Louise Le Meillour
email: llemeillour@mnhn.fr

Antoine Zazzo
email: zazzo@mnhn.frjolesur@mnhn.fr

Joséphine Lesur

Sophie Cersoy
email: scersoy@mnhn.fr

Arul Marie
email: armarie@mnhn.frszirah@mnhn.fr

Matthieu Lebon
email: lebon@mnhn.frdpleurd@mnhn.fr

David Pleurdeau

Séverine Zirah

Identification of degraded bone and tooth splinters from arid environments using palaeoproteomics

Keywords: bone diagenesis, Africa, arid environment, proteomics, ancient collagen, ATR-FTIR

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

potentially the most informative about past societies. Yet, the physical integrity of bone remains is often degraded due to the influence of the environment and burial conditions leading to a large amount of material described as "undetermined" in faunal analyses. New developments in biomolecular methods for the study of ancient remains enabled the archaeologists to use DNA analyses for solving number of archaeological questioning such as phylogenetic relationships [START_REF] Debruyne | Out of America: Ancient DNA Evidence for a New World Origin of Late Quaternary Woolly Mammoths[END_REF][START_REF] Enk | Complete Columbian mammoth mitogenome suggests interbreeding with woolly mammoths[END_REF][START_REF] Hänni | Tracking the origins of the cave bear (Ursus spelaeus) by mitochondrial DNA sequencing[END_REF] or the genetic impact of species domestication [START_REF] Bollongino | Early history of European domestic cattle as revealed by ancient DNA[END_REF][START_REF] Bollongino | Y-SNPs Do Not Indicate Hybridisation between European Aurochs and Domestic Cattle[END_REF][START_REF] Jaenicke-Després | Early Allelic Selection in Maize as Revealed by Ancient DNA[END_REF][START_REF] Zeder | Domestication and early agriculture in the Mediterranean Basin: Origins, diffusion, and impact[END_REF]. Even though major progresses has been made such as the amplification of the oldest DNA molecules ever identified [START_REF] Orlando | Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse[END_REF], it is still challenging to extract and amplify this type of biomolecules from arid environment [START_REF] Kimura | Ancient DNA from Nubian and Somali wild ass provides insights into donkey ancestry and domestication[END_REF][START_REF] Loosdrecht | Pleistocene North African genomes link Near Eastern and sub-Saharan African human populations[END_REF].

The analysis of protein remains in archaeological samples by palaeoproteomics has become a powerful tool for species identification [START_REF] Buckley | A method of isolating the collagen (I) α2 chain carboxytelopeptide for species identification in bone fragments[END_REF][START_REF] Buckley | Distinguishing between archaeological sheep and goat bones using a single collagen peptide[END_REF][START_REF] Buckley | Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods[END_REF][START_REF] Van Der Sluis | Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius. Palaeogeogr. Palaeoclimatol. Palaeoecol., Bone and enamel diagenesis: From the crystal to the environment -A tribute to Jean-François[END_REF], reconstructions of phylogenetic relations between extant and extinct species [START_REF] Buckley | Mammoth and Mastodon collagen sequences; survival and utility[END_REF][START_REF] Cleland | Peptide sequences from the first Castoroides ohioensis skull and the utility of old museum collections for palaeoproteomics[END_REF][START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF][START_REF] Welker | Middle Pleistocene protein sequences from the rhinoceros genus Stephanorhinus and the phylogeny of extant and extinct Middle/Late Pleistocene Rhinocerotidae[END_REF]Welker et al., , 2015a)), and sometimes even on unidentified splinters (Welker et al., 2015b). The oldest characterized protein of ostrich eggshell from Tanzania dates back to 3.8 My BP [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF], testifying the potential of palaeoproteomics. But most of the archaeological material analysed by palaeoproteomics comes from temperate climates and not from arid environments. Such environment generates different biochemical and physical constraints on bone matrix, related to differences in temperatures, hydrology, pH or even soil chemical composition.

Often, morphological alterations can come along with biochemical modifications of the organic matrix. Indeed, number of studies focus on both biological and chemical alteration of the bone organic matrix [START_REF] Collins | The survival of organic matter in bone: a review[END_REF][START_REF] Dobberstein | Archaeological collagen: Why worry about collagen diagenesis?[END_REF][START_REF] Hedges | Measurements and Relationships of Diagenetic Alteration of Bone from Three Archaeological Sites[END_REF][START_REF] Schroeter | Glutamine deamidation: an indicator of antiquity, or preservational quality?[END_REF][START_REF] Simpson | The effects of demineralisation and sampling point variability on the measurement of glutamine deamidation in type I collagen extracted from bone[END_REF], composed in majority of type I collagen. As the major component of bone extracellular organic matrix, this molecule is formed of two α1 chains and one α2 chain forming a triple helix, further arranged into fibrils [START_REF] Rich | The molecular structure of collagen[END_REF]. It contains the repetitive sequence Gly -Xaa -Yaa in which Gly is glycine, Xaa can often be either proline (Pro) or hydroxyproline (Hyp) and Yaa any amino acid including Pro or Hyp [START_REF] Jenkins | Insights on the conformational stability of collagen[END_REF][START_REF] Kadler | Collagens at a glance[END_REF][START_REF] Shoulders | Collagen Structure and Stability[END_REF]. The N-and C-terminal regions of the α1 and α2 chains are connected through hydroxylysine cross-links [START_REF] Eyre | Advances in collagen cross-link analysis[END_REF]. Those structural characteristics allow the three chains to associate and create empty regions described as gap zones [START_REF] Xu | Structure analysis of collagen fibril at atomic-level resolution and its implications for intra-fibrillar transport in bone biomineralization[END_REF], where calcium phosphate mineralization occurs, leading to the formation of subsequent hydroxyapatite crystals [START_REF] Wilson & Deann | The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors[END_REF]. Diagenetic processes inherent to archaeological material such as collagen hydrolysis or biochemical modifications (such as deamidation) can increase difficulties associated with biomolecules analyses [START_REF] Tuross | Alterations in fossil collagen[END_REF]. Thus, variations occurring in the collagen helix or additional modifications can highly hindered the characterization of biomolecules contained in remains from arid environment.

Despite the increasing number of collected information, no study has ever focused on the characterization of Later Stone Age fauna proteins originating from tropical zones in Africa. To our knowledge, only two papers rely on continental African remains [START_REF] Demarchi | Protein sequences bound to mineral surfaces persist into deep time[END_REF][START_REF] Prendergast | Reconstructing Asian faunal introductions to eastern Africa from multi-proxy biomolecular and archaeological datasets[END_REF]): yet, the first one focused on ostrich eggshells and the second on bone historical material.

In Africa, the frequent absence of sufficiently preserved DNA in archaeological material restricted molecular studies [START_REF] Colson | The preservation of ancient DNA and bone diagenesis[END_REF][START_REF] Gifford-Gonzalez | Domesticating Animals in Africa: Implications of Genetic and Archaeological Findings[END_REF] and protocols suited for arid environments need to be developed, in order to bring new insights about African faunas recovered in archaeological sites using palaeoproteomics.

The aim of this study was to develop a protein extraction protocol for palaeoproteomics analysis adapted to highly degraded remains from arid environment. The protocols were tested on bone splinters from the 2000 years-old site of Toteng, Botswana. Organic preservation was first estimated using attenuated total reflectance Fourier transform infra-red spectroscopy (ATR-FTIR) and the selected samples underwent three different decalcification protocols. A bottom-up proteomic approach was used to identify digests of the extracted proteins. Finally, protein identification and chrono-biogeographical repartitions of mammals in Africa enabled to propose species attribution of certain of the bone splinters. meters above sea level. First digs revealed a rich sequence [START_REF] Campbell | Southern Okavango integrated development study: archaeological survey of proposed Maun reservoir[END_REF][START_REF] Robbins | Test excavations and reconnaissance palaeoenvironmental work at Toteng, Botswana[END_REF][START_REF] Sadr | Kalahari archaeology and the Bushman debate[END_REF] with typical Later Stone Age lithic industry and a large spectrum of faunal remains, especially the presence of domestic bovid species, associated with Bambata sherds [START_REF] Huffman | Toteng pottery and the origins of Bambata[END_REF].

Material

In 2003, a new campaign was organized and the two sites delivered new artefacts and ecofacts that enriched the archaeological assemblage [START_REF] Robbins | Recent archaeological research at Toteng, Botswana : Early domesticated livestock in the Kalahari[END_REF]. Zooarchaeological analyses performed by S. Badenhorst revealed the presence of domestic sheep (O. aries) in Later Stone Age levels, directly AMS dated to 2020 ± 40 BP (2σ, [START_REF] Robbins | The Advent of Herding in Southern Africa: Early AMS Dates on Domestic Livestock from the Kalahari Desert[END_REF]. This early AMS date increased the interest of the site regarding the quest for the earliest domestic caprines in Southern Africa. We accessed the faunal remains (n=150) excavated during the 2003 campaign, stored at the National Museum of Botswana, and we temporarily exported them under authorization. Exported remains were examined by one of the authors (JL) and 12 bone fragments and 6 teeth were selected for this study.

Most of them were too fragmented and altered by taphonomic processes to allow precise identification (Fig. 2 and Table 1). The identified remains were attributed to bovids and equids. 3. Methods

Sample preparation

The surface of the sample was first cleaned with a diamond drill. Less than 1 mg was sampled for ATR-FTIR analyses and between 20 and 150 mg for palaeoproteomics. Fragments for proteomics were roughly crushed into chunks and then divided into three equal subsamples. The first two subsamples were placed in 2 mL Eppendorf tubes (Protein LoBind, Eppendorf, Hambourg, Germany) while the third one was placed in 5 mL glass tubes.

ATR-FTIR

Prior to palaeoproteomics analyses, we estimated the organic content of the samples using attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy following the method established in [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF] We applied approximately 0.5 mg of powder on the diamond (Golden Gate Single Reflection Diamond ATR accessory (Specac, France) with KRS-5 lens) of a Vertex 70 FTIR spectrometer (Bruker Optics, France). ATR-FTIR spectra were obtained by accumulation of 64 scans with a resolution of 4 cm -1 in the 4000-400 cm -1 wavenumber range.

During acquisition, anvil pressure on sample was adjusted to normalise ν3PO4 peaks to an absorbance of 0.5. Resulting data were treated using the OPUS software (Bruker Optics, France) and linear baselines were drawn to measure the area of amide I (between 1710-1590 cm -1 ) and phosphate ν3(PO4) (between 1110-940 cm -1 ) bands (Fig. S1). Then, the amide I/PO4 ratio (standard deviation taken into account) was used to estimate nitrogen and collagen weight percent content (%wt) using the equations presented below:

%N (wt) = 20.6 Amide I PO ! + 0.31 %collagen (wt) = 113.13 Amide I PO ! + 1.69
More detailed information on data calculation can be obtained in [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF]. Three different decalcification protocols were tested (Fig. 3) and an extraction blank performed for each, consisted in the same steps of chemical preparation without any bone powder.

Demineralization and protein extraction

The first protocol (Fig. 3, left) follows a previously published method by [START_REF] Buckley | A method of isolating the collagen (I) α2 chain carboxytelopeptide for species identification in bone fragments[END_REF].

Samples were placed in 1 mL 0.6 M HCl for 4 h at 4 °C. Solutions were replaced every hour after mixing and 10 min of 13 000 rpm centrifugation. After 4 hours, samples were washed 5 times with MilliQ water to reach neutrality.

The second protocol (Fig. 3, center) is an in-house protocol for collagen extraction [START_REF] Bocherens | Preservation of bone collagen sulphur isotopic compositions in an early Holocene river-bank archaeological site[END_REF]Cersoy et al., 2017a). Samples were immersed in 1 mL HCl 1 M for 30 min under 350 rpm agitation at room temperature (ThermoMixer, Eppendorf, Germany). Then, all samples were centrifuged at 13 000 rpm for 10 min and then washed 5 times with MilliQ water to reach neutrality.

In the third protocol (Fig. 3, right), samples were immersed in 1 mL 0.05 M tris(hydroxymethyl)aminomethane (Tris, Sigma Aldrich, Missouri) and 0.5 M ethylendiaminetretraacetic acid (EDTA) buffer, final pH 7.4 as proposed by [START_REF] Balasse | Early Weaning of Neolithic Domestic Cattle (Bercy, France) Revealed by Intra-tooth Variation in Nitrogen Isotope Ratios[END_REF] and [START_REF] Tuross | Variability in the preservation of the isotopic composition of collagen from fossil bone[END_REF]. The solution was changed daily for 4 days, then every two days.

Decalcification was visually and manually checked until a remaining pale-yellow transparent phantom and varied between 1-7 days depending on the sample (Table S1).

Enzymatic hydrolysis

Following demineralization, all samples were washed out with MilliQ water. They were then dried under vacuum (SpeedVac Concentrator™, Savant™, ThermoFisher Scientific, Massachusetts).

Dried fractions of organic material were solubilized in 100 μL 50 mM ammonium bicarbonate for 3 h at 65 °C under 650 rpm agitation (ThermoMixer, Eppendorf, Germany). Supernatants were collected after centrifugation, placed into clean tubes and stored on lab bench until they reached room temperature. Proteomics grade trypsin (Trypsin Gold, Promega, Wisconsin) was added to each sample, 1:100 proportions. They were incubated at 37°C under 650 rpm agitation overnight. Tryptic digestion was stopped adding 1 μL 10% trifluoroacetic acid (TFA, SigmaAldrich, Missouri). Protein concentration of each extract was measured using a microvolume spectrophotometer (NanoVue, GE HealthCare, France) using the pre-set parameters for protein concentration measurement. The concentration values were distributed between 0.5 and 2.3 μg/μL. Hz) to maximize the number of MS/MS spectra. Ion selection targeted m/z range 300-2200 with charge states 1 + to 5 + . The mass spectrometer was calibrated with sodium formate clusters at the beginning of each analysis (between 0 and 0.5 min) and data were automatically calibrated.

MS analysis

Data analysis

The LC-MS/MS data were converted to mgf files using DataAnalysis software (Bruker Daltonics, Massachusetts). Database search was carried out against public protein databases (SwissProt and NCBI simultaneously) using the online software Mascot (MatrixScience.com). We restricted the search to « Mammalia (mammals) » protein sequences and allowed 2 missed cleavages for the enzyme. We considered deamidation (N or Q), oxidation (M or P) and phosphorylation (S or T) as variable post-translational modifications (PTM), as these type of PTM were previously reported in the literature for archaeological samples [START_REF] Buckley | Proteome degradation in ancient bone: Diagenesis and phylogenetic potential. Palaeogeogr. Palaeoclimatol. Palaeoecol., Bone and enamel diagenesis: From the crystal to the environment -A tribute to Jean-François[END_REF][START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF]. 1 + , 2 + and 3 + peptides were considered with 10 ppm tolerance, MS/MS fragments with 0.1 Da tolerance and the statistic decoy applied to avoid false positive match.

MS/MS data were also analysed using PEAKS DB Studio software (7.5 version, BioInformatics Solutions, Waterloo, Canada, hereafter PEAKS) which permits to propose database search assisted de novo sequencing. PEAKS searches (including de novo, PEAKS PTM and SPIDER algorithms) were set using the following parameters: 10 ppm precursor mass (monoisotopic) and 0.1 Da fragment ion tolerances; a maximum of 5 PTM per peptide was authorized. PTM set in PEAKS searches were the same as for Mascot, together with the addition of pyroglu from Q and amino acid substitutions. Searches were performed using a restrained collagen database containing sequences described in Table S2. The database was constructed using NCBI protein sequences of alpha 1 and alpha 2 chains of collagen type I (COL1A1 and COL1A2) of the species of interest. The obtained results were filtered using the following parameters: ≤ 1% false discovery rate (FDR) for peptide spectral matches, ≤ 1% FDR and at least one unique peptide for proteins. Finally, peptides that were attributed to a unique species either by Mascot or PEAKS (MS/MS spectra) and all de novo peptides were verified using Geneious software (Biomatters, New Zealand) by aligning protein sequences of all species used in the construction of PEAKS restrained database (Table S2). We then made sure that amino acids substitutions between species were not due to any transcription mistake by verifying all codons manually.

Results

ATR-FTIR pre-screening

The collagen contents (collagen wt%) of the samples are summarized in Table 2. They vary between 1.9 and 5.7%, which corresponds to 8.6% to 25.8% of the organic content of modern bone (22.2 %, "B. taurus bone (modern reference)" in Table 2). Examples of the best and least preserved samples spectra are presented in Figure 4. We observed a decrease in the absorbance of the amide I band absorbance in the archaeological samples compared to the modern sample. Nine samples covering the entire range of collagen preservation were selected for palaeoproteomics analyses (Table 2).

2. LC-MS analyses

Four out of nine remains (TOT_09, TOT_11, TOT_14 and TOT_15) contained identifiable peptides. The other five (TOT_03, TOT_06, TOT_07, TOT_18 and TOT_26) presented a single peptide corresponding to trypsin autolysis, VATVSLPR ([M+2H] 2+ , m/z 421.75, rt 7.6-7.8 min).

Depending on the sample, 4 to 342 MS/MS spectra were generated. All identified peptides and corresponding proteins are presented for each sample in Table S3. Protocols using Tris-EDTA, 0.6 M HCl and 1 M HCl allowed the identification of 240, 89 and 97 peptides, respectively. Low errors (between 0 and 3.83 ppm) between the observed and calculated m/z for the peptides increased the confidence in the peptide match. All the identified peptides in the samples were assigned either to COL1A1 or COL1A2, corresponding to the α1 and α2 chains of type I collagen. No other proteins were identified in any sample. No contamination that could be attributed to peptide signals were detected in the preparation or the solvent mixture blanks, and no keratins or other human protein were detected, indicating the reliability of the protocol used in this work. The analysis suggest that peptide identification depends both on the preservation of the sample and of the decalcification protocol (Fig. 5). Among the samples, TOT_14 yielded the greater number of peptides and TOT_11 the least and all of them presented more identified peptides with Tris-EDTA than with 0.6 and 1 M HCl. PTM were identified in all the protocols with deamidation (NQ) and oxidations (MP) (Table S3). No phosphorylation (ST) was detected. Sequence coverages per sample and protocol range between 3 and 40% (Table 3). Samples decalcified using Tris-EDTA presented a larger coverage (between 18 and 40%) than the ones decalcified using 0.6 and 1 M HCl solutions (respectively ranged between 3-17% and 4-22%). Finally, all samples but TOT_11 presented more than 200 unassigned spectra after Mascot analyses (Table S3), denoting that proteins were hydrolysed at non-tryptic sites or harboured additional PTMs, and/or that the database used was incomplete. Table 3.

Proteins sequences coverages realised after using Mascot and PEAKS. Each protein sequence coverage is presented by sample (lines) and protocol (columns).

Species identification -PEAKS results

In order to refine Mascot identifications, we analysed raw data using PEAKS software. The use of PEAKS in this study was mainly to take into consideration additional PTMs and amino acid substitution and to compensate the potential lack of information in the databases. PEAKS algorithm is designed to reconstruct database search assisted de novo peptides from the MS/MS data and match the generated sequences against a protein database [START_REF] Zhang | PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification[END_REF]. PEAKS PTM and SPIDER tools were used after de novo reconstruction. We created a database from the type I collagen sequences reported within bovids, equids and suids in NCBI (Table S2). The limited database was constructed to restrain the search against species that were suspected after morphological identifications (Table 1).

Sequence coverages with PEAKS search covered up to 44% of protein sequence and slightly higher 13/24 times (Table 3). Likewise, samples decalcified using Tris-EDTA presented the highest coverage (between 9 and 44%), while coverages were lower when samples were decalcified using HCl (between 0-22% and 0-26% with 0.6 and 1 M HCl, respectively). We observed an increasing number of PTM: in addition to the already identified oxidation (MP) and deamidation (NQ), we observed dimethylation, hydroxylation, phosphorylation (STY) and even P to Y substitution (Fig. S2). Furthermore, the most abundant PTM identified was P hydroxylation (up to 60% of P), as described by [START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF]. Species identifications made by Mascot were ascertained and improved with de novo peptides as both non-tryptic and modified peptides were reconstructed and assigned to the restrained collagen database. To ascertain the species identifications, we looked for unique collagen peptides of all family and species represented in the samples. Even though COL1A2 is commonly used for distinction because it presents more taxonomic variations, we report here COL1A1 peptides for species and genera identification. Figure 6 presents the MS/MS spectra corresponding to COL1A1 peptides for the samples decalcified using Tris-EDTA. Spectrum in Fig. 6a corresponds to a unique peptide of COL1A1 of suids, identified in sample TOT_09 (long mammal bone). Three PTM are reported here:

two P hydroxylations and one Q deamidation. Similarly, sample TOT_11 (morphologically identified as an equid tooth) revealed a unique peptide of equid COL1A1 (Fig. 6b). The same peptide was observed in TOT_15, the second sample identified as an equid both by morphological criteria and previous search against available databases (this paper, 4.2.). TOT_14, morphologically identified as a size 2-3 bovid metapodial, was attributed to the species Ovis aries (sheep) thanks to 51 unique peptides of the COL1A1 identified using Mascot (Table S3). The re-examination of this sample's data highlighted that only one out of the 51 previously identified peptides was indeed specific for the alpha 1 chain of collagen type I (Fig. 6c) and that 2 amino acids changes can be observed against other species (Table 4). Differences between sheep, goat, bovine, suid and human COL1A1 peptide 921-936.

Discussion

In the context of rare palaeoproteomics analyses of remains originating from Africa, this study aimed to identify a suitable chemical preparation protocol and then to exploit proteomics data for taxonomic identification of splinters from Botswana. Data analysis allowed to point Tris-EDTA as the best decalcification protocol and to refine taxa attributions of the splinters.

Influence of the decalcification protocols

This study explored the use of ATR-FTIR as pre-screening for palaeoproteomics analyses.

This method is indeed now used in routine to select samples suitable for radiocarbon dating (Cersoy et al., 2017b;[START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF]. Our results show that below 2.9% collagen, no peptide could be identified in the Toteng samples. This threshold is in the same range than that was reported for radiocarbon dating proposed by Cersoy et al. (2017b) and [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF], set at ~ 4 ± 1.2% wt of collagen, suggesting that it could also apply to palaeoproteomics studies. This conclusion, however, is based on a limited number of samples, and more work is needed to further confirmation.

Moreover, it is important to remind that the heterogeneity of bone material can introduce biases in the results and that several samplings are to be made to ascertain the reliability of this estimation [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF]. 5.

Synthetic table of results presenting the best sequence coverages for the two chains of collagen type I, the protocol that allowed best sequence coverage for the whole collagen type I and collagen preservation estimated by ATR-FTIR.

Table 5 presents a synthesis of the results obtained for the four samples that yielded peptide.

Here, the relationship between the sequence coverage and organic phase preservation is not as clear as we might have expected. While the sample with the poorest preservation (TOT_11, 2.94%) presented the lowest sequence coverage (18% and 23% for COL1A1 & A2, respectively) the highest coverage (36% and 44% for COL1A1 & A2, respectively) was obtained for a sample showing intermediate collagen preservation (TOT_14, 3.58%). The triple helix bond by hydroxylysine at their extremity making the collagen structure can be argued as an explanation [START_REF] Shoulders | Collagen Structure and Stability[END_REF]. We can assume that the best-preserved samples should present the best collagen integrity. The preservation of collagen crosslinks could limit sequence coverage, as crosslinked peptides would not match to databases. Even if the sequence coverages obtained are not directly comparable to proteomics studies on modern bones (Schroeter et al., 2016), it appears that the protocol used that allowed best sequence coverage is Tris-EDTA. Indeed, recent studies have highlighted the potential of Tris-EDTA for the analysis of bone collagen [START_REF] Cappellini | Proteomic Analysis of a Pleistocene Mammoth Femur Reveals More than One Hundred Ancient Bone Proteins[END_REF][START_REF] Procopio | Minimizing Laboratory-Induced Decay in Bone Proteomics[END_REF][START_REF] Sawafuji | Proteomic profiling of archaeological human bone[END_REF]Schroeter et al., 2016) and we here point out its efficiency for palaeoproteomics analyses of poorly preserved archaeological bones and teeth.

This study also demonstrates the advantage of combining diverse data analysis treatment. The combination of the results obtained with database search (Mascot) and database search assisted de novo sequencing (PEAKS) allowed increasing peptide reconstruction and protein assignation. The use of database search allowed to estimate which sample presented remaining peptides and the corresponding protein and, in some extent, permitted to propose taxonomic assignations. The use of database search assisted de novo sequencing allowed to increase the number of both identified peptides, sequence coverages and to compare the efficiency of the different protocols. A higher number of PTM were observed, especially Q deamidation and P hydroxylation. Glutamine deamidation are often reported in the case of ancient proteins, and are believed to be caused by diagenetic processes [START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF][START_REF] Schroeter | Glutamine deamidation: an indicator of antiquity, or preservational quality?[END_REF][START_REF] Van Doorn | Site-specific deamidation of glutamine: a new marker of bone collagen deterioration[END_REF].

Proline hydroxylation is inherent to type I collagen [START_REF] Kadler | Collagens at a glance[END_REF] and can also be induced both by acidic treatment and to a lesser extent diagenesis [START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF][START_REF] Procopio | Minimizing Laboratory-Induced Decay in Bone Proteomics[END_REF]. Identifications with Mascot based on the COL1A2 peptides and the database search assisted de novo sequenced peptides of COL1A1 in each positive sample allowed to strongly confirm the identifications previously made and thus giving clear genus and species attributions of the splinters.

The results showed that 4 out of 9 splinters presented conserved proteins. The presence of a tryptic peptide and the absence of other proteins in TOT_03, _06, _07, _18 and _26 suggest that they were too degraded and that no organic fraction could be detected by MS analysis. However, and as it was previously demonstrated by Schroeter et al. (2016), there is a possibility for the remaining protein to have been solubilized in decalcification solution (Tris-EDTA, 0.6 M or 1 M HCl). Surprisingly, one sample that presented over ~ 3%wt of collagen preserved (TOT_07, 3.11%collagen wt) did not yield any protein. One explanation would be the overestimate of the collagen content caused by the important content of carbonate (calcite) [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF]. In any case, the collagen content value of 3.11 is low and, in comparison to the threshold (2.9%), sensibly equivalent taking the error into account (1.4 maximum). Thus, we could argue that a risk of non-identifying preserved proteins does exist in samples with ATR-FTIR values between 2.9 wt% and 3.11 wt% of collagen for palaeoproteomics analyses. In other way, the absence of non-collagenous protein (NCP) in the sample is consistent with what was previously reported in 4 000 years archaeological remains from Cyprus [START_REF] Buckley | Proteome degradation in ancient bone: Diagenesis and phylogenetic potential. Palaeogeogr. Palaeoclimatol. Palaeoecol., Bone and enamel diagenesis: From the crystal to the environment -A tribute to Jean-François[END_REF].

Refining species identification using biogeographical and chronological repartitions of African mammals

Regarding the results presented here, palaeoproteomics analyses allowed the identification of two equids (TOT_11 and TOT_15), one suid (TOT_09) and one sheep (TOT_14). Those attributions remain to genus level, excepted for TOT_14 which was identified to the species level. This can mostly be explained by the lack of protein sequences of African animals in the modern databases. However, the available literature provides guidance regarding the biogeographical and chronological repartition of African mammals [START_REF] Kingdon | The Kingdon field guide to African mammals[END_REF] allowing to refine the proteomics attributions (Table 6).

Because Toteng dates from the first century before common era (BCE), the equid remains probably belong to the zebra species Equus burchellii (Mammals Species of the World, 2005). Indeed, domestic equids (donkey E. asinus and horse E. caballus) were brought by Europeans much later, around the XVII th century [START_REF] Blench | A history of donkeys, wild asses and mules in Africa[END_REF][START_REF] Lesur | Et la gazelle devint chèvre: pré-histoires africaines d'hommes et d'animaux[END_REF] and no remains of wild horse (E. caballus przewalski) has ever been mentioned on the African continent. Regarding the sample attributed to the suid family (TOT_09) the situation is less clear. Six species coming from four genera of suids do exist nowadays in Africa: Phacochoerus aethiopicus and P. africanus, Potamochoerus porcus and P. larvatus, Hylochoerus meinertzhageni and Sus scrofa. Only the first two genera can correspond to this remain in terms of localization and chronology. Indeed, Hylochoerus can be found in western, central and a part of eastern Africa [START_REF] Grimshaw | The giant forest hog Hylochoerus meinertzhageni in Tanzania records rejected[END_REF][START_REF] Kingdon | The Kingdon field guide to African mammals[END_REF]; Sus scrofa is endemic of northern Africa [START_REF] Kingdon | The Kingdon field guide to African mammals[END_REF] and its domestic form, Sus domesticus was not introduced in Africa before the 5 th millenium BCE and arrived in southern Africa mostly by first millennia CE importations [START_REF] Lesur | Et la gazelle devint chèvre: pré-histoires africaines d'hommes et d'animaux[END_REF][START_REF] Smith | The origins of domesticated animals of Southern Africa[END_REF]. In term of species, the geographic distribution thus suggests that the remain could either be attributed to the bushpig Potamochoerus larvatus or the common warthog Phacochoerus africanus [START_REF] D'huart | Distribution of the common warthog (Phacochoerus africanus) and the desert warthog (Phacochoerus aethiopicus) in the Horn of Africa[END_REF][START_REF] Grubb | The afrotropical suids (Phacochoerus, Hylochoerus and Potamochoerus). Taxonomy and description. Pigs Peccaries Hippos Status Surv[END_REF].

Conclusion

This study reports a first attempt at using palaeoproteomics for the species identification of archaeological bones from arid environment of Southern Africa. Estimation of preserved collagen content of bone samples prior to palaeoproteomics analysis using ATR-FTIR permits to propose a threshold of ~ 3%, similar to that previously proposed for radiocarbon dating. The comparison of three decalcification protocols suggests that the use of Tris-EDTA is best suited to poorly preserved samples (less than 5 wt% of collagen preserved as estimated using ATR-FTIR) rather than classical HCl based protocols. Associated with an efficient combination of software for MS data analyses, this study ascertain the use of combined bioinformatics tools for accurate analysis of palaeoproteomics data as previously proposed by [START_REF] Cleland | Biologically and diagenetically derived peptide modifications in moa collagens[END_REF]. Both database search (using Mascot search engine) and database search assisted de novo sequencing (using PEAKS software) were used to reconstruct and assign remaining peptides. But the use of de novo tools and manual examination allowed to ascertain taxonomic identifications. Finally, this study enables to improve morphological identifications for splinters from the Later Stone Age site of Toteng, Botswana. About half of them contained enough proteins to propose a taxonomic attribution. This attribution was further refined by combining proteomics data with biogeographical and chronological distributions of mammals. We then suggest that species identification using palaeoproteomics of remains from Africa should be combined, when possible, with chronological and biogeographical repartitions data. Finally, this study permits the identification of two zebras, one Phacochoerus or Potamochoerus and one domestic sheep. The latter may potentially have important archaeological implications regarding the introduction of the first domestic caprines in Southern Africa.

Fig. 1 .

 1 Fig. 1. Africa map with Botswana localization (dark grey) in Austral Africa and country map with Toteng 1 and Toteng 3 sites localization. Print in color

Fig. 3 .

 3 Fig. 3. Flow chart illustrating the three decalcification protocols employed in this study and further steps of extraction, enzymatic hydrolysis and mass spectrometry analyses. Print in color

  & data treatment 3.5.1. Mass spectrometry Tryptic digests were analysed by mass spectrometry (UHPLC-MS). The chromatographic separation was conducted on an Ultimate 3000-RSLC (Thermo Scientific, Massachusetts) using a RSLC Polar Advantage II Acclaim column (2.2 μm, 120 Å, 2.1 × 100 mm, Thermo Scientific, Massachusetts) with a flow rate of 300 μL/min using a mobile phase gradient with A: H2O + formic acid (FA) 0.1 % and B: acetonitrile (ACN) + FA 0.08 %. Each analysis consisted in 1 μL of sample injected and a blank (5 μL of A-B mix -20:80) every 5 samples. The mass spectrometer (electrospray ionization, quadripole, time of flight instrument -ESI Q-TOF, Maxis II ETD, Bruker Daltonics) was used in positive mode on m/z range 200-2200 using an accelerating tension of 3 500 V. Analysis were performed in data dependent auto-MS/MS mode with a cycle time option fixed at 3 sec. and MS acquisition frequency of 2 Hz. Two series of experiments were conducted both with collision induced dissociation (CID): one with low acquisition frequencies to boost MS/MS spectra quality (0.5 Hz for low-abundance ions, 2 Hz for intense ions) and a second with high acquisition frequencies (4 Hz, 16

Fig. 4 .

 4 Fig. 4. ATR-FTIR spectra of Toteng best preserved (TOT_09, red curve) and least preserved sample (TOT_26, blue curve) in comparison to modern sample (B. taurus bone, yellow curve here). Print in color

Fig. 6 .

 6 Fig. 6. MS/MS spectra of COL1A1 distinguishing peptides identified. Letters in bold represent the variant amino acid against other species proteins; letters underlined represent amino acid with PTM. Discriminating ions are represented on each sequence above the corresponding spectrum. Each species sequence is represented by animal shape (horse represents equids, pig represents suids, cow represents bovids except for spectrum (c) where both goat and bovids are represented).(a) TOT_09, 457… GEPGPTGVQGPPGPAGEEGK …473 ([M+2H 2+ ], m/z 925.9250, rt 6.3 min). Here, 3 PTM have been identified: two P hydroxylations and one Q deamidation. This peptide corresponds to suid COL1A1.

Sample code Site Morphological identification Anatomical part Location on the site Surface alterations visible on the remain

	TOT_01	Toteng	Unidentified bovid	Ulna diaphysis	Square K, depth 75-80 cm.	Small mammals munching (SMM), rhizomes traces (RT)
	TOT_02	Toteng	Unidentified bovid	Ulna diaphysis	Square K, depth 75-80 cm.	SMM, RT, burnt?
	TOT_03	Toteng	Unidentified mammal	Unidentified	Square K, depth 80-85 cm.	SMM, RT
	TOT_04	Toteng	Medium-sized mammal	Unidentified	Square K, depth 80-85 cm.	SMM, RT, burnt?
	TOT_05	Toteng	Bos taurus or Syncerus caffer	Upper molar	Square K, depth 90-95 cm.	Large concretions, use wear
	TOT_06	Toteng	Bos taurus or Syncerus caffer	Upper molar	Square H, depth 115-120 cm.	Large concretions, use wear
	TOT_07	Toteng	Unidentified	Unidentified	Square L, depth 100-105 cm.	SMM, RT
	TOT_09	Toteng	Unidentified mammal	Long bone	Square L, depth 100-105 cm.	SMM, RT, crackled
	TOT_10	Toteng	Small-sized bovid	Lower left molar 3	Square L, depth 110-115 cm.	Concretions, use wear
	TOT_11	Toteng	Equus sp.	Tooth	Square 7, depth 30_35 cm.	Large concretions, use wear, RT
	TOT_12	Toteng	Large-sized mammal	Unidentified	Square 7, depth 30_35 cm.	Deep SMM and RT
	TOT_13	Toteng	Large-sized mammal	Long bone	Square 7, depth 30_35 cm.	Deep SMM, crackled
	TOT_14	Toteng	Medium-sized bovid	Metapodial	Square 7, depth 30_35 cm.	SMM, RT, crackled
	TOT_15	Toteng	Equus sp.	Tooth	Square 7, depth 38 cm.	Large concretions, use wear, several pieces
	TOT_16	Toteng	Equus sp.	Tooth	Square 7, depth 38 cm.	Large concretions, use wear, several pieces
	TOT_18	Toteng	Large to medium-sized bovid	Proximal phalanx 1	Square 7, depth 30-40 cm.	SMM, RT
	TOT_25	Toteng	Unidentified mammal	Long bone	Square 7, depth 45-47 cm.	SMM, RT, small diaphysis
	TOT_26	Toteng	Bos taurus or Syncerus caffer	1 st molar (upper or	Square 9, depth 45-50 cm.	Large concretions, use wear
				lower?)

Table 1

 1

Table 2 .

 2 ATR-FTIR results. Amide I/ν3PO4 ratio was calculated with OPUS software (Bruker Optics, France) on raw spectra. N wt% and collagen wt% were estimated after the ratio calculation. Selected samples and their corresponding collagen wt% for palaeoproteomics analysis are presented in bold black.

	Sample code	Amide I/PO4	%N wt	% Collagen wt
	B. taurus bone (modern reference)	0.18	4.04	22.17
	TOT_09	0.04	1.04	5.72
	TOT_15	0.03	0.98	5.38
	TOT_14	0.02	0.65	3.58
	TOT_12	0.01	0.61	3.34
	TOT_07	0.01	0.57	3.11
	TOT_16	0.01	0.57	3.10
	TOT_11	0.01	0.54	2.94
	TOT_10	0.01	0.50	2.73
	TOT_04	0.01	0.49	2.66
	TOT_03	0.01	0.47	2.57
	TOT_05	0.01	0.45	2.45
	TOT_25	0.01	0.45	2.44
	TOT_13	0.01	0.44	2.40
	TOT_02	0.00	0.41	2.21
	TOT_06	0.00	0.40	2.18
	TOT_18	0.00	0.37	2.03
	TOT_01	0.00	0.36	1.99
	TOT_26	0.00	0.35	1.91

  Number of assigned peptides per sample regarding the available protein databases by Mascot software. In black, number of assigned peptides using Tris-EDTA; in light grey using 0.6 M HCl and in dark grey using 1 M HCl. Preserved collagen content (collagen wt%) are presented above the corresponding sample.

	Fig. 5.
														3.58%
														94
															5.38%
										5.72%				62
										49
												2.94%
										28	32	30		29	33
	2.57%	2.18%	3.11%					23	20	2.03%	1.91%
	1	1	1	1	1	1	1	1	1			7	4			1	1	1	1	1	1
	TOT_03	TOT_06	TOT_07	TOT_09	TOT_11	TOT_14	TOT_15	TOT_18	TOT_26
										EDTA	0.6 M HCl	1 M HCl

Table 4 .

 4

	Species	COL1A1 peptide (921-936)	[M+H] + m/z monoisotopic	[M+2H] 2+ m/z
	Ovis aries; O. aries musimon	AGEVGPPGPPGPAGEK	1416.7118	709.3559
	Capra hircus	P...............	1442.7274	722.3637
	Bos taurus; B. mutus; B. indicus;	P...............	1442.7274	722.3637
	B. bison
	Equus caballus; E. asinus; E.	P..A............	1414.6961	708.3480
	caballus przewalskii
	Sus scrofa; S. scrofa domesticus	P..A............	1414.6961	708.3480
	Homo sapiens	P...............	1442.7274	722.3637

Table 6 .

 6 Synthetic table of morphological attributions of Toteng remains refined with the combination of palaeoproteomic and biogeographical and chronological data.

	Sample code	Morphological attribution	Palaeoproteomics identification	Biogeochronological attribution
	TOT_09	Long bone unidentified mammal	Suidae	Phacochoerus or Potamochoerus
	TOT_11	Tooth Equus sp.	Equidae	Equus burchellii
	TOT_14	Metapodial medium size bovid	Ovis aries	---
	TOT_15	Tooth Equus sp.	Equidae	Equus burchellii

Archaeological remains selected for the study from the 2003 campaign[START_REF] Robbins | The Advent of Herding in Southern Africa: Early AMS Dates on Domestic Livestock from the Kalahari Desert[END_REF][START_REF] Robbins | Recent archaeological research at Toteng, Botswana : Early domesticated livestock in the Kalahari[END_REF].

Acknowledgments

This work was supported by a grant from Agence Nationale de la Recherche under the LabEx ANR-10-LABX-0003-BCDiv, in the program « Investissements d'avenir » ANR-11-IDEX-0004-02 and a grant Action Thématique Muséum (ATM) from the Muséum national d'Histoire naturelle under the name « ProtéArch ».

The authors would like to thank the National Museum of Botswana for access to the Toteng remains and expressly M. Mvimi for obtaining the exporting permit, without whom this study will not have been possible. The authors also want to thank the Plateau Technique de Spectrométrie de Masse Bio-Organique and the Plateau de Spectrométrie Infrarouge of the MNHN for accessing the instruments and X. Gallet for his help for FTIR analyses.

Finally, the authors would like to thank the two anonymous reviewers for their time and comments that contributed to enrich this manuscript.


L E Meillour

C Le Danvic

Nagnan-Le Meillour

o In Austral Africa, no remains of domestic caprines (sheep Ovis aries and goat Capra hircus) has never been found in archaeological assemblages. This suggests that those domestic animals have been introduced from the Near East to East Africa and then to the southern part of the continent (MacHugh, Larson & Orlando 2017, Vigne 2011).

o Classical morphological determination of caprines from bone remains is often tricky due to the similarities between close related species as well as between domestic and wild species, fragmentation of the remains and bone diagenesis.

o Paleoproteomics has revealed as a powerful approach for the distinction of domestic caprines (Buckley et al. 2010). We used this technique on remains from two Austral African sites considered as the oldest pastoral sites of the region: Leopard Cave in Namibia (Pleurdeau et al. 2012) and Toteng in Botswana (Robbins et al. 2008). Our objective was to ascertain the presence or absence of domestic animals at these sites.

o Three different sample preparation protocols were tested in order to identify the best conditons for remains from arid environment. Bottom-up proteomics analysis was performed on an ESI-Q-TOF device with a data dependent autoMS/MS method. Modern samples from referential collections (MNHN) were also analyzed to constitute a database of unreported diagnostic peptides for species identification.

INTRODUCTION Leopard Cave

Toteng

CONCLUSION

We would like to thank the LabEx BCDiv for funding a Master's grant on this topic; the MNHN for funding the analysis through the ATM grant « ProtéArch »; the CNRS and the MNHN for funding the ISABIO equipment project; the UMR 8576 for accessing the lab and providing equipment for molecular biology manipulations; the « Plateforme de Caractérisation des Matériaux » for FT-IR analysis facilities and the « Plateau technique de Spectrométrie de Masse Bio-Organique » of the MNHN for access to the UHPLC-ESI-Q-TOF instrument.

RESULTS

MATERIALS

o

  +MS2(799.4415), 41.0eV, 2.4 min #156 [Cmpd 55, +MS2(799.4415), 41.0eV, 2.4 min #156] +MS2(853.9018), 34.2eV, 3.2 min #216 [Cmpd 96, +MS2(853.9018), 34.2eV, 3.2 min #216+MS2(799.4408), 41.0eV, +MS2(799.4408), 41.0eV, 2.3 min #157+MS2(961.8263), 35.0eV, +MS2(961.8263), 35.0eV, 4.3 min #362+MS2(730.9245), 29.2eV, 4.6 min #409 [Cmpd 208, +MS2(730.9245), 29.2eV, 4.6 min #409spectrum of [M+H] + ion of GAAGIPEGK peptide (m/z 799.44, 2,4 min) of one Leopard Cave's sample, demineralized with Tris-EDTA. MS/MS spectrum of [M+H] + ion of LCPDCPLLANDSR peptide with a phosphorylation (S) (m/z 853.90, 3,2 min) of one Leopard Cave's sample, demineralized with Tris-EDTA. MS/MS spectrum of [M+H] + ion of TPIVGQPSVPGGPVR peptide (m/z 730.92, 4,6 min) of one Leopard Cave's sample, demineralized with Tris-EDTA. Identification of the best protocol for arid environments remains : Tris-EDTA o Identification of specific markers of unpublished species (Antidorcas marsupialis) on 2 different proteins o Attribution of 4 morphologically undetermined remains o Re-attribution of Leopard Cave remains ➜ wild species: no domestication at this site o Identification of sheep at Toteng ➜ confirmation of domestication at this site st c. BCE Pleurdeau et al. 2012, Robbins et al. 2008 ARCHAEOLOGICAL IMPLICATIONS -MacHugh, Larson & Orlando 2017, Annual Review of Animal Bioscience, 5, 329-51. -Vigne 2011, Comptes rendus biologies, 334(3), 171-181. -Buckley et al. 2010, Journal of Archaeological Science, 37(1), 13-20. -Pleurdeau et al. 2012, Plos One, 7(7), e40340. -Robbins et al. 2008, Journal of African Archaeology, 6(1), 131-149. -Lebon et al. 2016, Radiocarbon, 1-15. -Cersoy et al. 2017, Radiocarbon, 1-16. -Kingdon 1997, Princeton Univ. Press, London, 544 p.


Claire Mosnier

Patrick Veysset

Anne Jarousse

Evaluation of 16 European beef production systems contribution to food security

Beef production, an inneficient food production

Ruminant productions criticised for low conversion efficiency of natural resources into edible food ➢ Yet, they can use resources that cannot be consumed by human Globally, when considering only feed that is also human edible :

• In average, in the world, 6 kg DM of plant resources needed to produce 1kg of proteins from cattle (Mottet et al. 2017) • High variability between production systems What is the contribution of beef production systems to food safety in Europe? Net edible protein production per ha at farm scale Diversified systems (cash crops or milk production) produce more protein per hectare because of the higher yield per hectare of these productions.

FR-CC2 FR-DCC BE-CC1 BE-CC2 BE-CCF BE-D IT-F1 IT-F2 IR-CCF IR-F GE-F2 FR-CCF FR-CC1 GE-F1 GE-
Extensive grass based cow-calf systems produce less protein per hectare. Net protein efficiency at beef production level

• Cow-calf systems based on grass efficient but do not produce finished meat

• Cow-calf + fatteners and fatteners efficiencies <1 except for :

• Grass based system IR-CCF • Italian intensive system based on co-products IT-F1

• Cost of production of beef meat

• Impact of farm size on production costs (fixed costs and labor)

• Overall production cost is lower for Fatteners due to higher productivity per LU EAAP -1st December 2020

Conclusion

• Harmonized description and evaluation of a variability of systems at European level

• But data collected from a sample of systems

• Cow-calf phase :

• Net protein efficiency >1 and low use of arable land • Low protein productivity per hectare and high cost per Kg. carcass eq.

• Fattening phase :

• Lower production costs • Protein efficiency <1, except for fattening with grass and/or coproducts.

• Variable results and the compromises to be found over the whole production cycle • Need for further research on the impact of improvements on food security criteria on other dimensions of the sustainability EAAP -1st December 2020

Thanks for your attention

  -Réseaux d'Elevage Ireland : Irish National Farm Survey &Teagasc Other contries, selection of commercial farms by project experts: Belgium: CRA-W -Eleveo-AWE Italy: CREA Germany: Uni Bonn EAAP -1st December 2020 Various production and feeding systems • Part of animals finished on farm : • From 0 to 83% for Cow-calf systems • From 50 to 100% for Fatteners and Cow-calf + fattener systems • For fatteners, variability in age and weigh of animal purchased • Use of feed potentially in competition with human food: • Cow-calf: 0,2 to 4 kg DM / LU / Day • Fattener: 3,3 to 16,5 kg DM / LU / Day • Cow-calf + Fattener: 0,8 to 17,6 kg DM / LU / December 2020 Indicators used for food security analysis • HEP Human Edible Protein -Sources : Laisse et al 2018 ; INRA 2018 • UAA: area of the holding; • TL, nTL, LFP: resp. Tillable Land, non-Tillable Land and Land equivalent for the purchased feed -Sources : Ecoalim (Wilfart et al 2016) et Agribalise (Colomb et al 2015) • Me, Mi, C: resp. Meat, Milk and Crops sold 𝐹𝑎𝑟𝑚 𝑇𝐿 + 𝐿𝐹𝑃 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑝𝑟𝑜𝑑𝑢𝑐𝑒 𝑀𝑒 𝑘𝑔

  beef productionOn average, 78% of land used by cow-calf systems are non-tillable land vs.93% of tillable lands for fatteners EAAP -1st December 2020

  This research was made possible by funding from SusAn, an ERA-Net co-funded under European Union's Horizon 2020 research and innovation programme (www.era-SusAn.eu), under Grant Agreement n°696231 EAAP -1st December 2020