+1 mo, CH4 emissions by k+ kids were 52% and 59% less than untreated kids (in D+ and D- groups, respectively). However, at W+4 mo, only D+k+ kids remained lower (33%) emitters and exhibited greater daily BW gain (146 g/d) compared with the other 3 groups (121.8 g/d). The analysis of the archaeal community structure by Denaturing Gradient Gel Electrophoresis (DGGE)showed a strong effect of BCM treatment on does and kids that persisted only in D+k+ kids. The study showed that the application of BCM during early life of kids modified the archaeal population that colonized the rumen, which resulted in decreased CH4 emissions around weaning. The effect is influenced by the treatment applied to the doe and persisted 3 mo later in D+k+ kids.

Effects of partial replacement of concentrate with feed blocks on nutrient utilization, microbial N flow, and milk yield and composition in goats

Two experiments were carried out to evaluate the effect of partial replacement of concentrate with 2 types of feed blocks (FB) on rumen protozoa numbers, nutrient utilization, microbial N flow to the duodenum, and milk yield and composition in goats. The concentrate included oat, corn grain, barley, soybean meal, salt, and vitamin-mineral mixture. The FB (types I and II) were composed of crude 2-stage olive cake (120 or 100g/kg), fava beans (0 or 400g/kg), barley (320 or 200g/kg), beet molasses (220 or 100g/kg), sunflower meal (180 or 0g/kg), quicklime (70 or 90g/kg), salt (60g/kg), urea (0 or 20g/kg), and vitamin-mineral mixture (30g/kg). In experiment 1, 6 adult, dry, nonpregnant, rumen-fistulated Granadina goats (46.9+/-2.15kg of BW) were used and 3 trials were carried out. In each trial, 2 goats were randomly assigned to receive 600g of alfalfa hay and 400g of concentrate (diet AC), 600g of alfalfa hay, 200g of concentrate, and FB I (diet ACBI), or 600g of alfalfa hay, 200g of concentrate, and FB II (diet ACBII) with 6 replications per diet. The FB were supplied ad libitum. The ratio of purine bases to N was higher in solid- and liquid-associated bacteria for FB goats than for AC goats. In experiment 2, 18 Granadina goats (39.6+/-1.89kg of BW) in the middle of the third lactation were used, and 3 trials were carried out by following a 3 x 3 Latin square experimental design. In every trial, 6 animals randomly received 1.0kg of alfalfa hay supplemented with 1.0kg of concentrate (diet AC) or 0.5kg of concentrate and FB I and II (diets ACBI and ACBII) with 18 replications per diet. The FB were supplied ad libitum. The intakes of organic matter and fat were higher with the AC diet than with the FB diets. The intake of acid detergent fiber was higher for FB-containing diets than for the AC diet. The neutral detergent fiber digestibility of FB diets was higher than that of the AC diet. Energy intake was higher for diets AC and ACBII than for ACBI. Microbial N flow was affected by diet. Milk yield was higher in goats fed the AC diet than in those receiving the FB diets. Conjugated linoleic acid content was higher in milk from FB than in milk from AC goats. Our study suggests that FB type II based on local ingredients could be used advantageously to reduce half of the amount of concentrate without detrimental effects on nutrient utilization, N value of the diet, and milk composition. The decrease of milk yield with ACBII compared with that obtained with the AC diet could be compensated by better quality of milk, decreased cost of feeding, and environmental advantage derived of including by-products in FB.

Feeding management in early life influences microbial colonisation and fermentation in the rumen of newborn goat kids

The aim of this work was to study the colonisation of the rumen by the three main microbial groups over the first 4 weeks of life and to assess to what extent the type of feeding management (natural with the mother, NAT, or artificial with milk replacer, ART) exerts an effect. Thirty pregnant goats carrying two fetuses were selected. At birth, one kid was taken immediately away from the doe and fed milk replacer (ART), while the other kid remained with the mother (NAT). Groups of four kids (from ART and NAT experimental groups) were slaughtered at 1, 3, 5, 7, 14, 21 and 28 days of life, resulting in seven sampling times. On the sampling day, after slaughtering, the rumen was weighed full and empty and the content sampled and pH measured. Aliquots of rumen digesta were immediately frozen for DNA and volatile fatty acid (VFA) analyses. The weight of the rumen was higher in NAT kids from Day 21 onward, while no difference was observed for rumen content volume. The three microbial groups (bacteria, archaea and protozoa) were detected from Day 1 in both experimental groups. The concentration of bacteria was higher in the rumen of NAT kids on Days 3, 5, 7 and 14 and of protozoa from Day 3 onward. This was reflected in greater VFA concentrations and lower pH in the rumen of NAT kids from Day 3. Our results confirmed substantial microbial colonisation from the first day of life in the undeveloped rumen. The feeding management (natural vs artificial) before weaning had an effect on microbial colonisation and rumen fermentation and, therefore, it should be considered when designing nutritional intervention strategies in early life.

In vitro – in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

Diet composition at weaning affects the rumen microbial population and methane emissions by lambs

The aim of this experiment was to investigate whether different diets at weaning determine the microbial populations established in the rumen, together with its methanogenic capacity, and whether these differences are consistent over a longer time period. Twenty ewes with single lambs were used in two periods. Period I: 10 lambs had access only to grass hay whilst with the dam and for 8 weeks after weaning (group H). The other 10 lambs had free access to concentrate and grass hay whilst with the dam and were fed with a 60 : 40 mix of concentrate and grass hay for 8 weeks after weaning (group C). Eight weeks after weaning, methane emissions were measured in polycarbonate chambers over a 3-day period. After methane emission measurements, five lambs from each group were randomly selected and slaughtered and samples of rumen content collected for measuring rumen fermentation parameters and for microbial enumeration (total and cellulolytic bacteria and methanogenic archeaea) by most probable number. Period II: the remaining 10 lambs were grouped together and fed the same diet (grass and concentrate) for 4 months. After this period, all animals were fed concentrate and grass hay (60 : 40) for 2 weeks and introduced in to the chambers to measure methane emissions over a 3-day period. After measurements, they were slaughtered and rumen samples collected and analysed as in Period I. Lambs from group H produced more (P = 0.04) methane than group C lambs (26.0 v. 22.5 L/kg DM intake) in Period I. Group H lambs also had less total bacteria (10.2 × 1010 v. 61.6 × 1010 cells; P = 0.284) but more cellulolytic bacteria (40.6 × 109 v. 10.0 × 109 cells; P = 0.098) and methanogenic archaea (37.1 × 109 v. 19.0 × 109 cells; P = 0.113) than group C lambs in Period I. The acetate to propionate ratio tended to be higher (P = 0.089) in group H lambs than in group C lambs (3.00 v. 2.35). In Period II, methane produced was not different (P > 0.05) between the groups (26.6. v. 25.7 L/kg DM intake by group C and H lambs, respectively). Microbial numbers and fermentation parameters were also similar in samples collected from both experimental groups in Period II. Our results show that the differences observed as a result of providing different diets at weaning disappear in the long-term. It may be appropriate to study a wider range of dietary treatments to better understand the factors determining the microbial populations establishing in the rumen.

Effects of concentrate replacement by feed blocks on ruminal fermentation and microbial growth in goats and single-flow continuous-culture fermenters.

The effect of replacing concentrate with 2 different feed blocks (FB) on ruminal fermentation and microbial growth was evaluated in goats and in single-flow continuous-culture fermenters. Diets consisted of alfalfa hay plus concentrate and alfalfa hay plus concentrate with 1 of the 2 studied FB. Three trials were carried out with 6 rumen-fistulated Granadina goats and 3 incubation runs in 6 single-flow continuous-culture fermenters. Experimental treatments were assigned randomly within each run, with 2 repetitions for each diet. At the end of each in vivo trial, the rumen contents were obtained for inoculating the fermenters. For each incubation run, the fermenters were inoculated with ruminal fluid from goats fed the same diet supplied to the corresponding fermenter flask. The average pH values, total and individual VFA, and NH(3)-N concentrations, and acetate:propionate ratios in the rumen of goats were not affected (P >or= 0.10) by diet, whereas the microbial N flow (MNF) and efficiency were affected (P <or= 0.001), with the greatest values observed for the diet without FB. In fermenters, the diet affected pH (P<0.001), propionate concentrations (P=0.01), acetate:propionate ratio (P=0.03), carbohydrate digestibility (P >or= 0.05), and total (P=0.02), NH(3) (P=0.005), and non-NH(3) (P=0.02) N flows, whereas the efficiency of VFA production was not affected (P=0.75). The effect of diet on MNF and efficiency depended on the bacterial pellet used as a reference. An effect (P<0.05) of diet on the composition of solid- and liquid-associated bacteria was observed. The compositions of liquid-associated bacteria in the fermenter contents and effluent were similar (P=0.05). Differences (P<0.001) between in vivo and in vitro values for most fermentation variables and bacterial pellet compositions were found. Partial replacement of the concentrate with FB did not greatly compromise carbohydrate fermentation in unproductive goats. However, this was not the case for MNF and efficiency. Differences between the results obtained in vivo and in vitro indicate a need to identify conditions in fermenters that allow better simulation of fermentation, microbial growth, and bacterial pellet composition in vivo. Reduced feeding cost could be achieved with the inclusion of FB in the diets of unproductive goats without altering rumen fermentation.

A comparison of different legume seeds as protein supplement to optimise the use of low quality forages by ruminants

The potential of different legume seeds species, including recently new developed varieties (Vicia faba: a commercial variety and varieties Alameda, Palacio and Baraka; Lupinus angustifolius; Pisum sativum and Cicer arietinum: varieties Fardon and Zegrí) as protein supplements to low quality forages was evaluated. First, the chemical composition, in vitro digestibility, in situ degradability and in vitro/in situ intestinal digestibility of legume seeds were determined. The chemical composition was rather similar within genus. Vicia faba beans contained more condensed tannins (35.8–56.4 g/kg DM) and less ether extract (12.8–9.5 g/kg DM) than the other legumes. The rumen degradability and intestinal digestibility of the undegraded protein in the rumen was very similar among the seeds, with exception of lupins, having a much higher degradation rate than the rest. Second, the fermentation characteristics of diets, which were based on two low quality forages (olive leaves and barley straw) and feed blocks without or with supplementation of legume seeds or soybean meal, were investigated by using single-flow continuous culture fermenters. In this trial, the fermentation parameters (ammonia, pH and volatile fatty acids), the microbial protein synthesis and the degradation of olive leaves and barley straw promoted by the different diets were studied. Compared to soybean meal, beans and peas showed similar suitability as protein supplements for sustaining in vitro fermentation of low-quality forages. However, our results suggest a significant interaction between the type of legume used as supplement and the type of forage used, which need to be further studied in vivo.