**3.2 Analysis of aflatoxins**

HPLC analysis of aflatoxins showed the presence of all the major aflatoxins AF (B1), B2, G1 and G2 in the bulk of the samples indicated by the peaks in the

*Farming systems adopted by dairy farmers in Bulawayo peri-urban showing that most the farmers practise semi-intensive farming.*

#### **Figure 2.**

*Percentage utilisation of feed types by dairy farmers in peri-urban Bulawayo showing that the most common feed used by the farmers is the mixed ration.*

**41**

**Table 1.**

*used for feeding the dairy cows.*

*One-way ANOVA for all feed types.*

**Figure 3.**

**Figure 4.**

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe*

*2*

*Representative chromatogram showing four peaks indicating the presence of all major aflatoxins.*

*Average total aflatoxin concentrations in the feeds. A* p *value of 0.043 shows that there was significant difference in the aflatoxin concentrations in the different feeds with mixed ration had the highest contamination.*

> **ANOVA Total AF conc (ug/kg)**

Between groups 14860.674 3 4953.558 2.832 **0.043**

*A p value <0.05 indicates that there is a significant difference in the levels of aflatoxin in the different types of feeds* 

Within groups 159185.082 91 1749.287

Total 174045.756 94

Sum of squares df Mean square F *p* value

chromatograms as shown in **Figure 3**. The calibration curves gave good linearity

values of 1. Total aflatoxin concentration in

*DOI: http://dx.doi.org/10.5772/intechopen.88582*

the feeds ranged from 0 to 250.9 μg/kg.

for the different aflatoxins with *r*

chromatograms as shown in **Figure 3**. The calibration curves gave good linearity for the different aflatoxins with *r 2* values of 1. Total aflatoxin concentration in the feeds ranged from 0 to 250.9 μg/kg.

**Figure 3.**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

Most of the farmers who took part in the study were practising semi-intensive farming followed by extensive and lastly intensive farming as summarised by

The cows were mainly fed with concentrates, mixed ration, brewers' spent grain and grass ranging from 6 to 10 kg per animal per day. Only 36% of the farmers had some knowledge on aflatoxins. The most utilised feed was mixed ration as shown by

HPLC analysis of aflatoxins showed the presence of all the major aflatoxins AF (B1), B2, G1 and G2 in the bulk of the samples indicated by the peaks in the

**3. Results**

**Figure 1**.

**Figure 2**.

**3.1 Farmer survey**

**3.2 Analysis of aflatoxins**

**40**

**Figure 2.**

**Figure 1.**

*semi-intensive farming.*

*feed used by the farmers is the mixed ration.*

*Percentage utilisation of feed types by dairy farmers in peri-urban Bulawayo showing that the most common* 

*Farming systems adopted by dairy farmers in Bulawayo peri-urban showing that most the farmers practise* 

*Representative chromatogram showing four peaks indicating the presence of all major aflatoxins.*

#### **Figure 4.**

*Average total aflatoxin concentrations in the feeds. A* p *value of 0.043 shows that there was significant difference in the aflatoxin concentrations in the different feeds with mixed ration had the highest contamination.*


*A p value <0.05 indicates that there is a significant difference in the levels of aflatoxin in the different types of feeds used for feeding the dairy cows.*

#### **Table 1.**

*One-way ANOVA for all feed types.*

### **3.3 Aflatoxin distribution in feeds**

Mixed ration had the highest total AF concentrations with an average concentration of 29.8 μg/kg, and grass had the lowest concentrations as shown in **Figure 4**. The one-way analysis of variance (ANOVA) (**Table 1**) gave a *p* value of 0.043, meaning that at 95% confidence level (*p* < 0.05) there is enough evidence to conclude that there is a significant difference in the total mean concentration of aflatoxins across the feeds. However, looking at MR and CN (**Table 2**), *p* = 0.766; therefore there was no significant difference in the mean total aflatoxin concentrations.

The distribution of aflatoxins in the feeds showed that AFB1 was the most common aflatoxin across all feeds as shown by **Figure 5**. However, there was variation with individual feeds as shown in **Figure 6a**–**d**.

Looking at the distribution of total aflatoxins across the different farming systems, **Figure 7** shows that the semi-intensive system had the highest aflatoxins with an average of 21.6 μg/kg. One-way ANOVA (**Table 3**), however, indicated that there is no significant difference in the mean total aflatoxin concentration in the feeds from semi-intensive and intensive farming systems as *p* = 0.937 which is greater than *p* value of 0.05 at 95% confidence level.


#### **Table 2.**

*One-way ANOVA between the mixed ration and feed concentrate.*

#### **Figure 5.**

*Distribution of aflatoxins across all feed types. One-way ANOVA analysis gave a* p *value of 0.017, indicating a significant difference between the concentrations of the individual aflatoxins with AFB1 being the most dominant aflatoxin.*

**43**

**Figure 7.**

*farming systems.*

**Figure 6.**

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe*

Distribution of AFB1 in the feeds from the different dry and rainy seasons is shown in **Figure 8**, and ANOVA analysis showed that there is a significant differ-

*Distribution of aflatoxins across the three farming systems. Descriptive statistics shows that extensive farming has the lowest aflatoxin concentration and semi-intensive farming has more aflatoxin concentrations in their feeds. However, one-way ANOVA gave* p = *0.470, indicating no significant difference among the different* 

*Distribution of aflatoxins in the feeds, (a) feed concentrates, (b) mixed ration, (c) brewers' spent grains and (d) grass. One-way ANOVA gave a* p *value of 0.017, indicating a significant difference in the concentration of individual toxins across all feeds. AFB1 was the dominant aflatoxin in mixed ration and grass, whereas for* 

*concentrates and brewers' spent grains, AFB2 was the predominating aflatoxin.*

ence in AFB1 concentrations in the different seasons (**Table 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.88582*

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe DOI: http://dx.doi.org/10.5772/intechopen.88582*

**Figure 6.**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

with individual feeds as shown in **Figure 6a**–**d**.

than *p* value of 0.05 at 95% confidence level.

Total 159352.193 66

*One-way ANOVA between the mixed ration and feed concentrate.*

Mixed ration had the highest total AF concentrations with an average concentration of 29.8 μg/kg, and grass had the lowest concentrations as shown in **Figure 4**. The one-way analysis of variance (ANOVA) (**Table 1**) gave a *p* value of 0.043, meaning that at 95% confidence level (*p* < 0.05) there is enough evidence to conclude that there is a significant difference in the total mean concentration of aflatoxins across the feeds. However, looking at MR and CN (**Table 2**), *p* = 0.766; therefore there was no significant difference in the mean total aflatoxin

The distribution of aflatoxins in the feeds showed that AFB1 was the most common aflatoxin across all feeds as shown by **Figure 5**. However, there was variation

Looking at the distribution of total aflatoxins across the different farming systems, **Figure 7** shows that the semi-intensive system had the highest aflatoxins with an average of 21.6 μg/kg. One-way ANOVA (**Table 3**), however, indicated that there is no significant difference in the mean total aflatoxin concentration in the feeds from semi-intensive and intensive farming systems as *p* = 0.937 which is greater

> **ANOVA Total AF conc (μg/kg)**

Between groups 218.928 1 218.928 0.089 **0.766**

*Distribution of aflatoxins across all feed types. One-way ANOVA analysis gave a* p *value of 0.017, indicating a significant difference between the concentrations of the individual aflatoxins with AFB1 being the most* 

Within groups 159133.265 65 2448.204

*A p value >0.05 indicates that there is no significant difference in the levels of aflatoxin.*

**Sum of squares df Mean square F** *p* **value**

**3.3 Aflatoxin distribution in feeds**

concentrations.

**42**

**Figure 5.**

*dominant aflatoxin.*

**Table 2.**

*Distribution of aflatoxins in the feeds, (a) feed concentrates, (b) mixed ration, (c) brewers' spent grains and (d) grass. One-way ANOVA gave a* p *value of 0.017, indicating a significant difference in the concentration of individual toxins across all feeds. AFB1 was the dominant aflatoxin in mixed ration and grass, whereas for concentrates and brewers' spent grains, AFB2 was the predominating aflatoxin.*

#### **Figure 7.**

*Distribution of aflatoxins across the three farming systems. Descriptive statistics shows that extensive farming has the lowest aflatoxin concentration and semi-intensive farming has more aflatoxin concentrations in their feeds. However, one-way ANOVA gave* p = *0.470, indicating no significant difference among the different farming systems.*

Distribution of AFB1 in the feeds from the different dry and rainy seasons is shown in **Figure 8**, and ANOVA analysis showed that there is a significant difference in AFB1 concentrations in the different seasons (**Table 4**).


#### **Table 3.**

*One-way ANOVA results comparing the semi-intensive and intensive farming systems.*

#### **Figure 8.**

*Seasonal variation in the distribution of AFB1. There was a significant difference in AFB1 concentrations (*p = *0.003) with samples from the rainy season having more of AFB1 than dry season samples.*


*A p value <0.05 indicates that there is a significant difference in the levels of AFB1 concentration in feeds in the rainy season.*

#### **Table 4.**

*One-way ANOVA results for dry season and rainy season.*

#### **4. Discussion**

Feed quality is of great importance in animal husbandry as it affects both animal health and productivity [24]. Consumption of aflatoxin-contaminated feeds by dairy cows may result in the aflatoxins occurring in milk posing health risks to

**45**

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe*

there are no regulatory limits in terms of animal feeds [32].

form of hay stored at the farm or fresh grass in the grazing land.

different feeds and feed ingredients used to feed dairy cows in Pakistan.

From this study it was shown that grass samples had the least aflatoxin concentrations with an average total aflatoxin concentration of 2.5 μg/kg and 169 × 10<sup>−</sup><sup>3</sup> μg/ kg of AFB1. These results are similar to the finding by Gizachew et al. [25] who also had grass as the least contaminated feed. However, they got a minimum AFB1 concentration 7 μg/kg for their samples, higher than what was established in this study. Sassahara et al. [37] analysed feedstuffs supplied to dairy cows in North of Paraná state, Brazil, and did not detect any aflatoxins in the silage samples. Work done by Driehuis et al. [33] in the Netherlands also showed the absence of aflatoxins in silage samples used to feed dairy cows. These findings suggest that grass in the form of silage or pasture is not really prone to fungal infections which may result in aflatoxin production. In this study most of the aflatoxigenic strains were isolated from the grass, but it was the feed with the least aflatoxin concentration. Gonzalez Pereyra et al. [38] highlighted that the presence of aflatoxigenic fungi on a substrate does not mean that the toxin is present in that particular food/feed matrix, but there is a risk of toxin production if the environmental conditions become favourable

humans [18]. Research has shown that some feedstuffs used in formulating animal feeds can become infected by aflatoxin-producing fungi [25]. Researchers worldwide have been analysing dairy feed for aflatoxin contamination and have reported

This study also showed that 96% of feeds used in feeding dairy cows in periurban Bulawayo that were analysed were contaminated with at least one of the naturally occurring aflatoxins. The results also indicate that 21% of the samples analysed had total aflatoxin levels above the regulatory limit set by international governing bodies of 20 μg/kg for animal feeds. This concurs with the findings by Reddy and Salleh [30] who reported that 22.5% of their samples had aflatoxin concentrations above this regulatory limit. Zimbabwe reviewed the AFB1 regulatory limit to 20 μg/kg in 1990 [31] for food intended for human consumption. However,

The feeds that are used in feeding dairy cows by farmers in peri-urban Bulawayo included feed concentrates, mixed ration, grass and brewer's spent grains. This is in accordance with the requirements of the diets of dairy cows which should consist of a component that provides protein and energy and a component of roughage [33]. In this study, the protein and energy were supplied by the concentrates, mixed ration and the brewer's spent grains, whereas the roughage was provided in the

Mixed rations are considered a whole meal for the cow as they contain basically all the nutrients that are found in forages and concentrates. Formulation of a mixed ration involves combining forages, by-products of other processes such as whole cottonseed or cottonseed cake, grains, protein source, minerals and vitamins [34]. Findings of this study showed that mixed ration had the highest total aflatoxin concentrations with an average of 29.0 μg/kg. ANOVA also showed that at 95% confidence level, there was a significant difference in the mean total aflatoxins in the feeds with the mixed rations having the highest total aflatoxin mean. Findings from this study concur with Mozafari et al. [35] who detected the highest aflatoxin concentrations in mixed ration among the other feeds they analysed. The diversity of the components used could have been potential sources of aflatoxigenic fungi which result in contamination of this feed type with aflatoxins. Other researchers [25] also reported high aflatoxin concentration in noug cake, a product of oil processing industry used in feeding dairy cows. Cottonseed was the most utilised feed ingredient for mixed rations by the farmers who participated in this study. However, Chohan et al. [36] reported feed concentrate having the highest aflatoxin concentration followed by mixed ration in their study on aflatoxin contamination of

various findings with most feeds exceeding the regulatory limits [26–29].

*DOI: http://dx.doi.org/10.5772/intechopen.88582*

#### *Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe DOI: http://dx.doi.org/10.5772/intechopen.88582*

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

Total 171592.940 88

**Table 3.**

**Figure 8.**

*season.*

**Table 4.**

Within groups 171580.359 87 1972.188

*One-way ANOVA results comparing the semi-intensive and intensive farming systems.*

**ANOVA Total AF conc (μg/kg)**

Between groups 12.581 1 12.581 0.006 **0.937**

*A p value >0.05 indicates that there is no significant difference in the levels of aflatoxin concentration between.*

**Sum of squares df Mean square F** *p* **value**

**44**

**4. Discussion**

Feed quality is of great importance in animal husbandry as it affects both animal

health and productivity [24]. Consumption of aflatoxin-contaminated feeds by dairy cows may result in the aflatoxins occurring in milk posing health risks to

*Seasonal variation in the distribution of AFB1. There was a significant difference in AFB1 concentrations* 

**ANOVA AFB1 conc (μg/kg) × 10<sup>−</sup><sup>3</sup>**

Between groups 6747185610.100 1 6747185610.100 9.500 **0.003**

*A p value <0.05 indicates that there is a significant difference in the levels of AFB1 concentration in feeds in the rainy* 

**Sum of squares df Mean square F** *p* **value**

*(*p = *0.003) with samples from the rainy season having more of AFB1 than dry season samples.*

Within groups 66758340020.045 94 710195106.596

Total 73505525630.144 95

*One-way ANOVA results for dry season and rainy season.*

humans [18]. Research has shown that some feedstuffs used in formulating animal feeds can become infected by aflatoxin-producing fungi [25]. Researchers worldwide have been analysing dairy feed for aflatoxin contamination and have reported various findings with most feeds exceeding the regulatory limits [26–29].

This study also showed that 96% of feeds used in feeding dairy cows in periurban Bulawayo that were analysed were contaminated with at least one of the naturally occurring aflatoxins. The results also indicate that 21% of the samples analysed had total aflatoxin levels above the regulatory limit set by international governing bodies of 20 μg/kg for animal feeds. This concurs with the findings by Reddy and Salleh [30] who reported that 22.5% of their samples had aflatoxin concentrations above this regulatory limit. Zimbabwe reviewed the AFB1 regulatory limit to 20 μg/kg in 1990 [31] for food intended for human consumption. However, there are no regulatory limits in terms of animal feeds [32].

The feeds that are used in feeding dairy cows by farmers in peri-urban Bulawayo included feed concentrates, mixed ration, grass and brewer's spent grains. This is in accordance with the requirements of the diets of dairy cows which should consist of a component that provides protein and energy and a component of roughage [33]. In this study, the protein and energy were supplied by the concentrates, mixed ration and the brewer's spent grains, whereas the roughage was provided in the form of hay stored at the farm or fresh grass in the grazing land.

Mixed rations are considered a whole meal for the cow as they contain basically all the nutrients that are found in forages and concentrates. Formulation of a mixed ration involves combining forages, by-products of other processes such as whole cottonseed or cottonseed cake, grains, protein source, minerals and vitamins [34]. Findings of this study showed that mixed ration had the highest total aflatoxin concentrations with an average of 29.0 μg/kg. ANOVA also showed that at 95% confidence level, there was a significant difference in the mean total aflatoxins in the feeds with the mixed rations having the highest total aflatoxin mean. Findings from this study concur with Mozafari et al. [35] who detected the highest aflatoxin concentrations in mixed ration among the other feeds they analysed. The diversity of the components used could have been potential sources of aflatoxigenic fungi which result in contamination of this feed type with aflatoxins. Other researchers [25] also reported high aflatoxin concentration in noug cake, a product of oil processing industry used in feeding dairy cows. Cottonseed was the most utilised feed ingredient for mixed rations by the farmers who participated in this study. However, Chohan et al. [36] reported feed concentrate having the highest aflatoxin concentration followed by mixed ration in their study on aflatoxin contamination of different feeds and feed ingredients used to feed dairy cows in Pakistan.

From this study it was shown that grass samples had the least aflatoxin concentrations with an average total aflatoxin concentration of 2.5 μg/kg and 169 × 10<sup>−</sup><sup>3</sup> μg/ kg of AFB1. These results are similar to the finding by Gizachew et al. [25] who also had grass as the least contaminated feed. However, they got a minimum AFB1 concentration 7 μg/kg for their samples, higher than what was established in this study. Sassahara et al. [37] analysed feedstuffs supplied to dairy cows in North of Paraná state, Brazil, and did not detect any aflatoxins in the silage samples. Work done by Driehuis et al. [33] in the Netherlands also showed the absence of aflatoxins in silage samples used to feed dairy cows. These findings suggest that grass in the form of silage or pasture is not really prone to fungal infections which may result in aflatoxin production. In this study most of the aflatoxigenic strains were isolated from the grass, but it was the feed with the least aflatoxin concentration. Gonzalez Pereyra et al. [38] highlighted that the presence of aflatoxigenic fungi on a substrate does not mean that the toxin is present in that particular food/feed matrix, but there is a risk of toxin production if the environmental conditions become favourable

for aflatoxin production. Nonetheless, detection of aflatoxins in a sample means the substrate has been contaminated by toxigenic species which could either be present or absent at the time of sampling. This was the case with the feed concentrates which had aflatoxin concentrations higher than the grass samples, but fewer toxigenic strains were isolated.

The most dominant aflatoxin across all feeds was AFB1 with an average concentration of 9.0 μg/kg and was detected in all the samples that tested positive for aflatoxin contamination. This is above the EU 5 μg/kg set for lactating cows. Udom et al. [39] and Gizachew et al. [25] also reported their samples having AFB1 concentrations exceeding the EU regulatory limit. The high levels of AFB1 in most samples could be attributed to the fact that it was the most common and prevalent aflatoxin in most food matrices [40, 41]. Moreover, some authors have indicated that most toxigenic *Aspergillus* strains produce AFB1 and therefore it occurs more frequently than the other aflatoxins [10, 42, 43]. AFB1 was predominant in the rainy season (**Figure 8**). These results are in agreement with the findings by Chohan et al. [36] which also showed high concentrations of AFB1 during the rainy season. For aflatoxin production, high temperatures and high humidity are required, and these conditions prevail during the rainy season.

However, for brewers' spent grains (BSG), AFB2 was the predominant aflatoxin. The BSG are a product of beer brewing industry [44] and has been found to be of valuable use in the feedstock industry mainly because it is affordable and available throughout the year [45]. BSG used in this study were from the production of opaque beer. The presence of aflatoxins in beer production has been associated with contaminated malt. Malt production involves increasing the moisture content of the grains to allow partial germination of the grain. Aflatoxigenic fungi are known to contaminate cereal grains which are also used in the beer production process [46]. If the malt is not properly dried or stored, fungal growth may be promoted resulting in the production of aflatoxins. Research on the fate of mycotoxins during the beer fermentation process showed that recovery of AFB2 in BSG is higher than other aflatoxins [47]. Some researchers [48] showed that AFB2 is able to adsorb onto yeast cells during fermentation. The yeast cells and the grain particles that are removed through filtration are collectively known as brewers' spent grains. This could be the possible reason why AFB2 levels were higher in BSG samples. Nevertheless, Gonzalez Pereyra et al. [38] were not able to detect any AFB2 in barley malt and brewers' spent grains from Argentina breweries. AFB1 has been reported as the most common aflatoxin occurring naturally in feedstuffs, but for this study it was not the case for BSG as the concentration of AFB2 was higher than that of AFB1.

This study also showed that aflatoxin contamination of brewers' spent grains, a known source of nitrogen and roughage, and grass were within the regulatory limits making them safer when compared to the concentrates and mixed ration. However, nutritional composition of the grass will not meet the dietary demands of the cows.

#### **5. Conclusion**

Detection of aflatoxins in the feed samples used for this study is a cause of concern as this may be indicating the possibility of transfer into the milk by the dairy cows. Although most samples were within the acceptable limit for total aflatoxin, it was noted that concentrations of AFB1, the most potent of them, were above the regulatory limit. Moreover, research has shown that AFB1 can be carried over into milk as its hydroxylated metabolite AFM1 making milk a route through which humans are exposed to aflatoxins. High prevalence of AFB1 during the rainy season could be an indication of poor storage of the feeds which may result in increased

**47**

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe*

moisture content resulting in proliferation of aflatoxin-producing *Aspergillus*. Therefore, there is a need to educate the farmers and their personnel on the importance of proper feed storage facilities in order to control contamination of the feeds.

I would like to thank the farmers who participated in this research; without you I would not have done it. I am also grateful to the farm personnel who assisted in the collection and safe storage of the samples, thank you for your immense support. I also want to thank the NRF (Grant number 105882)and the NUST Research Board (Grant number RB No. 43/16) for the funds that were made available towards

and Mulunda Mwanza1

1 Department of Animal Health, Northwest University, Mafikeng, South Africa

\*Address all correspondence to: ndangwa@gmail.com; nancy.nleya@nust.ac.zw

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Department of Applied Biology and Biochemistry, National University of Science

*DOI: http://dx.doi.org/10.5772/intechopen.88582*

**Acknowledgements**

**Conflicts of interest**

**Author details**

Nancy Nleya1,2\*, Lubanza Ngoma1

and Technology, Bulawayo, Zimbabwe

provided the original work is properly cited.

The authors declare no conflict of interest.

this research.

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe DOI: http://dx.doi.org/10.5772/intechopen.88582*

moisture content resulting in proliferation of aflatoxin-producing *Aspergillus*. Therefore, there is a need to educate the farmers and their personnel on the importance of proper feed storage facilities in order to control contamination of the feeds.
