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Asian Journal of Dairy and Food Research

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Full Research Article

Stable Isotope Composition in Moroccan Milk: Feed  and Water Relationships

Nour Eddine Amenzou1,*, Fouad Taous1, Loubna Elmoqrani1, Mahmoud Eddabdouby1, Meryem Moustakim1, Ismail Hilal1, Moncef Benmansour1
1Centre National de l’Energie des Sciences et Techniques Nucléaires (CNESTEN) Rabat, Morocco.

ABSTRACT

Background: This study investigated the use of stable isotope ratio analysis to trace the geographical origins of Moroccan dairy products, as well as possible applications to other Moroccan agricultural products. Ten agro-ecological zones with different climates were selected, including the Garb, Middle Atlas, Meknes, Lukos, Agadir, Taroudant, Settat,  andCasablanca regions. Milk samples were collected directly from cows on modern  andtraditional farms, as well as the water they consumed.

Methods: Analytical methods such as isotope ratio mass spectrometry (IRMS)  andthe Piccaro system were used to determine stable isotope ratios in milk  andwater samples. Chemometric methods including principal component analysis (PCA)  andhierarchical cluster analysis (HCA) showed that stable isotope ratios (H, C, N, O) in milk were highly correlated with environmental factors specific to the region. Three distinct groups emerged, especially for oxygen isotopes, depending on altitude  anddistance from the ocean. The δ18O composition was particularly distinct, indicating regional differences: northern, low-altitude coastal areas  andhigh-altitude inl andareas.

Result: The study also showed that the isotopic composition of milk is influenced by the isotopic composition of drinking water. Nitrogen isotopes, especially δ15N, distinguished two groups of milk: one from an area with fertilizer use (low δ15N values, intensive agriculture)  andanother from an area with moderate agricultural practices (higher δ15N values). These results highlight the potential of stable isotope analysis as a tool to verify the geographical origin of dairy products in Morocco.

INTRODUCTION

Morocco is located in western North Africa and is charact-erized by a semi-arid climate and a rapidly growing population. Faced with population growth and changes in eating habits, Moroccan authorities launched an ambitious plan in the early 1970s to meet food needs. The revised livestock policy aimed to establish intensive livestock production based on imported dairy cattle breeds and expanded pasture areas. At the same time, the relevant state departments introduced a milk collection policy.
       
Dairy production plays an important social and economic role in Morocco. In Morocco, approximately 790,000 farms produce milk and calves, of which only about 5% are considered professional dairies (FAO, 2011). Milk and its derivatives have always been considered an important but expensive component of the Moroccan diet, which is mainly based on cereals (bread) and vegetables. Milk has a strong symbolic meaning in local traditions, as it is used together with dates to welcome guests. Milk comes mainly from cattle (2.7 million cows produce more than 96% of annual milk production) (FAO, 2011).
       
The globalization of the food markets and the facility of transportation of products through and between the countries, generates that the consumers are worried by the origin of the food which the consume.                                                                                                                                           
In recent years, geographical indications and appellation of origin have gained more importance in Morocco, which led to the establishment of the law #25-06 that was published in the Official Bulletin # 5640 on June the 19 (2008). This law regulates the use of distinctive signs of origin as they relate to the quality of food  andagricultural products, including labelling of geographical indication. To apply for labelling, geographical indication  andorigin appellation, producers associations  andfood processors have to file a request (cahier des charges) to a national commission created for this purpose (article 17 of the law). Food  andagricultural products that have been approved by this commission are registered  andpublished in the Official Bulletin.

To date, there are ten products that have been approved by The Moroccan National Commission for Geographical Indications  andAppellation of Origin among them:        

Geographical Indication « Chefchaouan Goat cheese; Minister of Agriculture decision that was published in the Official Bulletin # 5976 on September 8 (2011).
       
Many techniques proposed in this subject, but the stable isotope ratio and trace elements give a good result concerning the geographic origin of the products food. The 18O/16O ratio of milk depends on the water ingested and the proportion of fresh vs. dry fodder. Isotope ratios of precipitation and groundwater depend largely on temperature, latitude, altitude and distance from the sea (Moser and Rauert, 1980; Kern et al., 2020).
       
The13C/12C ratio for both milk fat and cheese protein gives information on the type of forage fed to the cows. The carbon isotope value of plants depends on their photosynthetic cycles for CO2 fixation (Smith and Epstein, 1971). C4 plants such as maize show higher δ13C values than C3 plants, which constitute the major part of a cow’s fodder (Potočnik et al., 2020). Differences in the 15N/14N ratio also result essentially from forage. The nitrogen isotopic compositions of animal manures, composts and other fertilizers permitted in organic production systems are significantly enriched compared to synthetic nitrogen fertilizers (Sáez et al., 2020; Bateman and Kelly, 2007). The mean d15N values of organic fertilizers cluster around +8‰ and some fertilizers (e.g., animal manures) can have values higher than +35‰, due to the preferential volatilization of 15N-depleted ammonia in the field or during storage (Tixier et al., 2022; Bateman and Kelly, 2007; Mie et al., 2022; Bhargav et al., 2025).
       
The 87Sr/86Sr ratio can also be useful for origin assignments as it is dependent only on the types of rocks and soils and not on human activity, climate or season of production (Rossmann et al., 2000).
       
In this study we decided to conduct a study on the variability of 13C/12C, 15N/14N, 18O/16O, D/H isotope ration and trace elements in milk in the order to determine the geographic origin of dairy products in Morocco. For that the sampling strategy will be based on nature of the rock and soil, climate of the region and irrigated areas or rainfall agriculture practices.

MATERIALS AND METHODS

Sampling
 
For sampling, we selected agro-ecological zones (Ducrotoy et al., 2015). The ten geographical units described by the soils can be considered important agro-ecological zones because they have a certain homogeneity in terms of geomorphology/subsoil, precipitation and growing season (bioclimate and its thermal zoning).
1- The Middle Atlas.
2- The Rif.
3- The Loukkos area.
4- The Rharb area.
5- The Sais plateau.
6- The Mamora and central plateau.
7- The Chaouia plain and casablanca.
8- The plains and plateau north of the Atlas.
9- The Argan zone.
10- The High Atlas.
       
The samples (cow milk and farm water) have been collected from various farms during the spring season for all agro-ecological zones, as climatic factors influence the isotopic composition of the samples (milk and water). Samples were collected early in the morning. In total, 49 samples of cow milk from various geographical regions were collected. We also collected the farm water that the cows drink.
       
Table 1 show the geographical information (longitude and latitude and some farms characteristics) of each farm where the samples have been taken.

Table 1: Background information of sampling sites.


 
Analysis of samples
 
In the laboratory the all samples will be kept frozen until the following analyses were performed. The ratios of 13C/12C, 15N/14N, 18O/16O, will be determined by spectrometer coupled to element analyzer for the stable isotope. Also the farm water will be collected for the isotope analysis.
       
Stable isotope data are expressed as d-values according to (Brand et al., 2014; Huang et al., 2013) :

 
Where,
R =  Ratio of the heavy to light isotope of the element (e.g., H2/H1.
       
In the laboratory, fresh milk samples were subjected to vacuum distillation to isolate the water for the analysis of isotopes 18O and 2H (Boito et al., 2021). Additionally, raw milk samples were directly freeze-dried to produce milk powder for the analysis of 13C and 15N (Ataro et al., (2008).

Statistical data analyses were carried out on the data obtained from the various environmental and chemical and isotopic data. The data will be  processed using the statistical software package Xstat using Principal component analysis  and Analytic hierarchy process (OECD, 2006). 

RESULTS AND DISCUSSION

In this section the results obtained from isotopic ratio mass spectrometer will be discussed.
 
Stable isotope of water farm
 
The first parameter examined in this study concerns the stable isotopes in the drinking water of dairy cows, focusing on the stable isotopes of oxygen (δ18O) and hydrogen (δ2H) (Fig 1). The δ18O and δ2H values of the drinking water source samples are below the global atmospheric water line (GMWL) proposed by Craig, (1961) and are consistent with the local atmospheric water line (LMWL) proposed by Raibi et al., (2004) findings. This scenario suggests that the water undergoes some form of evaporation, resulting in isotopic depletion, as the lighter isotopes of oxygen and hydrogen are preferentially lost in this process. This observation is consistent with previous work by Bailey et al., 2018 and recent work by Gallart et al., (2024), who highlighted that evaporation enriches the remaining water with heavier isotopes, causing its isotopic distribution to change along the δ18O and δ2H axes. Detailed inspection of the graphs reveals clear differences in isotopic signatures from different regions, reflecting local environmental and climatic factors that influence the isotopic composition of meteoric water. The Local Meteoric Water Line (LMWL) relationships help distinguish water from different regions and provide valuable insights into the local water cycle and potential sources of evaporation and contamination.

Fig 1: The plot of δ2H vs. δ18O values of watering water from farms.



Oxygen and deuterium isotopes of fresh milk
 
On the other hand, when we compared the diagram on the Dairies waters with the diagram of the water coming from milk, we can observe enrichment on the milk (Fig 2). The δ2H and δ18O values of water extracted from dairy milk samples were found to be isotopically enriched relative to cow drinking water (Chesson et al., (2010); Gregorčič et al., (2020). This enrichment is attributed to the fractionation processes that occur during the metabolism and physiological processes in the cow’s body, leading to the enrichment of the milk with heavier isotopes of hydrogen and oxygen compared to the drinking water Boito et al., (2021). Therefore, the stable isotope analysis of hydrogen and oxygen in dairy milk can be used to authenticate the geographical origin of the milk and to detect any potential adulteration or fraudulent practices in the dairy industry Ehtesham et al., (2015). All milk samples exhibit enrichment with respect to water samples presenting a slope that reminds waters that have undergone evaporation process.

​

Fig 2: The plot of δ2H vs. δ18O values of liquid Milk.


       
There was a significant linear relationship between between δ18O of farm water and δ18O milk water shows that  isotopic composition if milk is influenced by drinking water (r2 = 0.5 p<0.05) (Fig 3) . The oxygen isotope ratios in milk samples were found to have a clear relationship with the water from the farm, which aligned with the expected effects of climate on 18O/16O fractionation Boito et al., (2021); Huang et al., (2017)). The δ18O water values in milk water show seasonal variability and are enriched in 18O compared to cow milk, reflecting the isotopic composition of the drinking water source and the effect of differences in the water uptake by the animals (Gregorčič et al., 2020; Crittenden et al., 2007). However, clear correlations between geography and isotopic fractionation of the other elements examined were difficult to discern, due to limited available knowledge of the main factors influencing isotope abundance for these elements at the various locations.

​

Fig 3: Relation between δ18O fresh milk and δ18O of farm water.


       
The data presented shows a significant negative correlation between the isotopic value of fresh milk and altitude (Fig 4), as well as between the isotopic value of fresh milk and distance from the Atlantic Ocean. The correlation can be attributed to progressive rainout or the ‘continental effect’ (Gremaud et al., 2004). Other studies have also found negative correlations between milk isotopic values and latitude, δO Rain and δH Rain (Shima et al., 2020). Additionally, elevation has been found to be positively correlated with one component of milk fatty acid composition and negatively correlated with two other components (Shima et al., 2020). Environmental factors such as solar radiation, temperature, relative humidity and precipitation have also been found to have varying degrees of correlation with milk quality parameters such as fat content, total solids and mesophilic count (Vélez-Terranova et al., 2023; Enrique et al., 2020).

​

Fig 4: Relation between δ18O of fresh milk, distance from the sea and Altitude for the studied farms.


       
The composition of stable isotope milk (Fig 5, Fig 6) can be distinguished into three groups based on the influence of different geographical and environmental factors.

​

Fig 5: δ18O versus δ2H in liquid milk from different regions.



​

Fig 6: Dendrogram for the origin of fresh milk.


 
Milk from the north influenced by the Mediterranean Sea
 
This group of milk has a specific isotopic composition due to the influence of the Mediterranean Sea, which affects the water isotope composition in the region.
 
Milk from continental regions with low altitude and far from the Atlantic Ocean
 
This group of milk has a different isotopic composition compared to the first group, as it is not directly influenced by the Mediterranean Sea. The isotopic composition of milk in these regions is more related to the continental climate and the altitude of the area.
 
Milk from regions near the Atlantic sea and low altitude
 
This group of milk has a distinct isotopic composition due to the proximity to the Atlantic Ocean and the low altitude of the region. The isotopic composition in these areas is influenced by both the ocean and the continental climate.

These differences in isotopic composition can be used to trace the origin of milk and verify its provenance, as well as to study the effects of different environments on the isotopic composition of milk (Gregorčič et al., 2020).
 
Carbon and nitrogen isotopes of powder milk
 
The dominant geographical phenomena influencing isotopic fractionation vary for each element. In the case of carbon, the differing photosynthetic CO2 fixation pathways used by plants (C3 or C4) leads to characteristic δ13C in the plant tissues, which is then rapidly reflected in the milk of cattle eating those plants Roffet-Salque et al., (2017), Wijenayake et al., (2020); Boutton et al., (1988). Carbon isotope tissue ratios vary from 16% to 7% in C4 plants and from 35% to 20% in C3 plants (Kirkels et al., 2022); Pate and Noble, (2000); Nandini et al., (2018).
       
According to 13C (Fig 7), we have two groups the first one ranged from -23,98 to -30,27% corresponding to C3 plant and second one from -16,5 to -21,9% corresponding to mixture of C3 and C4 plant respectively (Kirkels et al., 2022).

​

Fig 7: 13C versus 15N in powder Milk.


       
However, there is an understanding of at least some of the parameters that influence the fractionation of 15N in soils and in plants. The intensity of agricultural practices, fertilization regimes, crop types, tillage practices and diet can all impact the 15N levels of tissues in animals and plants (Chanda et al., 2024;). These factors can influence the nitrogen cycle and affect the δ15N values in soil, plants and animal products (Caio et al., 2020); Kornexl et al., 1997). According to 15N signature (Fig 7), we have two group the first one ranger from 7,2 to 9,8% corresponding to region with moderate agriculture, second one ranger from 1,8 to 6,4‰ corresponding to intensive agriculture.

CONCLUSION

The results show that the H, C, N, O stable isotope ratios in milk are related to the region, especially the vegetation type and environment. 18O and H isotopes distinguish three groups according to altitude and distance from the Atlantic Ocean. The correlation between 18O in milk and dairy water confirms the influence of drinking water. 13C isotopes distinguish milk from C3-only equipment from milk from equipment with a mixture of C3 and C4. 15N isotopes distinguish milk from intensive agriculture (low 15N) and moderate agriculture (high 15N).
       
These results emphasize that geographical, climatic and agricultural factors affect the stable isotope ratios incorporated into animal tissues through diet, water and environmental exchanges. Therefore, stable isotope analysis of H, C, N and O offers great potential for determining the geographical origin of milk.

ACKNOWLEDGEMENT

The authors are thankful to the IAEA for the financial and technical support provided under Research Contract MOR-18051.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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