Effect of storage time on chemical, physical and microbial composition of total mixed rations for lactating dairy cattle

Kwikiriza J, Fred K¹ and Hussein K W

Department of Agricultural Production, School of Agricultural Sciences, College of Agricultural and Environmental Science, Makerere University, P O Box 7062, Kampala, Uganda
kwikirizajustus1@gmail.com
¹ National Livestock Resources Research Institute, National Agricultural Research Organization, P O Box 5704, Kampala, Uganda

Abstract

Total mixed ration (TMR) is a method of feeding that has been adopted in large scale cattle enterprises primarily to facilitate the distribution of feed to large numbers of animals. However, aerobic spoilage during storage and distribution can lead to o reduce feed quality. The purpose of this study was, therefore, to assess the dynamic changes in chemical, microbial, and physical quality of a TMR that occur during air exposure to deepen the understanding of aerobic deterioration, the cause of spoilage and feed quality changes overtime. Maize silage, Chloris gayana hay, wheat bran, molasses, maize grain, soybean meal, rice bran, common salt, bentonite, limestone, di-calcium phosphate, and mineral powder were mixed) to formulate a TMR ration. Without compaction, 3 kg were sampled from a TMR produced at the National Livestock Resources Research Institute (NaLiRRI) and kept in sterile polythene sample bags with the upper surface exposed to air. Samples for analysis were collected every 6 hours for the first day and every 12 hours thereafter for the subsequent two days. Samples collected at 0, 6, 12, 18, 24, 36, 48, and 72 hours were used to assess the dynamic changes in chemical, microbial, and physical quality of the TMR. An aerobic stability test showed that TMR started to deteriorate at 5 h of aerobic exposure when the TMR temperature was 2℃ above ambient temperature. The pH linearly increased (p<0.0001) with increasing storage time. Dry matter (DM) and organic matter (OM) decreased at a decreasing rate (p<0.0001) with storage time. Moisture content and ash increased at a decreasing rate with increasing storage time. Significant pH and ash increases were observed at 12 and 18 h respectively, while OM and DM decreases were significant at 18 and 12 h respectively. Molds and enterobacteria linearly increased (P<0.05) with storage time while yeast, lactic acid bacteria, and aerobic bacteria increased at a decreasing rate (P<0.05) with storage time. Regression analysis showed lactic acid bacteria (LAB) to have the highest proliferation in the first 6 hours, followed by yeasts, aerobic bacteria (AB), molds, and lastly enterobacteria (EB) with simple linear regression coefficients of 0.84, 0.55, 0.33, 0.23, and -0.37, respectively. The regression coefficients of LAB, yeasts, molds, and aerobic bacteria showed a sharp increase (p<0.05) at 12 hours, while enterobacteria showed a sharp increase (p<0.05) at 36 hours. TMR, when exposed to air, deteriorates rapidly due to changes in physical, chemical, and microbial composition, resulting in high losses of nutritional components. Yeasts and aerobic bacteria were responsible for initiation of aerobic deterioration. Enterobacteria and molds tended to play a secondary role in TMR deterioration. Deterioration occurred within 5 hours of aerobic exposure. Therefore, yeast and aerobic bacteria are the major players in initiating aerobic spoilage in TMR.

Key words: aerobic bacteria, aerobic spoilage, enterobacteria, lactic acid bacteria, molds, yeasts

Introduction

Livestock contributes about 17% to agricultural GDP, and dairy farming contributes about 40-50% of livestock-related GDP (Balikowa 2011). Feeding accounts for more than 50-60% of the total cost of a livestock enterprise (Kishore et al 2017). However, the allocation of land for fodder cultivation has been decreasing tremendously because of urbanization and other land uses. Also, the cost of concentrate feed ingredients has gone up because of competition with the human food chain (Ramachandra et al 2007).

This leads to the exploration of alternate feed resources which do not compete with the human food chain. Various feeding systems have been developed for optimum utilization of feed resources for sustainable livestock production. The different crop residues and agro-industrial by-products that are available locally are of poor nutritional quality. Scientific utilization of these crop residues in combination with the conventional concentrate feed ingredients helps in the preparation of a quality feed for economical livestock production (Kishore et al 2017). Fibrous crop residues and agro-industrial by-products play an important role as a source of feed for ruminants, but the utilization of these feeds is limited because of poor nutrient content and digestibility.

The feeding value of these crop residues can be increased by incorporating them into total mixed rations (TMR) by fortifying them with required nutrients (Pattanaik et al 2010). By feeding a nutritionally balanced ration at all times, cows are allowed to consume as close to their actual energy requirements as possible, thus maintaining the physical or roughage characteristics required for proper rumen function (Bargo et al 2002).

However, when total mixed rations are exposed to oxygen during storage, aerobic microorganisms cause heating and spoilage. Yeasts that metabolize lactic acid are the primary spoilage organisms in total mixed rations, although acetic acid bacteria and other organisms can also cause spoilage (Kung et al 1998). These microbes reduce the nutritional value of feed as organic matter is lost as carbon dioxide gas from the feed. Total mixed rations that have spoiled because of exposure to air are undesirable because of a loss of nutrients and the potential for negative effects on animal performance or humans due to the development of molds that produce mycotoxins. (Driehuis and Elferink 2000; Woolford 1990). This creates a need to determine the factors for aerobic instability.

The objectives of the experiment described in this paper were to evaluate microbial, chemical, and physical changes with storage time in dairy TMR, thus assessing causes of spoilage and feed quality changes after formulation.

Materials and methods

Experimental site description

The experiment was conducted in Nakyesasa at the National Livestock Resources Research Institute (NaLiRRI) in Wakiso district, Uganda. NaLiRRI is located at 0°31′N, 32°35′E in central Uganda, at an elevation of 1127 m above sea level. The area receives a bimodal rainfall pattern, with the first rainfall peak lasting from March to June and the other from September to November. Annual average precipitation is 1270 mm and with temperatures ranging from 18 to 26 °C.

Experimental setup:

Maize silage, Chloris gayana hay, wheat bran, molasses, maize bran, soybean meal, rice bran, common salt, bentonite, limestone, di-calcium phosphate, and mineral powder were mixed in a ratio of (307.3:53.8:110.5:27.6:35.3:71.8:110.2:0.2:4.9:29.5:21.7:1.7) to formulate a total mixed ration for lactating dairy animals. Forage was mixed using Fimaks Mastermix self-propelled mixer track 14m3 (Organize Sanyi Cad. 5 No:216500 Mustafakemalpasa/ Bursa/ Turkey.)

Without compaction, 3kg of the TMR were kept in sterile polythene sample bags (24) and conventional thermometers (Digital max/min Thermo-hygrometers) with a probe were placed in the center of the TMR. 3 thermometers, each assigned to an individual replicate, were used. The bags were kept in a room with the upper surface exposed to air. Changes in the temperature were recorded every 2 hours.

The first set of samples were collected immediately after TMR formulation and every 6 hours for the first day and 12 hours thereafter for two consecutive days. 0, 6, 12, 18, 24, 36, 48, and 72 hours constituted the 8 treatments in three replicates. At 0 h, 3 samples were taken from the length of the sample bag i.e., the top, middle, and bottom. These were mixed thoroughly, and divided into 2 samples. The first sample, 200g of forage was oven dried at 65°C for 2 days to determine chemical parameters and the second sample for microbial analysis as follows.

Microbial enumeration

Populations of microorganisms were analyzed according to the method of Kizilsimsek et al (2007).The procedure was modified, where 25g of the samples were blended with 225 mL of sterilized distilled water for 1 min. The pH of this mixture was recorded using a pH meter (ez9901 water quality tester probe).

Liquid from the blended samples were serially diluted (10-fold) and analyzed for LAB, enterobacteria, aerobic bacteria, yeasts, and molds. Serial dilutions were made from 10^–1 to 10^–9 in 0.85% sterilized NaCl solution (9ml) using 1ml of diluent and 0.1ml was plated onto media and thoroughly spread using a sterile spreading lop. Each dilution factor was spread on the 5 different media thus each sample required 9 plates of each media and in total 45 plates. 1080 plates were used for the entire experiment.

Colonies were counted from the plates at appropriate dilutions and the number of colony forming units was expressed per gram of fresh matter. Lactic acid bacteria (LAB) were measured by a plate count on deMan, Rogosa, and Sharpe agar after incubation at 37°C for 48 h under anaerobic conditions. Aerobic bacteria were counted on nutrient agar medium under incubation for 24 to 48 h at 30°C under aerobic conditions. Yeasts and molds were counted on potato dextrose agar after incubation for 2 to 5 days at 30°C and were distinguished by colony appearance and cell morphology. Enterobacteria were counted on MacConkey agar incubated for 48 h at 30°C under aerobic conditions. Colonies were counted from the plates containing between 30 and 300 colonies.

Chemical Analysis

Dry matter (DM) content was determined by drying the samples in an oven at 65°C for 48 hours until a constant weight was reached as dry matter. The dry sample was ground to pass through a 1-mm screen for analysis of later parameters. Organic matter (OM) was calculated as the weight loss upon ashing. Ash was the weight of residue after ashing.

Data collection

The pH was recorded using a pH meter (ez9901 water quality tester probe). Dry matter was the weight after oven drying and was transformed into a percentage of fresh matter. Organic matter (OM) was calculated as the weight loss upon ashing. Ash was the weight of residue after ashing and was transformed into a percentage of dry matter. Plates were counted using hand lenses on plates containing between 30 and 300 colonies. The number of cells in each gram of the original sample was then log transformed.

Aerobic stability test

The probes of the conventional thermometers placed in the center of the bags were used to monitor both ambient and TMR temperatures as the TMR samples were exposed to air for the aerobic stability test. Aerobic deterioration was judged to have started when the temperature of the TMR reached 2°C above the ambient temperature (Yuan et al 2016).

Data analysis

Table 1. Variation in means of chemical parameters with storage time in TMR

Parameter

Treatments in hours

SEM

p-values

0

6

12

18

24

36

48

72

Lin

Qua

Cub

pH

3.91d

3.99d

4.80bc

5.06bc

4.64c

5.19b

5.05bc

5.76a

0.13

0.001

0.179

0.042

DM*

48.9a

48.0a

46.8ab

46.2ab

45.5b

44.8b

43.7c

42.8c

0.32

0.001

0.001

0.546

MC*

51.1c

52.0c

53.2b

53.8b

54.5ab

55.2ab

56.4a

57.2a

0.32

0.001

0.001

0.546

Ash$

12.9c

13.3c

13.5bc

13.8bc

14.4b

15.5a

15.6a

15.6a

0.24

0.001

0.002

0.040

OM$

87.1a

86.8a

86.5ab

86.2ab

85.6b

84.5c

84.1c

84.4c

0.25

0.001

0.002

0.040

SEM, standard error of mean; P-value, standard probability; pH, potential of hydrogen; DM, dry matter; MC, moisture content; OM, organic matter; *, % of fresh matter; $, % of DM. Lin; linear. Qua; quadratic. Cub; cubic a–d Values in the same row with different superscript letters differ (p<0.05); Data are means of triplicate analyses.

All microbial counts were log10 transformed to obtain log-normal distributed data. One-way analysis of variance (ANOVA) was used to determine the effect of storage time on chemical, physical, and microbial composition of the total mixed ration using SAS software package (SAS, 2004). Duncan’s multiple range test (P<0.05) was used to interpret any significant differences among the mean values. Polynomial regression was done to show the relationship of parameters to storage time.

Results

Effect of storage time on chemical parameters of the total mixed ration

The changes in the chemical quality of the TMR during exposure to air are shown in Table 1. The pH linearly increased (p<0.0001) during aerobic exposure with the lowest pH recorded at 0 hours (3.91) and increased to 5.76 after 72 hours. Dry matter decreased (p<0.05) at a decreasing rate 48.9% of fresh matter at 0 h treatment to 42.8% of fresh matter at 72 hours.

The moisture content of the TMR increased (p<0.05) at a decreasing rate from 51.1% of fresh matter at 0 h to 57.2% of fresh matter at 72 h. Ash content of the TMR showed an increase (p<0.0001) at a decreasing rate with the lowest at 0 h 12.9% to 15.6% of dry matter at 72 h. OM of the TMR decreased (p<0.0001) at an decreasing rate from 87.1% to 84.4% of dry matter in the 0 and 72 h treatments, respectively.

Simple linear regression analysis of variation of means of chemical parameters with storage time

Regression analysis coefficients for the independent categorical variable (storage time) showing chemical changes are presented in Table 2. pH increase within 6 hours was not significant. However, it increased (p<0.05) at 12 hours and the subsequent treatments. 82% of changes in pH were due to storage time effect.

Dry matter (DM) decrease within 6 hours was not significant, however, it decreased (p<0.05) at 12 hours and the subsequent treatments. 98% of changes in DM were due to storage time effect.

Organic matter (OM) decrease within 6 and 12 hours was not significant, however, it decreased (p<0.05) at 18 hours and the subsequent treatment hours. 87% of changes in OM were due to storage time effect. As expected, ash content increase within 6 and 12 hours was not significant, however, it increased (p<0.05) at 18 hours and the subsequent treatment hours. 87% of changes in OM were due to storage time effect.

Effect of storage time on temperature of the total mixed ration

The changes in ambient and TMR temperatures are presented in Figure 1. The temperature of the TMR was initially 2.3℃ above ambient temperature at 0 h and then decreased to 0.2℃ below ambient temperature at 4 hours. The temperature of the TMR then started to increase and was 3.3℃ at 6 hours. TMR temperature then increased drastically to 25.5 ℃ above ambient temperature at 16 hours but thereafter, started to decline.

Changes in; TMR temperature (●), ambient temperature (○) during aerobic deterioration

Figure 1. Effect of storage time on average temperature of the TMR

Subsequent TMR temperature decreased gradually to 42.6℃ at 40 hours and started to increase again to a second pick of 44.9℃ at 56 h. TMR temperature then decreased gradually to 41.5℃ at 72 h.

Effect of storage time on microbial populations of the total mixed ration

The changes in the microbial diversity of the TMR during exposure to air are shown in Table 3.

Yeast population at the beginning of the experiment was 5.62 log cfu/g FM and was the lowest throughout the experiment. The population, however, increased (p<0.0001) to 8.39 log cfu/g FM at 18 hours and gradually decreased to 7.95 log cfu/g FM at 72 hours following a cubic trend (P<0.05). Mould population was 3.46 log cfu/g FM at 0 h and increased linearly(p<0.0001) to 10.99 log cfu/g FM within 72 hour of the experiment.

Lactic acid bacteria increased (p<0.0013) from initial population of 6.56 log cfu/g FM to 10.2 log cfu/g FM at 0 h and 24 h respectively. LAB then decreased to 8.70 log cfu/g FM at 72 hours following a quadratic trend (P<0.05). Aerobic bacteria increased (p<0.0001) at a decreasing rate throughout the experiment from 5.80 to 11.3 log cfu/g FM at 0 and 72 hours respectively. Enterobacteria population was initially 3.61 log cfu/g FM (0 hours) remained relatively the same in the first five treatments and then increased linearly (p<0.0001) to 7.72 log cfu/g FM at 48 hours and decreased 6.79 log cfu/g FM at 72 hours.

Simple linear regression analysis on microbial proliferation with storage time

Regression analysis coefficients for the independent categorical variable (storage time) showing microbial changes are presented in Table 4. Lactic acid bacteria showed the highest regression coefficient in the first 6 hours (0.84) followed by yeast, aerobic bacteria, molds and enterobacteria with 0.55, 0.33, 0.23 and - 0.37 coefficients, respectively.

At 12 hours, lactic acid bacteria, yeasts, aerobic bacteria and molds showed increase (P<0.05) in regression coefficients with 2.29, 1.82, 1.75 and 1.02 respectively. There was no increase in enterobacteria (p>0.05).

At 18 hours, lactic acid bacteria, Yeasts, aerobic bacteria and molds had increases (P<0.05) in coefficients with 2.82, 2.77, 2.55 and 2.23, respectively. There was no significant increase in enterobacteria regression coefficient. At 24 hours, lactic acid bacteria still showed the highest coefficient (3.63 (P<0.05)) followed by molds, aerobic bacteria, yeasts and then enterobacteria with 3.34, 3.26, 2.49 and 0.21, respectively.

At 36 hours, molds overtook lactic acid bacteria in increase rate with coefficient of 4.27 followed by aerobic bacteria, lactic acid bacteria, enterobacteria and lastly yeasts with coefficients of 4.19, 2.68, 2.15 and 2.12, respectively. At 48 hours, Aerobic bacteria had the highest regression coefficient 4.83 followed by molds, aerobic bacteria, lactic acid bacteria and yeasts with 4.57, 4.11, 3.39 and 2.29, respectively. At 72 hours, molds have the highest regression coefficient of 7.53, then aerobic bacteria, enterobacteria, yeasts and lactic acid bacteria with coefficients of 5.52, 3.18, 2.33 and 2.14, respectively.

Discussion

Microbiological data

Aerobic spoilage is the primary problem associated with maintaining the nutritive value of TMR after formulation. Fermentation acids and other substrates are metabolized by aerobic bacteria, yeasts, and molds causing a rise in temperature. Therefore, monitoring chemical and microbiological dynamics is essential to evaluate TMR aerobic stability (Yuan et al 2016).

The conditions in deteriorating silage are continuously changing and, as a result, the composition of the microflora will change accordingly (Lindgren et al 1985). Lactic acid bacteria of over 10⁶ cfu/g were detected in fresh TMR. This was attributed to good fermentation quality in silages possibly with an accumulation of lactic acid as indicated by the low pH and inhibition of yeast in the initial period (Wang et al 2016). Soluble sugars like glucose are converted into pyruvate by glycolysis and then reduced to lactic acid. This is a single-step reaction carried out by lactic acid bacteria (LAB).

Lactic acid bacteria had the highest proliferation in the first 6 h and this is attributed to the properties of TMR ingredients, which could easily form a relatively anaerobic condition after being put back without compaction (Wang et al 2016). Also, once the aerobic microorganisms reach a sufficiently high number at the surface exposed, they use up the oxygen entering the TMR, therefore creating anaerobic conditions favorable for LAB proliferation.

Results of this study showed that yeast microbes were the first to propagate and their increase corresponded to the increase in temperature of the TMR. As a result, the temperature of the TMR started increasing above the ambient temperature, suggesting aerobic instability. Many researchers have confirmed that yeasts are primarily responsible for the onset of the aerobic spoilage of silage (McDonald et al 1991). Wilkinson and Davies (2013) reported that yeast counts in excess of 10⁵ cfu/g fresh weight (FW) are likely to be associated with reduced silage aerobic stability.

Also, Tabacco et al (2009) observed that the rise in the silage temperatures and pH levels only occurred when the yeast count was higher than 5 log10 cfu /g. These yeasts are able to grow at low pH conditions and possibly utilize lactic acid and WSC for growth.

Recent studies have shown that lactate assimilating yeasts are generally the main initiators of the aerobic spoilage of silages (Pahlow et al 2003). The observed increase in temperature (Fig 1) and pH (Table 1) with an increase in the yeast population is consistent with earlier observations which showed that under aerobic conditions, yeasts utilize lactic acid, causing an increase in silage temperature and pH (Pahlow et al 2003). The observed linear increase in pH of the TMR with time is indicative of bacilli and other aerobic bacteria growth that quickly increase the temperature further. Arguably, the second spike in temperature was possibly due to mold proliferation that completed the deterioration of the silage.

Results in Table 4 suggest that aerobic bacteria and yeast had closely related regression coefficients at 6 h. Therefore, yeast and aerobic bacteria were responsible for the initiation of aerobic deterioration. Bacteria belonging to the genus Bacillus have also been mentioned as microorganisms responsible for the initiation of aerobic deterioration (Woolford et al 1978). Courtin and Spoelstra (1990) reported that aerobic bacteria can also induce aerobic deterioration. Initially, there is a rise in the yeast population followed by an increase in mould numbers during the later stages of deterioration; both population increases are accompanied by a temperature rise, with the second rise also being associated with large increases in pH (Ohyama et al 1977).

The detection of high levels of aerobic bacteria (Table 3) in aerobically deteriorated TMR indicated the role of aerobic bacteria in the late deterioration, which agreed with the studies conducted by McDonald et al (1991) and Seppälä et al (2013). Enterobacteria that survive the ensiling stage may start growing again and reach numbers in excess of 10⁷ g-1 fresh matter (Table 3) when silage pH increases during the aerobic spoilage phase (Donald et al 1995). Furthermore, enterobacteria played less of a role in TMR with low pH as they were mostly observed in deteriorated TMR. It is consistent with the results that enterobacteria do not proliferate and their viability decreases at pH values lower than 4.5 to 5.0 (Heron et al 1993).

Temperature data

Aerobic stability is a term that nutritionists have used to define the length of time that silage remains cool and does not spoil after it is exposed to air (Kung 2010). It is common that aerobic deterioration is accompanied by a temperature increase, pH changes, and an increase in yeast counts. Possibly, different microflora including yeast, aerobic bacteria and mold growth during aerobic exposure metabolize various substrates, resulting in heat production (Lee et al 2019).

The results are consistent with previous studies on maize silages; the first temperature peak is associated with the development of yeasts and aerobic bacteria, and the second temperature increase is a reflection of mold development (Wilkinson and Davies 2013).

The increase in temperature by 2°C above the ambient temperature is mostly adopted to evaluate aerobic deterioration. It has been indicated that the temperature increase in TMR is associated with the microbial oxidation of acids and water soluble carbohydrates to carbon dioxide and water (Ranjit and Kung 2000). Deterioration occurred within 5 hours of aerobic exposure.

Chemical data

The pH of the TMR increased linearly with the time of aerobic exposure, which might be attributed to the decrease in lactic acid content. The increase in pH was an indicator of aerobic deterioration of the TMR because lactic acid might have been consumed by yeasts during aerobic exposure, and then the TMR became more suitable for the growth of other undesirable microorganisms such as mold and enterobacteria.

The significant decreases in organic matter (OM) and dry matter (DM) (Table 1) with aerobic exposure were possibly due to the fact that different micro flora including yeast, aerobic bacteria and mold growth results in microbial breakdown of OM and DM components into carbon dioxide and moisture, which are then lost from the TMR. Arguably, this accounts for the losses in DM and OM.

Conclusions

• TMR when exposed to air deteriorates rapidly, which causes high losses of nutritional components. Aerobic deterioration of TMR started at 5 h. Yeasts and aerobic bacteria were responsible for initiation of aerobic deterioration. Enterobacteria and molds were shown to play a secondary role in TMR deterioration.

Acknowledgements

I acknowledge the efforts of my academic Supervisor, Prof. Fred Kabi who offered his precious time to guide me through this research. I also acknowledge the administration of (NALiRRI) for allowing me to do all the research activities from the institute. And with sincere gratitude, I thank my field supervisor, Mr. Kato Walusimbi, a research officer at NALiRRI for his remarkable and critical input towards this research. Finally, I also acknowledge the technical staff of the nutrition and livestock breeding programs of NaLiRRI; Mr. Galinya Herbert and Mr. Emudong Patrick. Thank you for guiding me during field work. I would like to acknowledge all the scientists whose literature has been cited in this report.

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