# Alpaca FGF5: Hypothetical Post-Transcriptional Readthrough Regulation in Skin Biopsies

Pallotti S.<fnref n="1" />, Pediconi D.<fnref n="1" />, Morelli M.B.<fnref n="2" />, Subramanian D.<fnref n="3" />, Molina M.G.<fnref n="4" />, Antonini M.<fnref n="5" />, Renieri C.<fnref n="2" /> and La Terza A.<fnref n="1" />

**Abstract.** Two different phenotypes are described in alpaca, identified as Suri and Huacaya which differ for the type of the fleece. The Huacaya fleece is characterised by compact, soft and highly crimped fibres while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). Total RNA purified from alpaca Suri and Huacaya skin biopsies, was reverse transcribed to cDNA using oligo dT priming and subsequently amplified by using FGF5 specific primers. Further, the resulting amplicons were cloned and sequenced. As previously identified in others mammals, our results also show that the alpaca FGF5 gene, give rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e. a transition C>T at position 499 downstream of the ATG codon) able to generate a Premature Stop Codon (PSC). The highly conserved nucleotide and aminoacid sequence after PSC suggested, and western blot analysis confirmed, a Readthrough event (RT). The analysis of mRNA sequence revealed motifs and characteristics that correlate with mRNA that undergoing RT (i.e. the higher “leakyness” of UGA Stop Codon, leakyness” due to the position -1, -2 in respect the UGA PSC with purines and +4 with pyrimidines, presence of suzesky sequences, pseudoknots in 3'UTR and tandem repeat Stop Codon after the canonical TAA, presence of long intron in the gene and long 3'UTR). To the best of our knowledge this is the first case of readthrough event on PSC reported for FGF5 gene.

**Resumen.** Existen dos fenotipos diferentes de alpaca llamados Suri y Huacaya que únicamente difieren en el tipo de vellón. El vellón de Huacaya se caracteriza por sus fibras compactas, suaves y altamente rizadas, mientras que el vellón de Suri es más largo, recto, menos rizado y más brillante. En nuestro estudio, el factor de crecimiento de fibroblastos 5 (FGF5) se investigó como un posible gen candidato para la longitud de la fibra de alpaca (Vicugna pacos). El ARN total purificado obtenido de biopsias de piel de alpaca Suri y

<footnote n="1">¹School of Bioscience and Veterinary Medicine, University of Camerino Italy.
Stefano.pallotti@unicam.it</footnote>

<footnote n="2">²School of Pharmacy, University of Camerino, Italy</footnote>

<footnote n="3">³Department of Marine Life Science, Jeju National University, South Korea</footnote>

<footnote n="4">⁴Facultad de Ciencias Agropecuarias .Universidad Nacional de Córdoba.
Universidad Católica de Córdoba, Argentina</footnote>

<footnote n="5">⁵ENEA C.R. Casaccia Biotec-Agro, S.M. di Galeria, 00060 Roma, Italy</footnote>
Huacaya, se transcribió de manera inversa a ADNc utilizando primers oligo(dT), y posteriormente se amplificó utilizando cebadores específicos de FGF5. Los amplicones resultantes fueron posteriormente clonados y secuenciados. Al igual que ne otros mamíferos los resultados también mostraron que el gen FGF5 de alpaca da lugar a una isoforma corta (FGF5S) y otra larga (FGF5). Curiosamente se observó una mutación puntual (es decir, una transición C> T en la posición 499 por debajo del codón ATG) en la isoforma larga, capaz de generar un codón STOP prematuro (PSC). Las secuencias de nucleótidos y de aminoácidos altamente conservados después del PSC sugirió, y el análisis de transferencia Western confirmó, un evento de lectura (RT). El análisis de la secuencia del ARNm reveló motivos y características que se correlacionan con el ARNm relativo al RT (es decir, la mayor "fuga" de UGA del codón STOP, "fuga" en la posición -1, -2 con respecto al UGA PSC en bases púricas y +4 en pirimidínicas, presencia de secuencias suzesky, pseudonodos en el extremo 3'UTR y repeticiones en tándem del codón STOP después del TAA canónico, presencia de un intrón largo del gen y un largo extremo 3'UTR). Hasta donde conocemos, éste es el primer caso de lectura del evento PSC citado para el gen FGF5.

**Keywords:** alpaca, suri, huacaya, FGF5

## Introduction

This paper contains the results of the research on FGF5 gene of the alpaca carried out at the Italian University of Camerino. The results are published in Pallotti et al. (2018).

Regulation of gene expression is found at different stages, one of which operates at the level of termination in protein synthesis. Sometimes translation continues reading through stop codons due to different kinds of events such as misreading by some natural non-cognate tRNAs or natural suppressor tRNA. This phenomenon, called readthrough (RT), allows the ribosome to pass through the termination codon in mRNA to continue translation until the next stop codon. Translational readthrough is widespread in viruses, fungi and Drosophila. However, its prevalence in mammals is not clear. (Schueren, F., & Thoms, S. 2016).

Fibroblast growth factor 5 gene (FGF5) belongs to the FGF family composed by at least 23 members that binds four receptors and perform different biological functions (Zhang et al., 2006; Beenken and Mohammadi, 2009; Sasaki et al., 2011). FGF5, first identified as human oncogene (Zhan et al., 1988), is expressed in different tissues such as the brain, heart, liver, spleen, muscle, rumen and skin (He et al., 2014; Zhang et al., 2015).

In mouse, FGF5 mRNA is highly expressed in the hair follicle as two isoforms, identified as FGF5 and FGF5S (Suzuki et al., 2000), and the latter is due to the alternative splicing of exon 2 (Hattori et al., 1996). Both isoforms, through binding to FGF receptor 1 and 2, regulate the hair follicle growth cycle during the anagen stage: FGF5 actively inhibits cell proliferation and the synthesis of hair fibers, while FGF5S antagonizes the inhibitory effects of FGF5 through competi-
tively binding the FGF receptors (Suzuki et al., 2000; Ota et al., 2002; He et al., 2016).

Previous studies showed how FGF5 gene is a crucial regulator of hair length in a wide variety of mammals such as human (Higgins et al., 2014), rabbit (Mulsant, 2010), cats (Kehler et al., 2007), dogs (Cadieu et al., 2009), cetaceans (Chen et al., 2013) and sheep (Hu et al., 2017; Li et al., 2017).

Alpaca (Vicugna pacos) is a South American camelid specialized in fiber pro-
duction (Bonavia, 1996). Based on the fleece type, two different phenotypes are
identified for alpaca, known as huacaya and suri. While the huacaya coat consists
of compact, soft and highly crimped fibers, the suri coat consists of straight, less-
crimped, lustrous and silky fibers (Antonini, 2010). An important phenotypic fea-
ture is the longer staple length of the alpaca suri compared to huacaya (Lupton &
McColl 2011; Ferguson et al., 2012). Several studies on segregation analysis were
carried out in order to understand the genetic control of fleece characteristics in
alpaca and, in particular, the mode of inheritance of the suri phenotype (Ponzoni et
al., 1997; Renieri et al., 2009; Sponenberg, 2010; La Manna et al., 2011). In a recent
study by Presciuttini et al (2010), the data supported a genetic model in which two
linked loci must simultaneously be homozygous for recessive alleles in order to
produce the huacaya phenotype, while the suri phenotype is determined by the
presence of a dominant allele at either locus.

The primary aim of the study was to investigate FGF5 transcript variability in
suri and huacaya alpaca and to assess possible association between the identified
transcript variants and differences in hair length showed by the two phenotypes.
Studying these transcripts, we found evidence of post-transcriptional readthrough
regulation.

**Materials and Methods**

A total of 20 animals, consisting of 10 suri and 10 huacaya, were sampled from the Quimsachata Experimental Station, Instituto Nacional de Innovacion Agraria (INIA), Peru. The same animals were used in two studies by Chandramohan et al., (2013; 2015).

The sample was structured in order to obtain two sets of records. The first set was represented by 3 families (9 individuals) and the second set composed of 11 genetically unrelated animals (five huacaya and six suri).

For collection of skin biopsies, trichotomy was performed using disposable
stainless-steel blades. Skin biopsies were obtained after antisepsis, and local anes-
thesia with 2% lidocaine was injected at the border of the sampling site. Two
samples were collected from each animal by disposable biopsy punch (8 mm diam-
eter) and were stored in All Protect (Qiagen). Then, the skin fragments were re-
moved from preservative reagents and stored in liquid nitrogen.

RNA and proteins were extracted using a AllPrep DNA/RNA/Protein Mini Kit (Quiagen, Germany), and the first strand cDNA was synthesized using Prim
ScriptTM Reverse Transcriptase (Takara Biotech, Japan), according to the manu-
facturer's protocol. To identify both FGF5 isoforms, 9 primers were designed
according to orthologous sequences published for other mammals retrieved from
the NCBI GenBank. Primers were designed to amplify the complete FGF5
isoforms. The first strand cDNA was synthesized with 1 µg of total RNA using
10 pmol oligo dt primer, 0.5 mM dNTPs, 1X RT buffer, 20 U RNase inhibitor and
200 U PrimScriptTM Reverse Transcriptase (Takara Biotech, Japan) in 20 µl total
reaction volume according to the manufacturer's instructions. The reaction mix-
ture was incubated for 45 min at 50 °C and then at 70 °C for 15 min.

The PCR reactions were performed using Bio-Rad thermal cycler (Bio-Rad) in
a 25-µl reaction final volume consisting of 0.5 U TaqDNA polymerase (Thermo
Scientific, Milano, Italy), 50 ng DNA, 1X GC Buffer (Takara, Saint-Germain-en-
Laye, France), 2 µM dNTPs, 10 µM each of forward and reverse primer, and
RNase free water. The cycle conditions were set as follows: initial denaturation at
95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing
for 20 s (at different T° depending on primers used) and extension at 72 °C for a
time depending on the length of expected amplicon. Amplicons were then separat-
ed by agarose gel electrophoresis and purified using Nucleospin columns (Ma-
cherey Nagel, Germany). The purified amplicons were cloned using CloneJET
PCR Cloning Kit (Takara, Saint-Germain-en-Laye, France) and sequenced by
StarSEQ GmbH (Mainz, Germany).

ExactSTART Eukaryotic mRNA 5'- & 3'-RACE Kit (Epicentre Bio) was used for the identification of the 5'UTR while the amplification of 3' UTR was performed using modified oligo (dt). Sequences were visualized with sequencing chromatogram trace viewer FinchTV v. 1.4.0 (Biosoft, Cambridge). The FGF5 nucleotide sequences were determined using NCBI BLAST (www.http://blast.ncbi.nlm.nih.gov/Blast). The amino acid sequence was deduced by the Translate tool of ExPASy (http://www.web.expasy.org/translate). Alignment of nucleotide and amino acid sequences was conducted using MAFFT version 7 (http://mafft.cbrc.jp/alignment/server).

The protein concentration was determined by Bradford assay (Bradford, 1976). Equal amounts of protein lysates (30 µg) were electrophoretically separated on SDS-PAGE 12 % PA gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5 % non-fat dried milk for 1 h at room temperature and then incubated at room temperature for 1 h with a monoclonal anti-FGF5 antibody No. H00002250-M01 produced in mouse (Abnova), binding the epitope AA 159-268 of FGF5 protein. After three washes, the blots were incubated with an anti-Mouse IgG anti-body produced in rabbit (1 µg/ml, Sigma) for 1 h at room temperature and subsequently visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA), according to the manufacturer's instructions.

To predict eukaryotic selenoproteins and SECIS elements along FGF5 nucleo-
tide sequences, we used Seblastian tool on Selenoprotein Prediction Server
(http://seblastian.crg.es/) (Mariotti et al., 2013). To predict and visualize the FGF5 RNA secondary structures and pseudoknot, the online versions of DotKnot (Sperschneider and Datta 2010; Sperschneider et al., 2011 (http://dotknot.csse.uwa.edu.au/) and PseudoViewer (Byun and Han 2006) (http://pseudoviewer.inha.ac.kr/PVWebService/WSPV_input.asp) were used.

## Results and Discussion

### cDNA analysis of the FGF5 gene

We amplified and sequenced the complete coding region by PCR using skin RNA from ten suri and ten huacaya.

The alpaca FGF5 gene gave rise to two transcripts similar to those of other mammals represented by the short (FGF5S) and the long isoform (FGF5), which were present in both suri and huacaya phenotypes.

In both phenotypes, the FGF5S transcript presented an open reading frame of 375 bp, encoding a protein of 125 amino acids (Fig. 1), while the FGF5 transcript presented an open reading frame of 498 bp due to a premature termination codon (PTC), which was predicted to be translated into a protein of 166 amino acids (Fig. 2). The FGF5s showed two 3'UTRs of 713 and 542 bp, respectively. In contrast, a single 3'UTR of 713 bp has been identified for the FGF5 isoform. FGF5s showed a 166 bases 5'UTR while FGF5 showed two different 5'UTRs of 166 and 205 bp (see supplementary material published in Pallotti et al., 2018).

Concerning the FGF5 transcript in both phenotypes, downstream to the premature termination codon in position 499, the sequence was highly conserved until the next stop signal in position 811, as also seen in other mammals (Fig.3). The highly conserved and functional sequence of amino acids downstream to the premature termination codon (PTC) suggested RT phenomenon and the synthesis of an entire FGF5 protein. To evaluate the role of the FGF5 gene for the hair length in alpaca, we compared the cDNA sequences from suri and huacaya. This analysis could not detect polymorphisms in either the coding regions or in the UTRs, which could explain the different hair length in the two phenotypes.
<table><caption>**Figure 1:** FGF5 Short isoform nucleotides and amino acids sequence. The sequence was taken from accession MF497582. The start codon and the stop codon are highlighted in gray.</caption>
  <tbody>
    <tr>
      <td>gttcgggtggcctctctctctctcccccctctccagtcctctttcccaaggctatgtccac</td>
      <td>-106</td>
    </tr>
    <tr>
      <td>ccggtcggcgagggggcagccagaggtacgcagccgcgcgggggctgcvggacccca</td>
      <td>-46</td>
    </tr>
    <tr>
      <td>gaaccagccctacaagatgcgcttaggaccccccgcggctggaagaATGagcttgtccttc</td>
      <td>15</td>
    </tr>
    <tr>
      <td>MSLSF</td>
      <td>5</td>
    </tr>
    <tr>
      <td>ctcctcctcctcttgctcagccacctgatcctcagcgcctgggctgcggggagaagcgc</td>
      <td>75</td>
    </tr>
    <tr>
      <td>LLLLLSHLILSASWARGEKR</td>
      <td>25</td>
    </tr>
    <tr>
      <td>ctcgtccccaaagggcaaccgggaccggctgccactgctaggaaccgggagagccgcagc</td>
      <td>135</td>
    </tr>
    <tr>
      <td>LVPKGOFGPAATARNPGGAS</td>
      <td>45</td>
    </tr>
    <tr>
      <td>agcagctggagcagcagaagcacgacgtcttcctcttcttcctctgcctcctcctcccc</td>
      <td>195</td>
    </tr>
    <tr>
      <td>SSWSSRSTTSSSSSASSSP</td>
      <td>65</td>
    </tr>
    <tr>
      <td>gcggctctctggaagccaaggaagcggcttggagcagaccagtttccagtggacccccc</td>
      <td>255</td>
    </tr>
    <tr>
      <td>AASLGSGSLGLEQTSFQWSF</td>
      <td>85</td>
    </tr>
    <tr>
      <td>tcggggccgggaccggcagcctctactgcagagtgggcatcggtttccatctgcagatc</td>
      <td>315</td>
    </tr>
    <tr>
      <td>SGRRTGSLYCRVGIGFHLQI</td>
      <td>105</td>
    </tr>
    <tr>
      <td>taccggatggcaaagtcaatggctccccacgaagccaatatgttaagccagatttacaga</td>
      <td>375</td>
    </tr>
    <tr>
      <td>YPDGVNGSHEANMLSQIYR</td>
      <td>125</td>
    </tr>
    <tr>
      <td>AGctgcaagttcagggagtgatttcaagagaacagctataaacacctatgcctcagtgtat</td>
      <td>435</td>
    </tr>
    <tr>
      <td>stop</td>
      <td></td>
    </tr>
    <tr>
      <td>acacagaaactgagaacacggggcgggagtgtacgtggccctgaacaagagggggaaagc</td>
      <td>495</td>
    </tr>
    <tr>
      <td>taaacggggctgcagccccccgggttaaaccccagcacgtctccactcactttctgccaag</td>
      <td>555</td>
    </tr>
    <tr>
      <td>atttaagcaatcgagcagccggaaactttctttcacagttactgttcctgaaaagaaaaa</td>
      <td>615</td>
    </tr>
    <tr>
      <td>accacctaaccccatcaagccaaaggtccccctttccacatctcggagaagccccagcac</td>
      <td>675</td>
    </tr>
    <tr>
      <td>ggtgaagtcagactcaagtttcgctttggataattatcatcttggccgtgaggaaccgc</td>
      <td>735</td>
    </tr>
    <tr>
      <td>gccgtccccctcaagagtttccctaggtgttttttagtgccctgaagacagagtttcggaca</td>
      <td>795</td>
    </tr>
    <tr>
      <td>cagctcatctctactacactgcatgaagccaggtcatttgttcagtctgactgaaac</td>
      <td>855</td>
    </tr>
    <tr>
      <td>aaaaacgttttctgataggaactgaaactggaattctttgtaccaacatgaagagcgcact</td>
      <td>915</td>
    </tr>
    <tr>
      <td>gcttcagttcagtgccacataaagccttttgctttatgcttgacgcatcctggtactctg</td>
      <td>975</td>
    </tr>
    <tr>
      <td>gttcagaaagttgaatgatgggaaactatgtacttttctgacttttacagatcaacctga</td>
      <td>1035</td>
    </tr>
    <tr>
      <td>aaaaacatacatggtcacattttttatttttggtttccaaagaatattttgcagataa</td>
      <td>1095</td>
    </tr>
    <tr>
      <td>aaatattttattaacttttgttttttgtttttttaaaaaaacatacctctgcattgagca</td>
      <td>1155</td>
    </tr>
    <tr>
      <td>tattttcttacttttattattttaattaataagacataaaacaaccattgaaatacagtt</td>
      <td>1215</td>
    </tr>
    <tr>
      <td>tatgaattataaatatgtttatagcccatttgaacaagtggatttccctttcatttttc</td>
      <td>1275</td>
    </tr>
    <tr>
      <td>ttattcattgcacttttaatttttattattattattatttttaccatacctcagatcatg</td>
      <td>1335</td>
    </tr>
    <tr>
      <td>taagtttagtttacatcttaaaatgtttaaattctctcatagcaaaaaaaaaaaaaa</td>
      <td>1395</td>
    </tr>
  </tbody>
</table>
<table>
  <tbody>
    <tr>
      <td>actggcgttataaatccagtgccagccgcgagatcgttcggtggtcctctctctctc</td>
      <td>-145</td>
    </tr>
    <tr>
      <td>tccccctctccagtcctctttcccaaggctatgtccacccgtgoggcaggggggcag</td>
      <td>-85</td>
    </tr>
    <tr>
      <td>cgccagaggtcacgcagccgcgoggggctgoggcagcccagaaccagccctacaagatgcg</td>
      <td>-25</td>
    </tr>
    <tr>
      <td>cttaggaccccccgcgctggaagaatAGagcttgtccttcctcctcctcctcttgtcagc</td>
      <td>36</td>
    </tr>
    <tr>
      <td>M S L S F L L L L S</td>
      <td>12</td>
    </tr>
    <tr>
      <td>cacctgatcctcagcgcctgggctcgcgggagaagcgccctgtccccaaagggcaacc</td>
      <td>96</td>
    </tr>
    <tr>
      <td>H L I L S A W A R G E K R L V P K G Q P</td>
      <td>32</td>
    </tr>
    <tr>
      <td>ggaccgctgccactgctaggaaccggaggcgcagcagcagctggagcagcagaagc</td>
      <td>156</td>
    </tr>
    <tr>
      <td>G P A A T A R N P G G A S S S W S S R S</td>
      <td>52</td>
    </tr>
    <tr>
      <td>acgacgttcctcctcttcctctgcctcctcctcccccgcggcttctctggaagccaa</td>
      <td>216</td>
    </tr>
    <tr>
      <td>T T S S S S S A S S P A A S L G S Q</td>
      <td>72</td>
    </tr>
    <tr>
      <td>ggaaccgcttggagcagaccagttccagtggagcccctcgggocgccgaccggcagc</td>
      <td>276</td>
    </tr>
    <tr>
      <td>G S G L E Q T S F Q W S P S G R R T G S</td>
      <td>92</td>
    </tr>
    <tr>
      <td>ctctactgcagagtgggcatcggttccatctgcagatctaccggatggcaagtcaat</td>
      <td>336</td>
    </tr>
    <tr>
      <td>L Y C R V G I G F H L Q I Y P D G K V N</td>
      <td>112</td>
    </tr>
    <tr>
      <td>ggctccacgaagccaaatatgtaagtatttttggaatatttgctgtctcaggggatt</td>
      <td>396</td>
    </tr>
    <tr>
      <td>G S H E A N M L S I L E I F A V S Q G I</td>
      <td>132</td>
    </tr>
    <tr>
      <td>gtaggaatacaggagttttcaccaacaatttttacgatgtcaaaaaaaggaaaactc</td>
      <td>456</td>
    </tr>
    <tr>
      <td>V G I R G V F S N K F L A M S K K G K L</td>
      <td>152</td>
    </tr>
    <tr>
      <td>catgcaagtgccagatttacagatgactgcaagttcagggagTGatttcaagagaacagc</td>
      <td>516</td>
    </tr>
    <tr>
      <td>H A S A R F T D D C K F R E - F Q E N S</td>
      <td>172</td>
    </tr>
    <tr>
      <td>tataacacctatgcctcagtgatacacagaactgagaacacgggogggagtgtgacagtg</td>
      <td>576</td>
    </tr>
    <tr>
      <td>Y N T Y A S V I H R T E N T G R E W Y V</td>
      <td>192</td>
    </tr>
    <tr>
      <td>gccctgaacaagaggggaaagctaaacgggctgcagccccccggtttaaacccccagcac</td>
      <td>636</td>
    </tr>
    <tr>
      <td>A L N K R G K A K R G C S P R V K P Q H</td>
      <td>212</td>
    </tr>
    <tr>
      <td>gtctccactactttctgccaagatittaagcaatcggagcagccggaactttctttcac</td>
      <td>696</td>
    </tr>
    <tr>
      <td>V S T H F L P R F K Q S E Q P E L S F T</td>
      <td>232</td>
    </tr>
    <tr>
      <td>gttactgttcctgaaaagaaaaaacacctacaaccccatcaagccaaaggtccccctttcc</td>
      <td>756</td>
    </tr>
    <tr>
      <td>V T V P E K K K P P N P I K P K V P L S</td>
      <td>252</td>
    </tr>
    <tr>
      <td>acatctcggagaagccccagcacggtaagtacagactaagtttgcctttggaTAAatta</td>
      <td>816</td>
    </tr>
    <tr>
      <td>T S R R S P S T V K Y R L K F R F G stop</td>
      <td>270</td>
    </tr>
    <tr>
      <td>tcatcttggccgtgaggaaccgcgccgtccctcaagagtttccctaggtgtttttatgtg</td>
      <td>876</td>
    </tr>
    <tr>
      <td>ccctgaagacagagtttcggacacagcttcatctctactacactgcatgaagccaggtc</td>
      <td>936</td>
    </tr>
    <tr>
      <td>atttgttcagtcctgactgaaacaaaaacgttttctgataggaactgaaactggaattctt</td>
      <td>996</td>
    </tr>
    <tr>
      <td>tgtaccaacatgaagagcgcactgccttcagttcagtgccacataaagccttttgctttat</td>
      <td>1056</td>
    </tr>
    <tr>
      <td>gcttgacgcatcctggtactctggttccagaaagttgaatgatgggaaactatgtactttt</td>
      <td>1116</td>
    </tr>
    <tr>
      <td>ctgacttttacagatcaacctgaaaaaacatacatggtacatttttttatttttggtttcc</td>
      <td>1176</td>
    </tr>
    <tr>
      <td>aaagaatattttgatgcagataaaaatatatttattaactttttgttttttgtttttttaaa</td>
      <td>1236</td>
    </tr>
    <tr>
      <td>aaaacatacctctgcattgagcatatttttctacttttattattttaattaataagacat</td>
      <td>1296</td>
    </tr>
    <tr>
      <td>aaaacaccattgaatacacagtttatgaattataaatatgtttatagcccatttgtaaca</td>
      <td>1356</td>
    </tr>
    <tr>
      <td>agtgatttccctttcattttttctattcattgcacttttaatttttattattattatta</td>
      <td>1416</td>
    </tr>
    <tr>
      <td>tttttaccatacctcagatcatgtaagtttagtttacaaatctttaaaatgtttaaattct</td>
      <td>1476</td>
    </tr>
    <tr>
      <td>cttcatagcaaaaaaaaaaaaa</td>
      <td>1501</td>
    </tr>
  </tbody>
</table>

**Figure 2:** FGF5 Long isoform nucleotides and amino acids sequence. The sequence was taken from accession MF497584. The start codon, the TGA PTC, the TAA canonical stop codon and the Skuzeski sequences of the form CARYYA and the “CAGCAGCA” sequence by Beier and Grimm (2001) are highlighted in gray (for the colored version see Pallotti et al., 2018). The (-) symbol at position 167 of the protein sequence indicates the unknown amino acid.
<table><caption>**Figure 3:** Alignment of FGF5 proteins sequences in different species. Identical amino acids among the two sequences are indicated by (*), whereas those with high or low similarity are indicated by (:) and (.), respectively. (A) FGF5 Long Sequences. The (-) symbol highlighted in gray at position 167 of the Vicugna pacos protein sequence indicates the unknown amino acid. (B) FGF5 Short Sequences.</caption><thead><tr><th></th><th></th><th></th></tr></thead><tbody><tr><td>A)</td><td></td><td></td></tr><tr><td>Ovis aries</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTVSSSSSP</td><td>60</td></tr><tr><td>Capra hircus</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTASSSSSP</td><td>60</td></tr><tr><td>Bos taurus</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTATSSSSSP</td><td>60</td></tr><tr><td>Vicugna pacos</td><td>MSLSFLLLLLSLHILSAWARGEKRLVPKGQPGPAATARNPGGASSWSSRSTTSSSSSP<br>*********.*********.********* ********* ** **..,****,</td><td>60</td></tr><tr><td>Ovis aries</td><td>ASSSSSAASRGGPGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Capra hircus</td><td>ASSSSSAASRGGPGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Bos taurus</td><td>ASSSSSAASRGGPGSLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Vicugna pacos</td><td>ASSSPAASLGSGSGLEQTSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML<br>****..** . .**..**..*********<br>*********.*********.********* *</td><td>120</td></tr><tr><td>Ovis aries</td><td>SILEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNYTAYSAI</td><td>180</td></tr><tr><td>Capra hircus</td><td>SILEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNYTAYSAI</td><td>180</td></tr><tr><td>Bos taurus</td><td>SILEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNYTAYSAI</td><td>180</td></tr><tr><td>Vicugna pacos</td><td>SILEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASARFTDDCKFRERFQENSYNYTAYSAI<br>*********<br>*********.*********.********* *</td><td>180</td></tr><tr><td>Ovis aries</td><td>HRTTEKTGREWYVALNKRGKAKRGCSPRVKQPHVSTHFLPRFKQSEQPELSFTVTPEKKK</td><td>240</td></tr><tr><td>Capra hircus</td><td>HRTTEKTGREWYVALNKRGKAKRGCSPRVKQPHVSTHFLPRFKQSEQPELSFTVTPEKKK</td><td>240</td></tr><tr><td>Bos taurus</td><td>HRTTEKTGREWYVALNKRGKAKRGCSPRVKQPHVSTHFLPRFKQLEQPELSFTVTPEKKK</td><td>240</td></tr><tr><td>Vicugna pacos</td><td>HRTENTGREWYVALNKRGKAKRGCSPRVKQPHVSTHFLPRFKQSEQPELSFTVTPEKKK<br>****..*********<br>*********.********* *</td><td>240</td></tr><tr><td>Ovis aries</td><td>PPNPLPKVPULSAPRRSPNTVKYRLKFRFG</td><td>270</td></tr><tr><td>Capra hircus</td><td>PPNPLPKVPULSAPRRSPNTVKYRLKFRFG</td><td>270</td></tr><tr><td>Bos taurus</td><td>PPNPLPKVPULSAPRRSPNTVKYRLKFRFG</td><td>270</td></tr><tr><td>Vicugna pacos</td><td>PPNPLPKVPULSTSRRSPSTVKYRLKFRFG<br>****..*********.********* *</td><td>270</td></tr><tr><td>B)</td><td></td><td></td></tr><tr><td>Ovis aries</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTVSSSSSP</td><td>60</td></tr><tr><td>Capra hircus</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTASSSSSP</td><td>60</td></tr><tr><td>Bos taurus</td><td>MSLSFLLLLFLSHLILSAWAQGEKRLAPKGQPGPAATERNPGGASSRRSSSSTATSSSSSP</td><td>60</td></tr><tr><td>Vicugna pacos</td><td>MSLSFLLLLLSLHILSAWARGEKRLVPKGQPGPAATARNPGGASSWSSRSTTSSSSSP<br>*********.*********.********* ** ********* ** **..,****,</td><td>60</td></tr><tr><td>Ovis aries</td><td>ASSSSSAASRGGPGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Capra hircus</td><td>ASSSSSAASRGGPGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Bos taurus</td><td>ASSSSSAASRGGPGSLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML</td><td>120</td></tr><tr><td>Vicugna pacos</td><td>ASSSPAASLGSGSGLEQTSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVGNSHEANML<br>****..** . .**..**..*********<br>*********.*********.********* *</td><td>120</td></tr><tr><td>Ovis aries</td><td>SQIYR</td><td>125</td></tr><tr><td>Capra hircus</td><td>SQIYR</td><td>125</td></tr><tr><td>Bos taurus</td><td>S</td><td>121</td></tr><tr><td>Vicugna pacos</td><td>SQIYR<br>*</td><td>125</td></tr></tbody></table>
<figure><img src="image_45.png" /><figcaption>**Figure 4:** Western blot analysis of FGF5. The gray arrowhead indicates the FGF5 protein. Lane M, molecular weight marker; Lane 1, huacaya; Lane 2, suri.</figcaption></figure>

### Analysis of the FGF5 protein

To test the possibility of the translation of the entire FGF5 protein in alpaca, we performed the western blotting analysis starting from the proteins extracted from the skin biopsies.

The western blotting result showed FGF5 proteins of approximately 30.4 kDa in both suri and huacaya, which was consistent with the predicted sizes of 267 aa based on the cDNA sequence data (Fig.4). The presence of the predicted 30.4 kDa protein was the main evidence of a stop codon readthrough in alpaca.

### Readthrough signals analysis

As shown in Figure 2, the alpaca FGF5 mRNA presents characteristics that make the transcript a good RT candidate. The efficiency of RT depends on a variety of factors, including the type of the termination codon and its surrounding mRNA sequence context (Dabrowski et al., 2015), the presence of sequence elements (Skuzesky et al., 1991; Harrell et al., 2002; Beier and Grimm 2001), a long 3'UTR (Jungreis et al., 2011), which reduces termination efficiency, and finally the secondary structure of the transcript (Bertram et al., 2001, Firth et al., 2011).

First, the identity of the stop codon is crucial (Dabrowski et al., 2015) for RT. In genes undergoing RT, the UGA PTC is present 10 times more than others stop codons, as their order of “leakiness” in eukaryotes is UGA>UAG>UAA (Firoozan et al., 1991; Jungreis et al., 2011; Dabrowski et al., 2015).
The efficiency of suppression of PTC varies with the nucleotides and sequence motifs surrounding the stop codon (McCaughan et al., 1995; Pedersen et al., 1991). The nucleotide immediately downstream the UGA PTC influences the translational termination in the expression of mammalian genes. This base compromises the efficiency of suppression and positively influences the RT with different forces: C > U > G > A (Jungreis et al., 2011). In alpaca, the FGF5 transcript U was the nucleotide at position +4 after UGA. Additionally, the nucleotides upstream to the PTC play an essential role in the termination of protein synthesis. According to Dabrowski et al. (2015), Bonetti et al. (1995), Tork et al. (2004) and Jungreis et al. (2011), the presence of adenine at -2 and purine at -1 positions immediately upstream to the PTC is a determinant for RT.

Special sequence elements are known to affect translation termination while positively influencing RT. The FGF5 mRNA presented four Skuzeski sequences (Skuzeski et al., 1991), a hexanucleotide sequences of the form CARYYA (R= purine; Y=pyrimidine), of which one is adjacent to the stop codon. According to Skuzeski et al. (1991) and to Harrel et al. (2002), these sequences could influence the binding of release factor or directly interact with the ribosome stimulating RT in different RNA viruses and eukaryotic organisms. Likewise, the “CAGCAGCA” sequence at 128 bp downstream to ATG is known to facilitate readthrough of a stop codon as proposed by the paper by Beier and Grimm (2001).

The physical separation of the PTC from the poly (A) tail due to a long 3' UTR can directly lead to RT mechanism (Jungreis et al., 2011). An increase in the distance between a PTC and the poly(A) leads to a reduction in the fidelity of termination due to reduced interaction between eRF3, a component of the termination complex, and the poly(A)-binding protein (Amrani et al., 2004; Kobayashi et al., 2004). In FGF5 transcript, the PTC was 1,050 bp upstream to the poly (A), likely leading to inefficient termination and consequent RT.

Finally, RNA secondary structure can play a major role in the regulation of translational termination. In eukaryotes, the UGA stop codons along nucleotide sequences can be translated as selenocysteines if mRNA harbors the secis sequence (selenocystein elements), which is a secondary structure involved in non sense codon suppression with cognate tRNAsec (Walczak et al., 1996). From the computational scanning of FGF5 mRNA, no secis elements were detected. RT can also be stimulated by the ability of the transcript to form pseudoknot structures that pause the ribosome to induce frameshift (Somogyi et al., 1993), or by distorting the mRNA structure to favor tRNA interaction over eRF1 binding at the A-site in the termination step (Bertram et al., 2001, Firth et al., 2011).

In FGF5 mRNA, seven hypothetical pseudoknots were found starting from position 28 downstream to UGA PTC (these results are available in Pallotti et al., 2018). The more stable pseudoknot is found from position 59 to 148 due to its lowest estimated free energy (-43.46 kcal/mol).
**Conclusions**

Taken together, our results demonstrate a post-transcriptional readthrough regulation in FGF5 gene of alpaca.

In a pool of FGF5 mRNA, RT allows synthesis of different proportions of functional extended polypeptide and truncated protein (Harrel et al., 2002), this phenomenon it may act as a crucial regulatory mechanism of FGF5 gene expression in the development of the different alpaca phenotypes. As suggested by Beier and Grimm (2001) and Roy et al. (2015), during the polypeptide synthesis, the UGA PTC may mediate translational termination or trigger incorporation of arginine, cysteine or tryptophan.

Differences in steric hindrance, charge and polarity of these three amino acids may affect the functionality of FGF5 in its role of binding and activating the FGFRs; in fact the Arg is the wild type amino acid at position 167 aa immediately upstream to the Glu residue which is the amino acid fundamental in the receptor binding (Plotnikov et al., 2000) of FGF family proteins. This regulation of FGF5 can in turn arrest the follicle development during the anagen phase or retard its progression, thus explaining in part the different hair length between suri and huacaya. Recently, the same mutation was described for the long-fiber llama (Daverio et al., 2017). These data suggest that a common domestic fiber-producing ancestor could be shared by the llama and alpaca. Further studies will be required to determine the real role of this mechanism in the translation of FGF5, in addition to the possible differences in the incorporation of the aa 167 between suri and huaca.

**Acknowledgments.** The authors would like to thank Loro Piana S.r.l. for their financial support. DS was financially supported by a "Young Indian Research Fellowship" through the Italian Minister of University and Research (MIUR) to ALT.

**References**

Amrani, N.; Ganesan, R.; Kervestin, S.; Mangus, D. A.; Ghosh, S.; Jacobson, A. 2004, A faux 3'-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay. Nature, 432(7013), 112-118

Antonini, M. 2010, Hair follicle characteristics and fibre production in South American camelids. Animal, 4(09), 1460-1471

Beenken, A.; Mohammadi, M. 2009, The FGF family: biology, pathophysiology and therapy. Nature reviews Drug discovery, 8(3), 235-253

Beier, H.; Grimm, M. 2001, Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic acids research, 29(23), 4767-4782

Bertram, G.; Innes, S.; Minella, O.; Richardson, J. P.; Stansfield, I. 2001, Endless possibilities: translation termination and stop codon recognition. Microbiology, 147(2), 255-269

Bonavia, D. 1996, Los Camelidos Sudamericanos (South American Camelids). IFEA-UPCH-Conservation International, Lima, Peru
Bonetti, B.; Fu, L.; Moon, J.; Bedwell, D. M. 1995, The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences insaccharomyces cerevisiae. Journal of molecular biology, 251(3), 334-345

Cadieu, E.; Neff, M. W.; Quignon, P.; Walsh, K.; Chase, K.; Parker, H. G.; Wong, A. 2009, Coat variation in the domestic dog is governed by variants in three genes. Science, 326(5949), 150-153

Chandramohan, B.; Renieri, C.; La Manna, V.; La Terza, A. 2013, The alpaca agouti gene: genomic locus, transcripts and causative mutations of eumelanic and pheomelanic coat color. Gene, 521(2), 303-310

Chandramohan, B.; Renieri, C.; La Manna, V.; La Terza, A. 2015, The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies. The Scientific World Journal, 2015. ISO 690

Chen, Z.; Wang, Z.; Xu, S.; Zhou, K.; Yang, G. 2013, Characterization of hairless (Hr) and FGF5 genes provides insights into the molecular basis of hair loss in cetaceans. BMC evolutionary biology, 13(1), 1

Dabrowski, M.; Bukowy-Bieryllo, Z.; Zietkiewicz, E. 2015, Translational readthrough potential of natural termination codons in eucaryotes-The impact of RNA sequence. RNA biology, 12(9), 950-958

Ferguson, M. B.; McGregor, B. A.; Behrendt, R. 2012, Relationships between skin follicle characteristics and fibre properties of suri and huacaya alpacas and Peppin Merino sheep. Animal production science, 52(7), 442-447

Firoozan, M.; Grant, C. M.; Duarte, J. A.; Tuite, M. F. 1991, Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay. Yeast, 7(2), 173-183

Firth, A. E.; Wills, N. M.; Gesteland, R. F.; Atkins, J. F. 2011, Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element. Nucleic acids research, gkr224

Firth, A. E.; Wills, N. M.; Gesteland, R. F.; Atkins, J. F. 2011, Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element. Nucleic acids research, gkr224

Harrell, L.; Melcher, U.; Atkins, J. F. 2002, Predominance of six different hexanucleotide recoding signals 3' of read-through stop codons. Nucleic acids research, 30(9), 2011-2017

Hattori, Y.; Yamasaki, M.; Itoh, N. 1996, The rat FGF-5 mRNA variant generated by alternative splicing encodes a novel truncated form of FGF-5. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1306(1), 31-33

He, X. L.; Yuan, C.; Chen, Y. L. 2014, Isolation, characterization, and expression analysis of FGF5 isoforms in cashmere goat. Small Ruminant Research, 116(2), 111-117

He, X.; Chao, Y.; Zhou, G.; Chen, Y. 2016, Fibroblast growth factor 5-short (FGF5s) inhibits the activity of FGF5 in primary and secondary hair follicle dermal papilla cells of cashmere goats. Gene, 575(2), 393-398

Higgins, C. A.; Petukhova, L.; Harel, S.; Ho, Y. Y.; Drill, E.; Shapiro, L.; Christiano, A. M. 2014, FGF5 is a crucial regulator of hair length in humans. Proceedings of the National Academy of Sciences, 111(29), 10648-10653. ISO 690

Hu, R.; Fan, Z. Y.; Wang, B. Y.; Deng, S. L.; Zhang, X. S.; Zhang, J. L.; Lian, Z. X. 2017, RAPID COMMUNICATION: Generation of knockout sheep via the CRISPR/Cas9 system. Journal of Animal Science, 95(5), 2019-2024
Jungreis, I.; Lin, M. F.; Spokony, R.; Chan, C. S.; Negre, N.; Victorsen, A.; Kellis, M. 2011, Evidence of abundant stop codon readthrough in Drosophila and other metazoa. Genome research, 21(12), 2096-2113

Kehler, J. S.; David, V. A.; Schäffer, A. A.; Bajema, K.; Eizirik, E.; Ryugo, D. K.; Menotti-Raymond, M. 2007, Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats. Journal of Heredity, 98(6), 555-566

Kobayashi, T.; Funakoshi, Y.; Hoshino, S. I.; Katada, T. 2004, The GTP-binding release factor eRF3 as a key mediator coupling translation termination to mRNA decay. Journal of Biological Chemistry, 279(44), 45693-45700

La Manna, V.; La Terza, A.; Ghezzi, S.; Saravanaperumal, S.; Apaza, N.; Huanca, T.; Renieri, C. 2011, Analysis of genetic distance between Peruvian Alpaca (Vicugna Pacos) showing two distinct fleece phenotypes, suri and huacaya, by means of microsatellite markers. Italian Journal of Animal Science, 10(4), e60

Li, W. R.; He, S. G.; Liu, C. X.; Zhang, X. M.; Wang, L. Q.; Lin, J. P.; Liu, M. J. 2017, Ectopic expression of FGF5s induces wool growth in Chinese merino sheep. Gene.

Lupton, C. J.; McColl, A. 2011, Measurement of luster in suri alpaca fiber. Small ruminant research, 99(2), 178-186

McCaughan, K. K.; Brown, C. M.; Dalphin, M. E.; Berry, M. J.; Tate, W. P. 1995, Translational termination efficiency in mammals is influenced by the base following the stop codon. Proceedings of the National Academy of Sciences, 92(12), 5431-5435

Mulsant, P. 2010, A note on linkage between the angora and fgf5 genes in rabbits. World Rabbit Science, 12(1), 01-06

Ota, Y.; Saitoh, Y.; Suzuki, S.; Ozawa, K.; Kawano, M.; Imamura, T. 2002, Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation. Biochemical and biophysical research communications, 290(1), 169-176

Pallotti, S.; Pediconi, D.; Subramanian, D.; Molina, M. G.; Antonini, M.; Morelli, M. B.; La Terza, A. 2018, Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca. Gene, 647, 121-128

Pedersen, W. T.; Curran, J. F. 1991, Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1. Journal of molecular biology, 219(2), 231-241

Plotnikov, A. N.; Hubbard, S. R.; Schlessinger, J.; Mohammadi, M. 2000, Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity. Cell, 101(4), 413-424

Presciuttini, S.; Valbonesi, A.; Apaza, N.; Antonini, M.; Huanca, T.; Renieri, C. 2010, Fleece variation in alpaca (Vicugna pacos): a two-locus model for the suri/huacaya phenotype. BMC genetics, 11(1), 70

Renieri, C.; Valbonesi, A.; La Manna, V.; Antonini, M.; Asparrin, M. 2009, Inheritance of suri and huacaya type of fleece in Alpaca. Italian Journal of Animal Science, 8(1), 83-91

Roy, B.; Leszyk, J. D.; Mangus, D. A.; Jacobson, A. 2015, Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3. Proceedings of the National Academy of Sciences, 112(10), 3038-3043

Sasaki, S.; Ohta, H.; Nakajima, Y.; Konishi, M., Miyake, A.; Itoh, N. 2011, The FGF Family in Humans, Mice, and Zebrafish: Development, Physiology, and Pathophysiology. In Human Genetic Diseases. InTech
Schueren, F.; Thoms, S. 2016, Functional translational readthrough: a systems biology perspective. PLoS genetics, 12(8), e1006196

Skuzeski, J. M.; Nichols, L. M.; Gesteland, R. F.; Atkins, J. F. 1991, The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. Journal of molecular biology, 218(2), 365-373

Somogyi, P.; Jenner, A. J.; Brierley, I.; Inglis, S. C. 1993, Ribosomal pausing during translation of an RNA pseudoknot. Molecular and cellular biology, 13(11), 6931-6940

Suzuki, S.; Ota, Y.; Ozawa, K.; Imamura, T. 2000, Dual-mode regulation of hair growth cycle by two Fgf-5 gene products. Journal of investigative dermatology, 114(3), 456-463

Tork, S.; Hatin, I.; Rousset, J. P.; Fabret, C. 2004, The major 5' determinant in stop codon read-through involves two adjacent adenines. Nucleic acids research, 32(2), 415-421

Walczak, R.; Westhof, E.; Carbon, P.; Krol, A. 1996, A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. Rna, 2(4), 367-379

Zhan, X. I.; Bates, B. R. I. A. N.; Hu, X. G.; Goldfarb, M. I. T. C. H. E. L. L. 1988, The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Molecular and Cellular Biology, 8(8), 3487-3495

Zhang, L.; He, S.; Liu, M.; Liu, G.; Yuan, Z.; Liu, C.; Li, W. 2015, Molecular cloning, characterization, and expression of sheep FGF5 gene. Gene, 555(2), 95-100

Zhang, X.; Ibrahimi, O. A.; Olsen, S. K.; Umemori, H.; Mohammadi, M.; Ornitz, D. M. 2006, Receptor specificity of the fibroblast growth factor family. The complete mammalian fgf family. Journal of Biological Chemistry, 281(23), 15694-15700
# Alpines Steinschaf (Alpine Stonesheep)

## Christian Mendel¹, Isabelle A. Ketterle²

¹Bavarian State Research Centre for Agriculture, Prof.-Dürrwaechter-Platz 1 D-85586 Poing-Grub

E-Mail: christian.Mendel@lfl.bayern.de

²Kollektion der Vielfalt, D-73119 Zell

E-Mail: isabelle.ketterle@gmx.de

**Abstract.** A breeding programme was established in 2010 to conserve the Alpine Stonesheep, a rare breed originating in the Neolithicum. Recently, an Alp index was developed as new selection criteria, involving different breeding lines in a cross-border project. The number of Alpine Stonesheep increased from 120 sheep in 2000 to 800 sheep in 2016. The conservation program also includes wool examinations, development of high-quality products made from the wool of this sheep breed, and the Alpine Stonesheep information centre in the Berchtesgardener Land /Bavaria, the natural habitat of the Alpine Stonesheep.

**Resumen.** En 2010 se estableció un programa de conservación genética para la raza autóctona Alpine Stonesheep, una raza que procede en origen de una raza neolítica. Recientemente, se ha desarrollado un nuevo índice de selección llamado índice Alp, para ser utilizado como criterio de selección para un proyecto transfronterizo con varias líneas de mejora. El número de ovejas Alpine Stonesheep aumentó de 120 en 2000 a 800 animales en 2016. El programa de conservación también incluye exámenes de lana, desarrollo de productos de alta calidad a base de lana de esta raza y el centro de información de la raza Alpine Stonesheep en Tierra de Berchtesgardener/Baviera, el hábitat natural de esta raza.

**Keywords:** conservation, sheep, traditional breeds

## Introduction

### *History of the breed*

The Alpine Stonesheep originated from the Neolithic Torfschaf, only a few breeds can be traced back directly to this sheep. The original stature of the Stonesheep is small, almost delicate with very thin, but very strong legs and firm claws. The breed is mainly found in Berchtesgarden, the surroundings of Traunstein and south-east of Rosenheim in the state of Bavaria in Germany.
## *Breed description*

Alpine Stonesheep are very smart animals, small to medium sized with a compact body. Within the breed, two types are defined: a larger and a smaller type. The face, the inner side of the thighs and legs are free from wool in these animals. The Alpine Stonesheep has short and pointed ears that are slightly hanging and stuck out sideways. The ears are longer when compared to the Carniolian Sheep. The Alpine Stonesheep has very hard claws with sharp edges. This makes it a confident climber despite the extremely thin legs. Another trademark of the Alpine Stonesheep is his long and woolly tail which is bended and mostly white at the end. Among both sexes, horned as well as unhorned animals exist. However, the male horn is considered stronger and mostly shaped in a spiral. The horns of ewes are smaller and slightly curved. Still, most ewes don't have horns at all. Typical for this kind of breed is the dual coated fleece with pithy, long coarse hair and fine wavy short bottom hair. In addition, the wool grease prevents the sheep from getting wet in snow and rainfall. The wool fineness is between 38-52 microns and depends on sex, age and the selection process of the breeder. Generally, the Alpine Stonesheep needs its wool clipped twice a year. The colour ranges from white to black and everything in between. The Alpine Stonesheep is well adapted to rough weather conditions. It is a great climber and is well suited for grazing on rocky alps. The sheep have an intensively developed herd instinct and will protect their offspring at any costs. Towards familiar persons they can show affection. Ewes weight around 45-60 kg while rams weight around 60-75 kg. In 2006, the preservation project to save the Alpine Stonesheep was started with 120 sheep. Today, in 2017, we can proudly count 680 sheep in the flockbooks.

## *Projects and research topics*

### *Breeding project*

A strong aim of ours was also to not only save the Alpine Stonesheep but also to establish a breeding program. The breeding program was started in 2010. The core part of the breeding program is the “Alp Index” together with wool examinations and the Alpine Stonesheep information centre.

Within the breeding program, we found eight different ram lines, based on data of old flockbocks: German ram lines A, M, B, H and N, and Austrian ram lines J, K and S. These ram lines help us to stop inbreeding and are an easy guideline for breeders to know which ram to buy for his ewes or at e.g. sheep shows.
The alp project – *Activating alpine pastures*

The Bavarian State Institute of Agriculture (LfL) at the Institute of Animal Breeding in Grub used the “Activating alpine pasture – new ways to diversity” of the Bavarian Academy for Nature Conservation and Landscape Management (ANL) to establish a special breeding project with the Alpine Stonesheep. The focus of the project is the preservation of the “Kleinrechenbergalm” by the grazing of young rams. The alp has not been used for over 20 years until the project started in 2012. The alp is located in the district of Traunstein in Upper Bavaria. The alp is located at approximately 1400 m above sea level and includes 5 hectares of land. The alp season starts in June and ends in the middle of September. The pasture of the alp provides enough food for 14-16 rams. In the end of May, all the selected rams are driven to Grub. At Grub all rams are weighted, dewormed and their claws are cut. After a short time for the rams to get used to each other, the breeders drive them to the alp and walk with them to the pasture for about an hour. After arriving at the alp pasture, the breeders need to install and repair the fence and then leave the rams on the alp till September. Every weekend another breeder goes up to the pasture to check on the rams. Beginning to Mid-September, the breeders hike up to the alp in the early morning, put down the fences for winter and then walk back into the valley with the rams. In the valley, other breeders that didn’t put a ram up the alp prepare the event called “Almabtrieb” – the rams come down after their time on the alp to get examined and graded. Also the “Almabtrieb” is a festivity at which people can buy products of the Alpine Stonesheep like woollen sweaters or get information about the breed and the preservation work. The rams get graded on their weight gain, claws, wool, muscularity and appearance. The alp index is based on this grading.

To know if the rams can still „survive“ in their natural habitat, we developed the Alp Index, a tool to track their development and changes while on the Alp. The alp index is calculated as follows:

* Weight gain during the summer on the alp (1-18 points; the more gain the better)

* Claws (grades 1-9, times 2)

* Wool (grades 1-9, times 2)

* Muscling (grades 1-9, times 2)

* Over all appearance (grades 1-9, times 2)

* Ranking of the rams (First place = 10 points ... 10th place = 1 point, further places 0 points)

Nine points is the highest grade a ram can score. The average alp index of all alp rams evaluated between 2013 and 2016 was 71.2. Further examples of alp index results (2014-2016) are given in Table 1.
<table><caption>**Table 1:** Examples of alp index results (2014-2016)</caption><thead><tr><th>Name</th><th>Line¹<br>(Country)</th><th>Weight 1<br>(kg)</th><th>Weight 2<br>(kg)</th><th>Weight gain<br>(kg)</th><th>Wool fineness<br>(µ)</th><th>Claw grade</th><th>Wool grade</th><th>Muscling grade</th><th>Appearance grade</th><th>Alp index</th><th>Year</th></tr></thead><tbody><tr><td>A-Bulgur</td><td>A (D)</td><td>59.4</td><td>63</td><td>3.6</td><td>41.8</td><td>11</td><td>16</td><td>16</td><td>14</td><td>72</td><td>2014</td></tr><tr><td>Helmut</td><td>H (D)</td><td>62.4</td><td>59</td><td>-3.4</td><td>43.2</td><td>10</td><td>14</td><td>12</td><td>12</td><td>49</td><td>2014</td></tr><tr><td>Michl</td><td>M (D)</td><td>59.9</td><td>65</td><td>5.1</td><td>40.7</td><td>8</td><td>10</td><td>14</td><td>10</td><td>51</td><td>2014</td></tr><tr><td>Josef</td><td>J (A)</td><td>63.2</td><td>66</td><td>2.8</td><td>47.0</td><td>14</td><td>14</td><td>16</td><td>12</td><td>65</td><td>2014</td></tr><tr><td>Sugar</td><td>S (A)</td><td>52.5</td><td>58</td><td>5.5</td><td>39.8</td><td>16</td><td>14</td><td>14</td><td>16</td><td>77</td><td>2015</td></tr><tr><td>Bartl</td><td>B (D)</td><td>68.5</td><td>74</td><td>4.7</td><td>43.2</td><td>15</td><td>14</td><td>16</td><td>16</td><td>80</td><td>2015</td></tr><tr><td>Hector</td><td>H (D)</td><td>45.0</td><td>51</td><td>6.0</td><td>48.5</td><td>12</td><td>16</td><td>16</td><td>16</td><td>80</td><td>2015</td></tr><tr><td>Nathan</td><td>N (D)</td><td>46.4</td><td>50</td><td>3.6</td><td>34.4</td><td>14</td><td>14</td><td>12</td><td>14</td><td>62</td><td>2015</td></tr><tr><td>Augustus</td><td>A (D)</td><td>47.0</td><td>57</td><td>10.0</td><td>36.0</td><td>7</td><td>8</td><td>7</td><td>8</td><td>83</td><td>2016</td></tr><tr><td>Bono</td><td>B (D)</td><td>60.0</td><td>66</td><td>6.0</td><td>51.1</td><td>8</td><td>8</td><td>7</td><td>8</td><td>78</td><td>2016</td></tr><tr><td>Nagid</td><td>N (D)</td><td>48.3</td><td>60</td><td>11.7</td><td>38.5</td><td>7</td><td>8</td><td>8</td><td>7</td><td>81</td><td>2016</td></tr><tr><td>Gustl</td><td>G (A)</td><td>69.2</td><td>72</td><td>2.8</td><td>44.0</td><td>5</td><td>7</td><td>7</td><td>6</td><td>51</td><td>2016</td></tr></tbody><tfoot><tr><td colspan="12">¹D = Germany A = Austria</td></tr></tfoot></table>
<table><caption>**Table 2:** The relation between the wool grade and the wool fineness (in microns)</caption><thead><tr><th>Wool grade</th><th>Number</th><th>Fineness (μ)</th><th>Comments</th></tr></thead><tbody><tr><td>9</td><td>2</td><td>40.1</td><td>40-44 μ</td></tr><tr><td>8</td><td>12</td><td>41.8</td><td>is ideal</td></tr><tr><td>7</td><td>22</td><td>43.5</td><td></td></tr><tr><td>6</td><td>8</td><td>39.9</td><td>wool is not</td></tr><tr><td>5</td><td>1</td><td>37.7</td><td>typical</td></tr></tbody></table>

*The wool project and its impact on the preservation of this breed*

Another huge impact on preserving a breed has the ability to find ways to use the breed and its products. With the Alpine Stonesheep the breeders association decided to remember old traditions and ways of using the Alpine Stonesheep. For example “Loden” is a product people used to make out of the Alpine Stonesheep wool. In 2006 we started with the wool project with 200 kg of greasy wool and today we have around 1 ton of greasy wool. At the start there were only 3 breeders involved today over 25 breeders bring their greasy wool to the annual breeders meeting in April (Fig.1). The main focus of the wool project is that not only the breeders bring their wool but also sell their products at fairs and in their farm shop. The Alpine Stonesheep wool project is part of “Kollektion der Vielfalt” (Fig.2).
<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /><figcaption>**Figure 1:** Development of number of breeders (above) and amount of greasy wool (below, kg) from the start of the project in 2004.</figcaption></figure>
<figure><img src="image_48.png" /><figcaption>**Figure 2:** Some of our wool products. (Photos: Isabelle Ketterle)</figcaption></figure>

**Conclusion and outlook**

We plan on continuing the alp project in order to monitor changes and development of the breed of the Alpine Stonesheep. Another future goal of ours is to focus even more on the different genetic lines and do more research into that area. Additionally, we are planing to organise wool workshops for breeders to disseminate more information and education on the topic of wool.

**References**

- Baumung R. 2003, Genetische Differenzierung von Schafrassen im Ostalpenraum. Arche Nova 2, 8-9

- Kaspar K. 1928, Studie über das Steinschaf im Chiemgau. Dissertation, TH-München, Germany
<figure><img src="image_49.png" /></figure>
Reproduction and Pathology

<figure><img src="image_50.png" /></figure>
<figure><img src="image_51.png" /></figure>
# The Alpaca Cria, Clinical and Immunological Aspects

P. Walter Bravo

Escuela de Medicina Veterinaria. Universidad Nacional San Antonio Abad. Cusco,
Peru

Email: pwbravo@gmail.com

**Abstract.** Alpaca crias are born after a long period of gestation and they are subjected to many noxas according to the place where they are born. In this paper, the author gives a brief summary of some key events of neonatology. Body temperature regulation, hypothermia, colostrum intake and its dynamics and failure to passive transfer are covered.

**Resumen.** Las crías de alpaca nacen después de un largo período de gestación y están sometidas a muchas noxas de acuerdo al lugar donde nacen. En este artículo, el autor hace un breve resumen de algunos eventos clave en neonatología. El resumen abarca la regulación de la temperatura corporal, la hipotermia, la ingesta del calostro y su dinámica, y el fallo de la transferencia pasiva.

**Keywords:** alpaca cria, neonatology, hypothermia, colostrum

Alpaca offspring, cria, as called in South America, is born in an advanced state of maturity, weighing from 4 to 10 kg. Heavy crias weigh 9 kg and come from 8-9 years old dams. At birth, neonates are covered by an extra membrane, the epidermal membrane, which covers the whole body with the exception of the natural openings. Rectal temperature of the neonate is similar to their dams, 37.5 °C; this temperature drops transiently to 35 °C during the first 30 to 45 minutes, then rises to 37.5 °C and then remains as such. Sixteen percent of crias remain at 35 °C, becoming hypothermic, and would need immediate assistance. Normal vital signs at birth include a heart rate of 60 to 90 beats per minute, and a respiratory rate of 10 to 30 per minute. Colostrum intake occurs an hour after birth, then occurs every 30 minutes for the first 4 hours of life, end then every hour. Colostrum is the primary source of antibodies, being IgG the main and represents 80% of all immunoglobulins. The highest IgG concentration is registered 24 to 48 hours after birth and is 2,500 to 3,000 mg/dL. Life for the neonate is better if the neonate sucks colostrum during the first 8 hours. However, if suckling occurs after 24 hours of birth, concentration of IgG is very poor,
175 mg/dL. Failure of passive transfer of IgG requires the administration of plasma from an adult animal. This transfer could be by intravenous and/or intraperitoneal transfusion. Crias are subject to different insults according to the country where they are born. Nonetheless, its care is vital and would have a positive impact on the number of weaned crias.

Alpaca crias are born in the morning hours wherein temperature conditions are suitable for survival. Eighty percent of crias are born between 8 and 12pm. Isolated cases of birthing are recorded between 4 and 6 pm, or even the next morning at 6 a.m. These last three cases may represent dystocia cases that went by unsuspected by the owner or caretaker. Weight of the cria is influenced by age of the dam. Heavier crias, 8.3 kg, are born from 8-9 year old dams, whereas crias weighing 6.9 kg are born from 2-3 year old dams (Bravo et al., 2009).

At birth neonates are covered by an extra membrane, the epidermal membrane. This membrane covers the entire body with the exception of natural openings like mouth, nostrils, eyes, ears, prepuce, penis and vulva. This membrane is also thick at the distal end of both toes, and is thin, transparent and facilitates fetal expulsion from the dam by uterus and abdominal contractions. It disintegrates as the cria begins to move and during the process of standing up.

Neonate rectal temperature is similar to its dam, 37.5 °C, and then drops to 35 °C by 30 minutes and then by three hours goes back to 38 °C. This rapid and transient drop in rectal temperature is normal, however, 16 % of crias develop a hypothermic state and in those crias, temperature does not reach 38 °C even by 24 hours of life. This is a reminder that when crias are born under adverse environmental conditions, they should be dried immediately. In this sense, crias dried with a hair dryer may survive; however, crias which are not dried may succumb to the process of hypothermia (Garnica et al., 1992).

Colostrum intake is vital for the newborn. Colostrum is the primary source of antibodies necessary to fight many diseases and because the epitelhiochorial placentation does not allow the passage of antibodies from the dam to the fetus. Studies done previously, indicate that 80 % of antibodies fall into the immunoglobulin type G, commonly known as IgG, and this IgG stays in the blood of the cria for longer period of time than other Igs, like IgA, IgM. As soon as crias stand up, they try to suckle the vital colostrum. It is not achieved on the first intent, but by the third attempt, the goal of suckling is reached. Crias are born without any IgG, i.e., agammaglobulinemic. IgG is absorbed and is present in the blood stream as soon as 1 hour after suckling, and it reaches a peak, 2,500 mg/dL, by 24-48 hours of life. It stays high for the next 10 days, and then starts to decline steadily to day-60 of life (Bravo et al., 1997). The time of first suckling is also important to consider. First successful suckling by 2-4 hours is far better than initiation of suckling by 6 hours. Recent data indicates that when neonates suckle by 2-4 hours after birth, IgG concentrations are greater than 1,200 mg/dL, by contrast when suckling occurs by 6 to 8 hours, IgG concentrations are 1,000 mg/dL. These values again indicate that suckling as soon as possible is more beneficial than delaying suckling
(Figure 1). A relationship between IgG concentrations and occurrence of suckling is observed naturally. The cria alpaca suckles every 30 minutes for the first 4 hours, and then suckling occurs every hour.

<figure><img src="image_52.png" /><figcaption>**Figure 1:** Concentrations of IgG in alpaca crias at 48 hours after colostrum suckling at 2, 4, 6, 8 and 24 hours of life.</figcaption></figure>

A second aspect is the time when a cria alpaca is able to produce its own IgG, since there is passive transfer from colostrum dam to the cria via colostrum. Results indicate that by day 90 the cria produces its own IgG, see Figure 2 (Quispe, 2009).

<figure><img src="image_53.png" /><figcaption>**Figure 2:** Time of alpaca IgG production in crias following cow colostrum administration at birth.</figcaption></figure>
The clinical consequence of a deficient IgG absorption or availability is called failure of passive transfer (FTP) and is depicted in Figure 3 wherein there is a clear difference of IgG concentrations between normal and crias with failure to transfer. In addition, FTP could be responsible for hypothermia, lack of gaining weight and cria mortality at least for the first 30 days.

<figure><img src="image_54.png" /><figcaption>**Figure 3:** Percentage of FTP in alpaca crias under clinical conditions. FTP was considered as concentrations of IgG were less than 900 mg/dL.</figcaption></figure>

Concentrations of IgG in milk of alpacas for the period of lactation have been also reported (Ampuero et al., 2008). Low IgG concentrations exists from the first through the fourth month of lactation, averaging 180 mg/dL, in contrast to a slight increase from the fifth through the eighth month, 300 mg/dL. The presence of IgG in milk and during lactation is unknown, but indicates that the mammary gland is still synthesizing this antibody.

In summary, alpaca crias are born in an advanced state of maturity. There is transient decrease in temperature which is common especially when climate conditions are adverse for cria survival. Those conditions could trigger hypothermia in 16 % of crias if colostrum is not suckled. Concentrations of IgG are vital for cria survival. As soon as the cria starts suckling, IgG increases, reaching a peak 24 to 48 hours after birth. It is better if first suckling begins 2-4 hours rather than 6-8 hours. A cria alpaca begins producing its own IgG at 2.5 months. FTP is a clinic entity present and it may affect up to 10 % of alpaca crias.
References

Ampuero, V.; Cornejo, D.; Pacheco, R.; Bravo, P.W. 2008, Determinación de la concentración de inmunoglobulina G en calostro y leche de llamas de las variedades Kcara y Chaku, y alpacas de las variedades Suri y Huacaya desde la parición hasta el destete. Thesis Biología, UNSAAC. 100 pp

Bravo, P.W.; Garnica, J.; Fowler, M.E. 1997, Immunoglobulin G concentrations in periparturient llamas, alpacas and their crias. Small Rumin Res 26:145-149.

Bravo, P.W.; Garnica, J.; Puma, G. 2009, Cria alpaca body weight and perinatal survival in relation to age of the dam. Anim Reprod Sci 111: 214-219

Garnica, J.; Chavez, L.; Bravo, P.W. 1992, Temperatura rectal y niveles de glucosa sanguínea alpacas crias. Allpak'a 2:29-33

Quispe, W. 2009, Determinación del tiempo de producción de IgG en crias alpaca. Thesis MV, Universidad Jorge Basadre, Tacna, Perú. 80 pp
<figure><img src="image_55.png" /></figure>
# Addition of Seminal Plasma to Frozen-Thawed Llama Spermatozoa does not Preserve Sperm Motility

Fumuso, F.G.<fnref n="1" />,<fnref n="2" />, Carretero, M.I.<fnref n="1" />,<fnref n="2" />, Chaves, M.G.<fnref n="1" />, Neild, D.M.<fnref n="1" />, Miragaya, M.H.<fnref n="1" /> and Giuliano, S.M.<fnref n="1" />

E-Mail: fernandafumuso@yahoo.com.ar

**Abstract.** In South American Camelids (SAC) sperm survival is low after thawing. The aim of this study was to determine the effect of the addition of seminal plasma (SP) on the survival of frozen-thawed llama spermatozoa. Sixteen ejaculates from four llama males were obtained by electroejaculation, under general anesthesia. Ejaculates were diluted 4:1 in 0.1 % collagenase in HEPES-TALP and incubated 4 minutes at 37 °C, then centrifuged and re-suspended in lactose, egg yolk and 7 % dimethylformamide, equilibrated at room temperature, and frozen in 0.50-ml straws. After thawing, samples were divided into three aliquots for addition of SP: 0 % (control), 10 % and 50 % (final concentrations) and then incubated 3 h at 37 °C. Sperm motility, viability, membrane function, acrosome status and DNA quality were evaluated in raw and post-thaw samples at 0, 1.5 and 3 h. A split-plot design was applied, blocking the males and using the treatment as one factor (levels: 0, 10 and 50 % SP) and time as the other factor, (levels: 0, 1.5 and 3 h). After thawing, all samples maintained sperm viability, membrane function, acrosome status, and DNA quality (p>0.05) over the incubation time. Sperm motility significantly declined (p≤0.05) at 3 h of incubation in all treatments. The rapid loss of motility of post-thaw llama spermatozoa while maintaining other seminal characteristics when in the presence of SP seems important to highlight and requires further studies. To conclude, post-thaw addition of 10 % and 50 % seminal plasma was unable to preserve sperm motility or improve the survival of llama frozen-thawed spermatozoa.

**Resumen.** La supervivencia de espermatozoides descongelados es baja en Camélidos Sudamericanos (CSA). Nuestro objetivo fue determinar el efecto de la adición de plasma seminal (PS) sobre la supervivencia de espermatozoides congelados-descongelados de llama. Dieciséis eyaculados de 4 llamas fueron obtenidos mediante electroeyaculación bajo anestesia general. Los eyaculados fueron diluidos 4:1 en colagenasa al 0,1 % en HEPES-TALP, incubados 4 minutos a 37 °C, centrifugados y resuspendidos en lactosa, yema de huevo y dimetilformamida al 7 %. Se equilibraron a temperatura ambiente y se criopres-

<footnote n="1">¹Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Av. Chorroarín 280, Buenos Aires, Argentina.</footnote>

<footnote n="2">²Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, Buenos Aires, Argentina.</footnote>
ervaron en pajuelas de 0,5 ml. Las muestras descongeladas fueron divididas en 3 para agregar PS: 0 % (control), 10 % y 50 % (concentraciones finales) y posteriormente incubados 3 h a 37 °C. En semen fresco y descongelado se evaluó movilidad, viabilidad, funcionalidad de membrana, estado acrosomal y calidad del ADN espermático a las 0, 1,5 y 3 h de incubación. Se aplicó un diseño de parcelas divididas en el tiempo bloqueando los machos, tomando el factor tratamiento (niveles: 0, 10 y 50 %) y el factor tiempo (niveles: 0, 1,5 y 3 h). Después del descongelamiento, todas las muestras mantuvieron la viabilidad, funcionalidad de membrana, estado acrosomal y calidad del ADN (p>0,05) a lo largo de la incubación. La movilidad espermática disminuyó significativamente a las 3 h de incubación (p≤0,05) en todas las muestras. Es de destacar la rápida pérdida de movilidad espermática después del descongelamiento, conservando las demás características en presencia de PS, requiriendo más estudios. En conclusión, la adición posdescongelado de 10 % y 50 % de PS no preservó la movilidad ni mejoró la supervivencia en espermatozoides de llama descongelados.

**Keywords:** llama, frozen-thawed semen, seminal plasma

**Introduction**

To date, no protocol for cryopreservation of SAC spermatozoa produces pregnancy rates that make application of this biotechnology commercially viable. Different diluents have been used, several cryoprotectants have been tried in different concentrations and diverse freezing curves have been tested (Morton et al., 2007; 2010; Santiani et al., 2005, 2013; Giuliano et al., 2010; Carretero et al., 2015). However, results are still discouraging when cryopreserved semen is artificially inseminated, obtaining between 0 and 26 % pregnancy rates (Bravo et al., 2000; Aller et al., 2003; Vaughan et al., 2003; Giuliano et al., 2012). Giuliano et al. (2012) demonstrated that to obtain pregnancy with refrigerated semen, it is necessary to inseminate female llamas after detecting ovulation. In 2015, Carretero et al. compared two cryoprotectants: glycerol (that is widely used in this species) and dimethylformamide, both at 7 %. The study revealed that glycerol causes severe damage to sperm DNA. These could be some of the possible reasons for the poor results obtained with this biotechnology and current research is trying to further elucidate this matter. It is acknowledged that SP is more than just a medium to transport spermatozoa through the reproductive tract. It has been proved that it has a role on osmotic stability of spermatozoa and works as a buffer, is implicated in sperm capacitation through its protein components, it is involved in female physiology through the ovulation factor it provides and has an important role in forming the sperm reservoir (Ratto et al., 2005; Adams et al., 2013; Apichela et al., 2014; Tribulo et al., 2015; Silva et al., 2015; Berland et al., 2016). Several studies had been carried out in productive species in order to elucidate the interaction between SP and spermatozoa, with differing results (Graham, 1994; Aurich et al., 1996; Maxwell et al., 1999; Moore et al., 2005; Domínguez et al., 2008; Leahy et al., 2010; Morrel et al., 2010; Kershaw-Young and Maxwell, 2011;
Carretero et al., 2015; 2016). In llama and alpaca there are studies that evaluated the effect of SP on raw sperm over time (Kershaw Young and Maxwell, 2011; Carretero et al., 2015). In the case of cryopreserved semen, one study has evaluated the effect of cooling llama spermatozoa to 5 °C for 24 h in the presence or absence of SP (Carretero et al., 2016). They observed that the samples refrigerated with SP showed a significantly lower total and progressive motility while preserving sperm viability. Most refrigeration and freezing techniques used so far in SAC have been adopted from those used in bulls and rams and most cryopreservation protocols dilute semen without removing SP before performing the freezing curves (Bravo et al., 2000; Vaughan et al., 2003; Aller et al., 2003; Santiani et al., 2005; Giuliano et al., 2010). Thus, while the presence of SP in the media could be interfering with the cryoprotectants reaching and protecting spermatozoa during temperature descent, on the other hand it may be needed at the time of AI to carry out its role in the female reproductive tract. Hence, the objective of our study was to evaluate over time the effect of adding, post-thaw, different dilutions of seminal plasma to llama spermatozoa that were cryopreserved without SP.

# Materials and Methods

## Animals and location

The study was carried out at the Faculty of Veterinary Sciences of the University of Buenos Aires, in Buenos Aires, Argentina. The city is situated at sea level, latitude 34°36' and longitude 58°26'. For the study, 4 male *Lama glama* ranging between 8 and 12 years of age were used. Animals were kept out at pasture in pens and supplemented with alfalfa; they also had free access to fresh water throughout the study.

## Semen collection

A total of 16 ejaculates (n=4; r=4) were collected, processed and evaluated. Semen collections were carried out using electroejaculation under general anesthesia, according to the technique described by Director et al. (2007). All procedures were approved by the Committee for the Use and Care of Laboratory Animals (CICUAL) of the Faculty of Veterinary Sciences of the University of Buenos Aires (protocol 2011/18).

## Semen evaluation

The sperm characteristics studied were: motility, membrane function, viability and DNA (condensation and fragmentation). Sperm motility was evaluated on a warm stage (37 °C) using a phase contrast microscope (100x); motility was classified as either oscillatory (OM) or progressive (PM) and total motility (TM) was also
estimated (Giuliano et al., 2010). Membrane function was evaluated using the HOS test carried out according to Giuliano et al. (2008). Sperm showing the characteristic swelling of the tail (HOS positive) were classified as having a functional plasma membrane. Fluorochromes 6-carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) were used for evaluating membrane integrity (viability) and this technique was carried out according to Giuliano et al. (2008). Spermatozoa that fluoresced green were classified as being viable (intact membrane) while sperm nuclei that fluoresced red were classified as nonviable (damaged membrane). For the DNA evaluation, two techniques were used: the Toluidine blue (TB) stain, to determine chromatin condensation, and the Sperm Chromatin Dispersion (SCD) assay, to analyze DNA fragmentation. The TB stain was carried out according Carretero et al. (2009). Briefly, each sample was smeared on clean, non-greasy slides and once dried, fixed with ethanol 96° and stained with a working solution of 0.02 % TB. Preparations were observed directly under immersion oil (1000x) evaluating a minimum of 200 spermatozoa per smear. Spermatozoa were classified into three groups according to the degree of chromatin condensation: light blue (negative, no chromatin decondensation), light violet (intermediate, some degree of decondensation) and dark blue-violet (positive, high degree of decondensation). Sperm in both positive and intermediate groups were considered to have altered chromatin condensation. Dithiothreitol (DTT) 1 % in distilled water was used as a positive control for the TB stain. The SCD assay to evaluate the degree of DNA fragmentation was carried out according to Carretero et al. (2012). Briefly, each sperm suspension was mixed with low melting point aqueous agarose and pipetted onto a glass slide. Each slide was incubated in different lysing solutions, dehydrated in sequential ethanol baths and stained with Giemsa. Preparations were observed directly under immersion oil (1000x) evaluating a minimum of 200 spermatozoa per smear. Spermatozoa were classified into four patterns according to the size of the halo: 1- nuclei with large DNA dispersion halos (LH); 2- nuclei with medium halos (MH); 3- nuclei with small halos (SH) and 4- nuclei with no halo (NH). The first two patterns (1 and 2) were considered spermatozoa without DNA fragmentation and the other two (3 and 4), spermatozoa with DNA fragmentation. Semen incubation with NaOH was used as a positive control of sperm DNA fragmentation.

*Freeze-thawing of llama semen*

With the objective of decreasing thread formation and facilitating manipulation of the samples, each ejaculate was diluted 4:1 in 0.1 % collagenase in HEPES-TALP (HP) medium and incubated 4 minutes at 37 °C according to Giuliano et al. (2010). Afterwards, the ejaculates were centrifugated at 800 g during 10 minutes to separate the spermatozoa from the seminal plasma and the enzymatic medium. Then the cryopreservation extender (lactose-EDTA-egg yolk with 7 % of dimethylformamide) was added to the pellet and the samples were equilibrated for
20 minutes at room temperature. Freezing was carried out according to the manual method described by Miragaya et al. (2001). Briefly, temperature descent was carried out in three phases by placing the straws, submerged in a mixture of ethanol: acetone (1:1), in a bronze canister with a graduated handle and holding them over liquid nitrogen vapors. Temperature phases were as follows: a) from room temperature to -15 °C, temperature descent was at a rate of 10-12 °C per minute; b) from -15 °C to -120 °C, temperature descent was at a rate of 25-40 °C per minute; and finally c) the straws were plunged into the liquid nitrogen at -196 °C and stored until evaluation. The samples were thawed at 37 °C in water bath for 60 seconds. After thawing, the samples were divided into three aliquots for the addition of the different concentrations of SP: 1) 0 % SP (control), 2) 10 % SP and 3) 50 % SP. Samples were then incubated 3 h at 37 °C and were evaluated at different time periods: 0, 1.5 and 3 h, using the same evaluations that were carried out in the raw semen.

### Statistical analysis

A Kruskal-Wallis test was carried out to evaluated motility (oscillatory, progressive and total) between treatments (0, 10 and 50 % SP) and times of incubation (0, 1.5 and 3 h). To evaluate the others seminal characteristics, a split-plot design was applied, blocking the males and using the treatment as one factor with three levels (0, 10 and 50 % SP) and time as the other factor, also with 3 levels (0, 1.5 and 3 h).

## Results

The seminal characteristics of raw semen were (media ± SD): volume: 1.9 ± 1.5 ml; oscillatory motility: 57.9 ± 17.4%; no progressive motility was observed in raw semen samples; membrane function: 31.8 ± 10.6% (HOS positive spermatozoa); membrane integrity: 56.9 ± 11.8% (live spermatozoa); normal chromatin condensation: 92.3 ± 7.8% and DNA integrity: 92.3 ± 7.8%. After treatment of raw semen with collagenase, total motility was 62.0 ± 9.7% (25.8 ± 14.8% was progressive motility and 36.2 ± 11.0% was oscillatory). A significant decrease of total motility, membrane function, viability and DNA integrity was observed in all post-thaw spermatozoa when compared to the raw semen samples (p<0.05).

Oscillatory motility was not observed or was almost null in all post-thaw samples (0, 10 and 50 % SP) at all incubation times (0, 1.5 and 3 h). A significant decrease in PM and TM was observed in all samples over time (p=0.001), but no significant differences (p>0.05) were observed in either PM or TM between post-thaw samples (0, 10 and 50 % SP) at any of the times of evaluation (Table 1; Figure 1). No significant differences (p>0.05) were observed in the percentages of live spermatozoa nor in the percentages of sperm with membrane function between post-thaw samples and both seminal parameters were conserved over time in all samples (Table 2). Nevertheless, a tendency to decrease the percentage
of spermatozoa with functional membranes was observed along the incubation period in all treatments (0, 10 and 50 % SP).

<table><caption>**Table 1:** Percentages of sperm with OM, PM and TM in raw llama semen, post-collagenase and post-thaw samples incubated at 37 °C with different percentages of SP (0, 10 and 50 %) and evaluated at different times (0, 1.5 and 3 h).</caption><thead><tr><th>Time of Incubation (h)</th><th>PS (%)</th><th>OM (%)</th><th>PM (%)</th><th>TM (%)</th></tr></thead><tbody><tr><td rowspan="4">0</td><td>0</td><td>0.0 ± 0.0 <sup>a</sup></td><td>29.5 ± 5.7 <sup>a</sup></td><td>29.5 ± 5.7 <sup>a</sup></td></tr><tr><td>10</td><td>0.0 ± 0.0 <sup>a</sup></td><td>28.0 ± 12.3 <sup>a</sup></td><td>28.0 ± 12.3 <sup>a</sup></td></tr><tr><td>50</td><td>0.0 ± 0.0 <sup>a</sup></td><td>18.9 ± 12.6 <sup>a</sup></td><td>19.1 ± 12.7 <sup>a</sup></td></tr><tr><td>0</td><td>0.0 ± 0.0 <sup>a</sup></td><td>16.1 ± 5.5 <sup>b</sup></td><td>16.1 ± 5.5 <sup>b</sup></td></tr><tr><td rowspan="3">1.5</td><td>10</td><td>0.0 ± 0.0 <sup>a</sup></td><td>17.2 ± 11.4 <sup>b</sup></td><td>17.2 ± 11.4 <sup>b</sup></td></tr><tr><td>50</td><td>0.2 ± 0.5 <sup>a</sup></td><td>9.9 ± 10.5 <sup>b</sup></td><td>9.9 ± 10.5 <sup>b</sup></td></tr><tr><td>0</td><td>0.0 ± 0.0 <sup>a</sup></td><td>8.7 ± 4.6 <sup>c</sup></td><td>8.7 ± 4.6 <sup>c</sup></td></tr><tr><td rowspan="2">3</td><td>10</td><td>0.0 ± 0.0 <sup>a</sup></td><td>8.4 ± 4.4 <sup>c</sup></td><td>8.4 ± 4.4 <sup>c</sup></td></tr><tr><td>50</td><td>0.6 ± 1.2 <sup>a</sup></td><td>4.6 ± 1.7 <sup>c</sup></td><td>4.9 ± 2.2 <sup>c</sup></td></tr></tbody></table>

No significant differences were observed (p>0.05) in sperm chromatin condensation between post-thaw samples at any of the evaluations over time (Table 2).

When DNA fragmentation was evaluated, no statistical differences (p>0.05) were observed between post-thaw samples (0, 10 and 50 % of SP). A significant increase (p<0.05) in the percentage of spermatozoa with DNA fragmentation was observed in all post-thaw samples (0, 10 and 50 % SP) when compared with raw semen. This increase was observed specifically in the category of nuclei with small halos in all post-thaw samples (Table 3).

<footnote><sup>a,b,c</sup> Different letters within columns indicate statistical differences between samples and evaluation periods for each motility pattern (p<0.05).</footnote>
<table><caption>**Table 2:** Percentages of sperm viability, membrane function and chromatin condensation in raw llama sperm and posthawed spermatozoa incubated with different amount of SP (0, 10 and 50%) evaluated in different times of incubation at 37 °C (0, 1.5 and 3 h).</caption><thead><tr><th>Time of Incubation (h)</th><th>SP (%)</th><th>Sperm viability (%)</th><th>Membrane function (%)</th><th>Normal chromatin condensation (%)</th></tr></thead><tbody><tr><td rowspan="3">0</td><td>0</td><td>19.9 ± 9.5 <sup>a</sup></td><td>21.5 ± 9.6 <sup>ab</sup></td><td>92.8 ± 6.7 <sup>a</sup></td></tr><tr><td>10</td><td>19.6 ± 11.1 <sup>a</sup></td><td>21.0 ± 9.4 <sup>ab</sup></td><td>92.1 ± 6.9 <sup>a</sup></td></tr><tr><td>50</td><td>16.8 ± 9.2 <sup>a</sup></td><td>20.0 ± 9.5 <sup>bc</sup></td><td>93.1 ± 6.6 <sup>a</sup></td></tr><tr><td rowspan="3">1.5</td><td>0</td><td>17.7 ± 11.8 <sup>a</sup></td><td>12.6 ± 6.4 <sup>bc</sup></td><td>92.2 ± 1.1 <sup>a</sup></td></tr><tr><td>10</td><td>16.8 ± 11.9 <sup>a</sup></td><td>14.1 ± 6.6 <sup>bc</sup></td><td>96.8 ± 1.2 <sup>a</sup></td></tr><tr><td>50</td><td>20.0 ± 8.9 <sup>a</sup></td><td>13.7 ± 10.4 <sup>bc</sup></td><td>96.0 ± 2.6 <sup>a</sup></td></tr><tr><td rowspan="3">3</td><td>0</td><td>15.0 ± 7.7 <sup>a</sup></td><td>13.1 ± 4.8 <sup>bc</sup></td><td>91.5 ± 9.4 <sup>a</sup></td></tr><tr><td>10</td><td>15.5 ± 7.5 <sup>a</sup></td><td>12.9 ± 4.5 <sup>bc</sup></td><td>92.1 ± 7.3 <sup>a</sup></td></tr><tr><td>50</td><td>15.7 ± 10.2 <sup>a</sup></td><td>12.6 ± 5.3 <sup>c</sup></td><td>91.8 ± 9.0 <sup>a</sup></td></tr></tbody></table>

<sup>a,b,c</sup> Different letters within columns indicate statistical differences between post-thaw samples and evaluation periods.

<table><caption>**Table 3:** DNA fragmentation of fresh llama semen and posthawed llama spermatozoa incubated with different percentages of seminal plasma (0, 10 and 50% of SP)</caption><thead><tr><th></th><th>LH</th><th>MH</th><th>LH+MH</th><th>SH</th><th>NH</th><th>SH+NH</th></tr></thead><tbody><tr><td>0% SP</td><td>32.7 ± 24.7 <sup>a</sup></td><td>40.8 ± 11.5 <sup>a</sup></td><td>73.6 ± 16.8 <sup>a</sup></td><td>23.4 ± 14.8</td><td>2.9 ± 2.4</td><td>23.4 ± 16.8</td></tr><tr><td>10% SP</td><td>30.5 ± 22.8 <sup>a</sup></td><td>45.6 ± 14.7 <sup>a</sup></td><td>76.1 ± 21.0 <sup>a</sup></td><td>19.8 ± 18.4</td><td>4.1 ± 3.7</td><td>23.9 ± 21.0</td></tr><tr><td>50% SP</td><td>27.2 ± 22.6 <sup>a</sup></td><td>49.7 ± 18.7 <sup>a</sup></td><td>76.9 ± 23.4 <sup>a</sup></td><td>21.2 ± 21.4</td><td>1.9 ± 2.4</td><td>23.1 ± 23.4</td></tr></tbody></table>

<sup>a,b,c</sup> Different letters mean statistical differences between treatments

Discussion

Poor reproductive results after AI with cryopreserved semen have been reported for several species (boar, donkey, camelids, etc.) and consequently this biotechnology can't be commercially applied. Active research is therefore currently being carried out to determine where the problem could possibly be. In the case of SACs, multiple roles have been attributed to SP in their reproductive physiology (Ratto et al., 2005; Adams et al., 2013; Apichela et al., 2014; Tribulo et al., 2015; Silva et al., 2015; Berland et al., 2016) but there are still various unanswered questions. Most cryopreservation protocols in camelids add the extenders to the whole ejaculate without removing SP (Bravo et al., 2000; Aller et al., 2003; Vaughan et al., 2003; Santiani et al., 2005; Giuliano et al., 2012; Carretero et al., 2015). In the present study, SP was removed before cryopreservation of llama semen and then different percentages of SP were added after freeze-thawing the samples to determine its effect on sperm parameters (motility, viability, membrane
function and DNA quality). We used different final dilutions of SP (0, 10 and 50 %) and further incubated the samples 3 h at 37 °C and observed that none of them were able to preserve total sperm motility over time (0 % SP: 29.5 ± 5.7 % vs 8.7 ± 4.6 %; 10 % SP: 28.0 ± 12.3 % vs 8.4 ± 4.4 %; 50 % SP: 19.1 ± 12.7 % vs 4.9 ± 2.2 % (values are mean ± SD)). Sabatini et al. (2014) evaluated the effect of the addition of SP to post-thaw donkey spermatozoa and they observed that total and progressive sperm motility significantly decreased (p<0.05) after 4 hours of incubation when 5 and 20 % SP was added compared to samples without SP (0 %). They also observed this effect at time zero when they added higher percentages of SP (70 %). When we added 50 % SP, at time zero we also observed lower percentages of sperm total motility compared to the other post-thaw samples (0 and 10 %), but this was not statistically significant (p>0.05). The study of Sabatini et al. (2014) established that motility of post-thaw donkey spermatozoa decreased when SP is added and that the effect is seen more rapidly when a higher final proportion of SP is used. These results are similar to ours with the difference that we observed this decrease progressively over the incubation time (0, 1.5 and 3 h) in all post-thaw samples, which could be a difference due to the differences between species.

Conversely, studies in rams obtained better sperm motility results since they reported that progressive and total motility were higher when they added SP after thawing (Ledesma et al., 2016). These authors also observed that intracellular Reactive Oxygen Species (ROS) and membrane stability (viability) were higher with the SP treatment compared to the controls (SP absent) and compared to the addition of seminal plasma vesicles. This contrast in results could be due either to the different physiology of ram spermatozoa or to differences in SP components, as it has been demonstrated that camelid SP differs from that of other species (Kershaw-Young and Maxwell, 2012). In addition, Ledesma et al. (2016) used a different final SP concentration in their study (25 %) from the ones that we used (10 and 50 %), a fact which also could have influenced the results.

Regarding llama sperm viability, membrane function and DNA quality, these parameters did not significantly change (p>0.05) between post-thaw samples (0, 10 and 50 % of SP) over the incubation. Zea et al. (2015) studied cryopreserved alpaca spermatozoa collected from a deviation of the deferent duct. They performed three treatments: without SP, with SP added before cryopreservation and with SP added after freezing-thawing. They observed a significant decrease in motility, membrane function and sperm viability in the samples where SP was added post-thaw. However, the authors did not mention the final proportions of diluent, spermatozoa and SP or the amount of time the samples were incubated. Similar to our study, the addition of SP from the sperm rich fraction of boar ejaculates to post-thaw sperm samples did not have an influence on sperm membrane integrity or lipid peroxidation after 2 h of incubation (Torres et al., 2016). With regard to DNA quality, the cryopreservation protocol did not affected chromatin condensation (p>0.05) but produced a significant increase in DNA
damage ($p \le 0.05$) compared to the raw semen. Other than this result, we did not observe a significant difference between post-thaw samples (0, 10 and 50 % of SP) regarding chromatin condensation and fragmentation. In donkey thawed semen, the degree of DNA fragmentation was not affected by the addition of SP immediately after dilution (Sabatini et al., 2014).

To conclude, post-thaw addition of 10 % and 50 % seminal plasma was unable to preserve sperm motility or improve the survival of llama frozen-thawed spermatozoa. The rapid loss of motility of post-thaw llama spermatozoa, while maintaining other seminal characteristics in the presence of SP, seems important to highlight and requires further studies.

## References

* Adams, G.P.; Ratto, M.H. 2013, Ovulation-inducing factor in seminal plasma: A review. Animal Reproduction Science 136: 148-156.

* Apichela, S.A.; Argañaraz, M.E.; Giuliano, S.; Zampini, R.; Carretero, I.; Miragaya, M.; Miceli, D.C. 2014, Llama oviductal sperm reservoirs: involvement of bulbourethral glands. Andrologia. 46: 290-5

* Aller J.F.; Rebuffi, G.E.; Cancino, A.K.; Alberio, R.H. 2003, Influencia de la criopreservación sobre la motilidad, viabilidad y fertilidad de espermatozoides de llama (Lama glama). Arch. Zootec. 52: 15-23.

* Aurich, J.E.; Kühne, A.; Hoppe, H.; Aurich, C. 1996, Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology 1996. 46: 791-797.

* Berland, M.A.; Ulloa-Leal, C.; Barria, M.; Wright, H.; Dissen, G.A.; Silva, M.E.; Ojeda, S.R.; Ratto, M.H. 2016, Seminal Plasma Induces Ovulation in Llamas in the Absence of a Copulatory Stimulus: Role of Nerve Growth Factor as an Ovulation-Inducing Factor. Endocrinology 157: 3224-3232.

* Bravo, P.V.; Callo, P.; Garnica, J. 2000, The effect of enzymes on semen viscosity in Llamas and Alpacas. Small Ruminant Research 38: 91-95.

* Director, A.; Giuliano, S.; Carretero, M.; Pinto, M.; Trasorras, V.; Miragaya, M. 2007, Electroejaculation and seminal characteristics in llama (Lama glama). Journal of Camel Practice and Research 14 (2): 203-206.

* Domínguez, M.P.; Falcinelli, A.; Hozbor, F.; Sánchez, E.; Cesari, A.; Alberio, R.H. 2008, Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm. Theriogenology 69: 564-573.

* Carretero, M.I.; Lombardo, D.; Arraztoa, C.C.; Giuliano, S.M.; Gambarotta, M.C.; Neild, D.M. 2012, Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test. Animal Reproduction Science 131(1-2):63-71

* Carretero, M.I.; Neild, D.M.; Ferrante, A.; Caldevilla, M.; Arraztoa, C.C.; Fumuso, F.G.; Giuliano, S.M. 2014, Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa. Andrologia 47: 685-693.

* Carretero, M.I.; Neild, D.M.; Ferrante, A.; Caldevilla, M.; Arraztoa, C.C.; Fumuso, F.G.; Giuliano, S.M. 2015, Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa. Andrologia 47: 685-93.
Carretero, M.I.; Giuliano, S.M.; Arraztoa, C.C.; Santa Cruz, R.C.; Fumuso, F.G.; Neild, D.M. 2016. Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium. Andrologia, doi: 10.1111/and.12691.

Giuliano, S.; Director, A.; Gambarotta, M.; Trasorras, V.; Miragaya, M. 2008, Collection method, season and individual variation on seminal characteristics in the llama (Lama glama). Anim Reprod Sci 104: 359-69.

Giuliano, S.; Carretero, M.; Gambarotta, M.; Neild, D.; Trasorras, V.; Pinto, M.; Miragaya, M. 2010. Improvement of llama (Lama glama) seminal characteristics using collagenase. Anim. Reprod. Sci. 118 (1): 98-102.

Giuliano, S.M.; Chaves, M.G.; Trasorras, V.L.; Gambarotta, M.; Neild, D.; Director, A.; Pinto, M.; Miragaya, M.H. 2012, Development of an artificial insemination protocol in llamas using cooled semen. Animal Reproduction Science 131; 204-210.

Graham, J.K. 1994, Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process. Theriogenology 1994. 41: 1151-1162.

Kershaw-Young, C.M.; Maxwell, W.M.C. 2011, The effect of seminal plasma on alpaca sperm function. Theriogenology 76: 1197-1206.

Kershaw-Young, C.M.; Maxwell, W.M.C. 2012, Seminal plasma components in Camelids and comparisons with other species. Reprod. Dom Anim 47: 369-375.

Leahy, T.; Marti, J.I.; Evans, G.; Maxwell, W.M. 2010, Seasonal variation in the protective effect of seminal plasma on frozen-thawed ram spermatozoa. Animal Reproduction Science 119: 147-153.

Ledesma, A.; Fernández-Alegre, E.; Cano, A.; Hozbor, F.; Martínez-Pastor, F.; Cesari, A. 2016. Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm. Anim Reprod Sci 173: 35-41.

Maxwell, W.M.; Evans, G.; Mortimer, S.T.; Gillan, L.; Gellatly, E.S.; McPhie, C.A. 1999, Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reproduction, Fertility and Development 11(2): 123-126.

Miragaya, M.H.; Chaves, M.; Neild, D.; Berretta, C.; Agüero, A. 2001, Proceedings of 3rd International Symposium on Stallion Reproduction. Fort Collins, Colorado, USA. pp 45.

Moore, A.I.; Squires, E.L.; Graham, J.K. 2005, Effect of seminal plasma on the cryopreservation of equine spermatozoa. Theriogenology 63: 2372-2381.

Morrell, J.M.; Rodriguez-Martinez, H.; Johannisson, A. 2010, A Single layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing. Reproductive BioMedicine Online 21: 429-436.

Morton, K.M., Bathgate, R., Evans, G., Maxwell, W.M. 2007, Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws. Reprod Fertil Dev 19: 792-6.

Morton, K.M.; Evans, G.; Maxwell, W.M. 2010, Effect of glycerol concentration, Equex STM supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm. 2010. Theriogenology 74: 311-6.

Ratto, M.H.; Huanca, W.; Singh, J.; Adams, G.P. 2005, Local versus systemic effect of ovulation-inducing factor in the seminal plasma of alpacas. Reproductive Biology and Endocrinology 3:29.
Santiani, A.; Huanca, W.; Sapana, R.; Huanca, T.; Sepúlveda, N.; Sánchez, R. 2005, Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. *Asian J Androl* 7: 303-9.

Sabatini, C.; Mari, G.; Mislei, B.; Love, C.; Panzani, D.; Camillo, F.; Rota, A. 2014, Effect of post-thaw addition of seminal plasma on motility, viability and chromatin integrity of cryopreserved donkey jack (Equus asinus) spermatozoa. *Reprod Domest Anim.* 49: 989-94.

Santiani, A.; Evangelista, S.; Valdivia, M.; Risopatrón J.; Sánchez, R. 2013, Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. *Theriogenology* 79:842-6.

Santiani, A.; Huanca, W.; Sapana, R.; Huanca, T.; Sepúlveda, N.; Sánchez, R. 2005, Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. *Andrology* 7: 303-309.

Silva, M.; Fernández, A.; Ulloa-Leal, C.; Adams, G.P.; Berland, M.A.; Ratto, M.H. 2015, LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of β-nerve growth factor of seminal plasma origin in female llamas. *Theriogenology* 84: 1096-102.

Torres, M.A.; Ravagnani, G.M.; Leal, D.F.; Martins, S.M.; Muro, B.B.; Meirelles, F.V.; Papa, F.O.; Dell'aqua, J.A.; Alvarenga, M.A.; Moretti, A.S.; De Andrade, A.F. 2016, Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing. *J Anim Sci.* 94: 1906-12.

Tribulo, P.; Bogle, O.; Mapletoft, R.J.; Adams, G.P. 2015, Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle. *Theriogenology* 83: 1394-401.

Vaughan, J. 2003, Artificial insemination in alpacas (Lama pacos). 2003. Kingston – ACT, RIRDC: 90.
<figure><img src="image_56.png" /></figure>
# Alpaca Semen Quality throughout the Breeding Period

P. Walter Bravo<fnref n="1" />, W. Garcia<fnref n="2" /> and V. Alarcon*<fnref n="1" />

E-Mail: pwbravo@gmail.com

**Abstract.** Semen characteristics were determined in twelve male alpacas (4 young, 4 adult and 4 old) during an entire breeding period, two months, at La Raya research station. Characteristics included volume (ml), motility (%), and concentration (million spz/ml). This center is located at 4,200 m sea level and on the Southern hemisphere. Males were separated at random from a group of 150 breeding males and were maintained under natural pastures, with no nutritional supplementation. Three-age males were considered: young (3 years old), adult (6-7 years old), and old males (10-12 years old). Semen was collected by vaginal aspiration following natural breeding to a receptive female. Semen volume was determined reading from the 15 ml collecting tube. Semen motility, percentage of spermatozoa with tail movement was determined on a microscopic field. Semen concentration, million spz/ml, was determined using the hemocytometer method. Data were analyzed using the NCSS software and with general linear method. There were some males, independent from age and number of breeding that did not yield a semen sample and were not considered in the analysis. Ejaculated volume increased from 0.5 ml at the beginning of breeding to 5 ml at the end of the breeding period; however, sperm motility showed an opposite trend, decreased from 50 to 17 % (P≤0.05). Sperm concentration started at 5 million spz/ml, increased to 70 million a month later, and by the end of breeding decreased to 16 million spz/ml (P≤0.05). The length of breeding, 2 months, affected differently to ejaculated volume, motility and concentration under field conditions which definitively could affect male fertility.

**Resumen.** Se determinaron las características del semen de 12 alpacas macho (4 juveniles, 4 adultas y 4 ancianas) durante un período completo de cría de dos meses de duración en la estación de investigación La Raya. Las caracteres estudiados fueron el volumen (ml), la motilidad (%) y la concentración (millones de esp/ml). Este centro se encuentra a 4.200 metros sobre el nivel del mar en el hemisferio sur. Los machos se escogieron aleatoriamente de un grupo de 150 machos reproductores y se mantuvieron en pastos naturales sin suplemento nutricional. Se hicieron tres grupos de edades: jóvenes (3 años), adultos (6-7 años) y ancianos (10-12 años). El semen se recolectó de hembras receptivas mediante as-

<footnote n="1">¹Escuela profesional de Medicina Veterinaria, Universidad Nacional San Antonio Abad. Cusco, Perú.</footnote>

<footnote>*Deceased.</footnote>

<footnote n="2">²Insituto veterinario de investigaciones tropicales y de altura, Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos. Lima, Perú.</footnote>
piración vaginal después de la monta natural. El volumen de semen se determinó mediante lectura de un tubo de 15 ml. Se utilizó un microscopio para la determinación de la motilidad del semen y del porcentaje de espermatozoides con movimiento en la cola. La concentración de semen en millones de esp/ml se determinó usando el método del hemocitómetro. Se utilizaron modelos lineales generalizados para analizar los datos mediante el software NCSS. Se eliminaron los dátos de machos que no produjeron muestra de semen independientemente del grupo de edad. El volumen eyaculado se incrementó desde 0.5 ml hasta 5 ml del comienzo al final del período reproductivo, pero la motilidad de los espermatozoides presentó la tendencia contraria, disminuyendo del 50 % a 17% ($p \le 0,05$). La concentración de esperma inicial fue de 5 millones de esp/ml, 70 millones un mes más tarde, y 16 millones de esp/ml al final del período ($p \le 0,05$). La duración de 2 meses del período reproductivo afectó el volumen eyaculado, la motilidad y la concentración en condiciones pudiendo afectar definitivamente a la fertilidad.

**Keywords:** semen, breeding period, motility, concentration

## Introduction

The breeding period, under South American conditions, lasts for about two months and takes place during the rainy summer days. When large females are kept, males are divided into two breeding teams, 2.5 % of females on each team. One team breeds for a week and the next team on the following week while the first team rests. This sequence of alternating males is maintained during a period of 2 to 2.5 months. Under this system, fertility is around 75 to 80 % (Novoa et al., 1973). An improved fertility rate (86 %) was also reported when males were exposed to females for 2 days and rest for 4 days (Condorena and Velasco, 1979).

Semen characteristics have been determined on field conditions and using the vaginal aspiration method immediately following a natural breeding to a receptive female (Neely and Bravo, 1998). This method avoids the use of an artificial vagina, stricture on the latex to simulate the female's cervix, and a heating pad to maintain the artificial vagina for the length of copulation. In addition, with this method suitable and comparable semen characteristics have been reported lately (Alarcon, Garcia and Bravo, 2012). The aims of this study were to follow semen characteristics throughout a breeding period of 2 months, as used in South America. Semen characteristics were volume, motility and concentration.

## Materials and Methods

This study was carried out at La Raya research center, located in the Peruvian highlands, at 4,200 m sea level, 70 °W longitude and 14 °S latitude.

Twelve males, four young three-years old, first time breeders; four adult 6-7 years old, and four old, 11-12 years old were separated at random from a group of 150 breeding males, and considered in this study. Males were maintained grazing natural pastures and with no nutritional supplementation. Females bred by the
males according to the management of the research station were located into four different groups, open from previous breeding periods, two lactating groups and a group of maiden females. The breeding period lasted two months, February and March.

Males were transported to the breeding females and allowed to a receptive female. A single breeding was allowed, and opportunities to breed were present all the time. Males accompanied the rest of 138 males to the different female groups. Generally, males bred females twice a week. Females bred (222) were marked and separated from males until the next scheduled breeding time.

Semen characteristics were determined following the protocol of Garnica, Achata and Bravo et al. (1993). Briefly, semen volume was read from the scale present of the 15-ml collecting tube. Motility was determined placing 10 µl de sample on a pre-warmed glass slide and under a 10X magnification using a microscope. It was read as percentage of motile spermatozoa on a microscopic field. Concentration was determined by the hemocytometer method. 50 µl of semen sample was mixed 1:100 with 3% saline solution. 10 µ of diluted sample was placed and a hemocytometer, allowed to rest for three minutes and then spermatozoa was counted using the grid of a Newbauer chamber.

Data was analyzed using the general linear model with the age of males as fixed factors and using the Number Crunching Statistical System, NCSS, software. Differences, if any, were considered at P<0.05.

## Results and Discussion

### *Semen characteristics by age*

General volume, motility and concentration were 2.3 ml, 44.6 %, and 29.9 million spz/ml respectively. The effect of age on the same semen characteristics are presented in fig. 1, 2, and 3, respectively. Semen volume recovered was significantly greater (P<0.05) in young males than in adult and old males. Spermatozoa motility was significantly higher (P<0.05) in adult and old males than in young males. Spermatic concentration was different (P<0.05) in old males than in adult and young males.

Semen characteristics by age did follow different trends. Volume decreased by age, adult and old males were similar, by contrast volume was higher in young males. Although previous reports were on limited days, most of them only for 12 to 24 days, our results on volume were similar values were reported earlier in alpacas (Bravo, Flores and Ordoñez, 1997; Urquieta et al., 2005; and Alarcon, Garcia and Bravo, 2012).

Spermatozoa motility was less in young males than in adult and old males. This difference could be a reflection of sexual maturity. Young males were introduced to receptive females for the first time and their motility was not equal to older
males. Similar trend was also observed in alpacas (Bravo, 2002; Urquieta et al., 2005).

<figure><img src="image_57.png" /><figcaption>Fig1
aspi</figcaption></figure>

<figure><img src="image_58.png" /></figure>

**Figure 2:** Mean and standard error of spermatozoa motility on semen samples recovered by vaginal aspiration following copulation to a receptive female on three different ages of male alpacas.
<figure><img src="image_59.png" /><figcaption>**Figure 3:** Mean and standard error of the mean of spermatic concentration in male alpacas of three different ages that bred for a period of two months.</figcaption></figure>

Spermatic concentration varied like spermatozoa motility, being higher in old males, than in adult and young males. Young males, as stated earlier, might have been reaching sexual maturity and then their sperm concentration was less than in older males. Comparable results were reported earlier in alpacas (Bravo, 2002).

*Semen characteristics by day of breeding*

Means of semen volume, motility and concentration are presented in figures 4, 5, and 6, respectively. Semen volume was different (P<0.05) at the beginning of the breeding period (0.5 ml) in contrast to 6 ml by the end of the same period. Spermatozoa motility started by 45 %, remained at the same value, but was significantly lower (17 %) by the end of breeding period (P<0.05). Spermatic concentration started at 6 million spz/ml, reached a peak to 65 million, and then dropped to 17 million by the end of the breeding period (P<0.05).

The effect of day on a breeding period of 60 days was variable and revealing. Semen volume collected started low, but to our surprise reached up to 6 ml by the end of the period of evaluation. Spermatozoa motility was maintained elevated for the first 45 days of breeding and then decreased dramatically by day 60 of breeding period. Sperm concentration was also different. It was low at the beginning of the breeding period, increased reaching a peak by day 30th, and then decreased slowly by the end of the evaluation period.
<figure><img src="image_60.png" /><figcaption>**Figure 4:** Mean semen volume recovered following breeding a receptive female for a period of 60 days.</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>**Figure 5:** Mean spermatozoa motility of male alpacas for a period of 60 days.</figcaption></figure>
<figure><img src="image_62.png" /><figcaption>**Figure 6:** Mean spermatic concentration of male alpacas that bred for a period of 60 days.</figcaption></figure>

It is the first time that alpaca semen was evaluated for 60 days; however, our results are comparable to previous work on alpacas (Bravo, Flores, and Ordoñez, 1997; Bravo, 2002) and llamas (vonBaer, 1998). Altogether, these results reflect that males may have a relative small pool of response to the length of breeding period. By the end, samples were less concentrated than at the beginning and volume increased. It should be remembered that alpacas have small testicles than other livestock species, and also may indicate a limiting factor on the testicular response to the number of receptive females.

Semen sample was not collected on 17 times, which represents 7 % of total of collecting times. There was no difference, P>0.05, on lack of semen collection between aged-group males, 4 times on young males, 8 times on adult males and 5 times on old males. The fact that sometimes semen samples were not collected was real and is not significant, neither this lack of semen collection is different in the three group of males used in this study. It is unknown why sometimes semen is not collected, but also was reported whenever an artificial vagina was used in alpacas (Bravo, Flores and Ordoñez, 1997).

## Conclusion

In summary, semen characteristics were different in the three group of males used in this study. In addition, the length of the breeding period has definitively an effect on semen volume, spermatozoa motility and sperm concentration. The results of this study may have to be taken into consideration to evaluate the fertility of the male alpaca.
References

Alarcon, V., Garcia, W., Bravo, P.W. 2012, Inseminación artificial de alpacas con semen colectado por aspiración vaginal y vagina artificial. Revista Investigaciones Pecuarias Peru 23:58-64

Bravo, P.W., Flores, D., Ordoñez, C. 1997, Effect of repeated collection on semen characteristics of alpacas. Biology of Reproduction 57:520-524

Bravo, P.W., 2002, Male reproduction. In: The reproductive process of South American camelids. Seagull Printing. Salt Lake City, USA. pp. 49-64

Condorena, N., Velasco, J. 1979, Comparación de dos sistemas de empadre en la alpaca. Revista Investigaciones Pecuarias 4:47-54

Neely, D.P., Bravo, P.W. 1998, Reproductive evaluation and infertility in the male llama and alpaca. In: Current therapy in large animal theriogenology. RS Youngquist, Editor. WB Saunders Co. Pp. 787-792

Novoa, C., Sumar, J., Leyva, V., Fernandez-Baca, S. 1973, Incremento reproductivo en alpacas de explotaciones comerciales mediante el método de empadre alternado. Revista Investigaciones Pecuarias 3:85-87

Urquieta, B., Flores, P., Muñoz C., Bustos-Ogregon, C., Garcia-Huidobro, J. 2005, Alpaca semen characteristics under free and direct mounts during a mating period. Animal Reproduction Science 90:329-339

Von Baer, L., Hellemann, M.V. 1998, Variables seminales en llama (Lama glama). Archv. Medicina Veterinaria Chile. 30:171-176
# The Sperm Chromatin Dispersion Assay (HALO Test) Correlates with the Tunel Technique in Llama Sperm

M.I. Carretero<fnref n="1" />,<fnref n="4" />, F.G. Fumuso<fnref n="1" />,<fnref n="4" />, S.M. Giuliano<fnref n="2" />, D.M. Neild<fnref n="1" />, P. Cetica<fnref n="3" />,<fnref n="4" />
and M.H. Miragaya<fnref n="1" />

Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal:

E-Mail: ignaciacarretero@fvet.uba,ar

**Abstract.** Evaluation of routine seminal characteristics is used to predict fertility but cannot always reveal defects in genome integrity. Evaluation of sperm DNA becomes important for assessing ejaculates prior to applying assisted reproduction techniques. To evaluate llama sperm DNA fragmentation, we set up the Sperm Chromatin Dispersion assay (SCD). This technique does not require costly equipment and can be prepared using lab supplies without the need to buy special kits. Due to its objectivity and reliability, the TUNEL (terminal deoxynucleotidil transferase mediated dUTP nick end labelling) technique has become the assay of choice to measure sperm DNA fragmentation. The objective of this study was to use SCD to evaluate llama sperm DNA fragmentation in raw semen and correlate this technique with TUNEL. Sixteen ejaculates were obtained from 5 males using electroejaculation under general anesthesia. The DNA fragmentation was evaluated using SCD and TUNEL. Semen incubation with 0.3 M NaOH during 5 min was used in both techniques as a control of DNA fragmentation. Pearson's correlation test was used for statistical analysis. Sperm DNA fragmentation values were 13.7±6.3% and 8.6±5.6% for SCD and TUNEL respectively and in both assays, NaOH produced DNA fragmentation in 100% sperm. Despite the greater fragmentation values in SCD compared to TUNEL, a highly positive correlation was observed between the techniques (r=0.80, p=0.0002). These results indicate that llama sperm DNA evaluation with SCD gives similar results to TUNEL and so could be used in andrology laboratories that don't have expensive equipment and even in high altitude labs, such as those in the Puna region.

**Resumen.** La evaluación de las características seminales de rutina es utilizada para predecir fertilidad. Sin embargo, puede no revelar defectos en la integridad del genoma. La evaluación del daño del ADN espermático constituye una herramienta potencial para eval-

<footnote n="1">¹Cátedra de Teriogenología,</footnote>

<footnote n="2">²Cátedra de Física Biológica,</footnote>

<footnote n="3">³Cátedra de Química Biológica,</footnote>

<footnote n="4">⁴Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
Argentina.</footnote>
uar los eyaculados antes del uso de técnicas de reproducción asistida. Para evaluar la frag-
mentación del ADN espermático de llama, se puso a punto la técnica de dispersión de la
cromatina espermática (Sperm Chromatin Dispersion assay, SCD). Esta técnica no requiere
de equipamiento costoso y puede realizarse mediante insumos de laboratorio, sin la nece-
sidad de la compra de kits costosos. Debido a su objetividad y confiabilidad, la técnica de
TUNEL (terminal deoxynucleotidil transferase mediated dUTP nick end labelling) se ha
convertido en el ensayo de elección para medir la fragmentación del ADN espermático. El
objetivo de este estudio fue utilizar el SCD para evaluar la fragmentación del ADN de
espermatozoides de llama y correlacionar esta técnica con el TUNEL. Un total de dieciséis
eyaculados fueron obtenidos de 5 machos utilizando electroeyaculación bajo anestesia
general. La fragmentación del ADN fue evaluada a través de SCD y TUNEL. La incu-
bación del semen con NaOH 0,3 M durante 5 min fue utilizada como control de frag-
mentación del ADN en ambas técnicas. Para el análisis estadístico se utilizó la correlación
de Pearson. Los valores de fragmentación del ADN fueron 13,7±6,3 % y 8,6±5,6 % para
SCD y TUNEL, respectivamente. En ambas técnicas el NaOH produjo un 100 % de
espermatozoides con fragmentación del ADN. A pesar de observar mayores valores de
fragmentación en el SCD en relación al TUNEL, se observó una correlación altamente
positiva entre ambas técnicas (r=0,80; p=0,0002). Estos resultados indican que se observan
porcentajes de espermatozoides con ADN fragmentado similares con el SCD y el TUNEL,
y por lo tanto, podría utilizarse en laboratorios de andrología que no posean equipamiento
costoso e incluso en laboratorios situados a altas latitudes como la región de la Puna.

Keywords: llama, sperm, SCD, TUNEL

**Introduction**

Evaluation of routine seminal characteristics (concentration, motility, morphology and membranes) is used to predict fertility but they do not assess all aspects of sperm function. Poor sperm DNA integrity can negatively affect sperm fertilizing capacity in vitro (Henkel et al., 2004). Thus, evaluation of DNA becomes important for assessing ejaculates prior to applying assisted reproduction techniques. Cortés-Gutierrez et al., (2007) classified the techniques for evaluating sperm DNA fragmentation into two groups. The first group includes the methods used to mark single and double strand DNA breaks, for example, when enzymes are used to incorporate nucleotides in situ, such as Terminal dUTP Nick-End Labelling (TUNEL) or *in situ* Nick Translation (ISNT). Taking into account that DNA fragmentation increases chromatin susceptibility to denaturation, the second group of methods include those that measure this susceptibility after treatment. This group includes the Sperm Chromatin Structure Assay (SCSA), DNA Breakage Detection-Fluorescence *in situ* Hybridization (DBD-FISH), Single-Cell-Gel Electrophoresis (SCGE) or Comet assay and the Sperm Chromatin Dispersion test (SCD) or Halo test. Many of these techniques are laborious, some depend on the irregular activity and accessibility of enzymes to DNA ruptures (Fernández et al., 2005) while others are costly and require complex equipment. To evaluate llama
sperm DNA fragmentation, we previously set up the SCD by optical microscopy (Carretero et al., 2012) and the TUNEL assay by flow cytometry and fluorescence microscopy (Carretero et al., 2015). The SCD technique does not require costly equipment and can be prepared using lab supplies without the need to buy special kits. On the other hand, due to its objectivity and reliability, the TUNEL technique has become the assay of choice to measure sperm DNA fragmentation.

The objective of this study was to use SCD to evaluate llama sperm DNA fragmentation in raw semen and correlate this technique with the TUNEL assay.

# Materials and Methods

## Animals and location

The study was carried out at the Faculty of Veterinary Sciences of the University of Buenos Aires, in Buenos Aires, Argentina. The city is situated at sea level, latitude 34° 36' and longitude 58° 26'. For the study, 5 male *Lama glama* ranging between 8 and 10 years of age and weighing 147.5 ± 14.10 kg (mean ± SD) were used. Animals were kept out at pasture in pens and supplemented with bales of alfalfa; they also had free access to fresh water throughout the study. All males were shorn during the month of November.

## Semen collection

Semen collections were carried out between the months of April and October. Sixteen ejaculates were obtained from 5 males using electroejaculation under general anesthesia according to the technique described by Director et al. (2007). All procedures were approved by the Committee for the Use and Care of Laboratory Animals (CICUAL) of the Faculty of Veterinary Sciences, University of Buenos Aires (Protocol 2010/24).

## Seminal characteristics evaluation

The following routine seminal characteristics were evaluated in raw semen: volume, thread formation, concentration, sperm motility, membrane function and membrane integrity (viability). The ejaculate volume and thread formation were evaluated with a micropipette. Sperm numbers were calculated using a Neubauer hemocytometer. Sperm motility was evaluated using a phase contrast microscope (100×) and a warm stage (37 °C). The hypoosmotic swelling (HOS) test was used for assessing membrane function, and the fluorochromes 6-Carboxyfluorescein Diacetate (CFDA) and Propidium Iodide (PI) were used for assessing membrane integrity (viability). These techniques were carried out according to Giuliano et al. (2008).
*Sperm DNA evaluation*

To reduce thread formation and improve handling, each ejaculate was diluted 4:1 in HEPES-TALP medium with 0.1 % collagenase and incubated at 37 °C for 4 min. Then, samples were centrifuged for 8 min at 800 × g and the pellets were resuspended in PBS (Giuliano et al., 2010).

The SCD assay was carried out according to Carretero et al. (2012) to evaluate the degree of DNA fragmentation. Briefly, each sperm suspension was mixed with low-melting-point aqueous agarose and pipetted onto a glass slide. Each slide was incubated in different lysing solutions, dehydrated in sequential ethanol baths and stained with Giemsa. A minimum of 200 sperm heads were observed under bright-field microscopy (1000x). Sperm were classified into four patterns according to the size of the halo: i) nuclei with large DNA dispersion halos; ii) nuclei with medium halos; iii) nuclei with small halos and iv) nuclei with no halo. The first two patterns (i and ii) were considered sperm without DNA fragmentation and the other two (iii and iv) sperm with DNA fragmentation.

The TUNEL assay was carried out using the Roche In Situ Death Detection Fluorescein® kit according to Carretero et al. (2015). Briefly, samples were fixed in 4% paraformaldehyde in PBS during 15 min at 4 °C, then were centrifuged and permeabilized in 0.1 % Triton X-100 in 1 % sodium citrate for 30 min at room temperature. After that, samples were centrifuged and incubated with the TUNEL reaction during 1 h at 37 °C in darkness. A minimum of 200 spermatozoa were evaluated per sample using an epifluorescence microscope with a rhodamine and standard fluorescein filter set. Sperm showing bright green fluorescence represented damaged cells (TUNEL positive), in which dUTP was incorporated to DNA breaks, in contrast to non-stained cells representing non-damaged sperm (TUNEL negative). Semen incubation with 0.3 M NaOH during 5 min at room temperature was used in both techniques as a control of DNA fragmentation.

*Statistical analysis*

Pearson's test was used to correlate the SCD and TUNEL assays. All statistical analyses were performed using the R 2.2.1 Program (2005).

*Results*

The percentages of routine seminal characteristics were (median ± SD): volume: 3.4 ± 2.7 ml; oscillatory motility: 23.7 ± 18.2%; concentration: 56.1 ± 52.1 x 106 sperm/ml; membrane function: 29.8 ± 7.4% and membrane integrity (viability): 47.5 ± 13.2%. All samples showed thread formation, which disappeared after incubation with collagenase.

Sperm DNA fragmentation values were 13.7 ± 6.3 % and 8.6 ± 5.6 % for SCD and TUNEL, respectively. In both assays, 0.3 M NaOH produced DNA
fragmentation in 100 % sperm. A highly positive correlation was observed between
the SCD and TUNEL techniques (r= 0.80; p= 0.0002).

Discussion

Many of the techniques that evaluate sperm DNA are laborious and costly, requiring complex equipment such as flow cytometers and/or fluorescence microscopes. However, more simple techniques, that don't require complex equipment, exist. Two such are the Toluidine blue stain for evaluating the degree of chromatin condensation and the SCD assay for evaluating DNA fragmentation. In our laboratory, both these techniques have been adapted for use in the South American Camelids as well as in horses, dogs and cats (Sardoy et al., 2008; Carretero et al., 2009; 2010a; 2010b; 2010c; 2012; Monachesi et al., 2014; Allera et al., 2016). However, due to its accuracy and dependability, the TUNEL technique has become one of the assays of choice to measure sperm DNA fragmentation. For this reason, our laboratory adapted the TUNEL assay by flow cytometer and fluorescence microscopy to evaluate llama sperm DNA (Carretero et al., 2015). Various studies have been carried out to correlate different sperm DNA evaluation techniques in human andrology laboratories (Chohan et al., 2006; Zhang et al., 2010; Feijó et al., in press) and our aim was to do likewise for llama sperm.

The results for all raw semen parameters were within the normal range report-
ed for raw ejaculates in this species (Bravo et al., 2000; Aller et al., 2003; Giuliano
et al., 2008; Casaretto et al., 2012; Apichela et al., 2014; Carretero et al., 2015).

The percentages of DNA fragmentation observed with both techniques and in
the controls, were comparable to those obtained previously (Carretero et al., 2012;
2015). Despite the greater fragmentation values obtained using the SCD assay as
compared to TUNEL, a statistically significant positive correlation was observed
between the two techniques for llama sperm DNA fragmentation. Similarly, in
human sperm a significant correlation between TUNEL and SCD had also been
observed (Zhang et al., 2010; Feijó et al., in press). Chohan et al. (2006) not only
observed correlation between TUNEL and SCD, but also between these two tech-
niques and the Sperm Chromatin Structure Assay (SCSA) in human spermatozoa.
In addition, Fernández et al (2003) confirmed SCD test results for sperm DNA
fragmentation in porcine, using breakage DBD-FISH. Sperm with a very small or
absent halo showed extensive DNA fragmentation using DBD-FISH. Unlike the
SCSA, TUNEL and DBD-FISH, the Sperm Chromatin Dispersion assay can be
performed without complex or expensive instrumentation such as flow cytometry
or fluorescence microscopy. We have shown that SCD can be carried out using
either fluorescent or bright-field microscopy. In our laboratory we adapted the
technique for evaluating equine and llama sperm (Carretero et al., 2010; 2012) and
found that bright-field microscopy was more reliable than fluorescence microsco-
py. The reason for this is because bright-field microscopy allows one to distinguish
more clearly the limits of the chromatin dispersion halos, permitting a more precise
classification of the different patterns (large, medium, small or no halos) (personal observations). In addition, evaluation with bright-field microscopy (as opposed to use of fluorescence) allows observation of the flagellum, thus confirming that in effect the halo belongs to a sperm cell and not any other DNA (Fernández et al., 2005).

The present study also demonstrated that the SCD test identified more sper-
matozoa with DNA damage than the TUNEL assay (13.7 ± 6.3 % and 8.6 ±
5.6 %, respectively) suggesting that the SCD test may be more sensitive. Similar
results were observed by Zhang et al. (2010) in sperm from infertile men (25.2 ±
10.2 % for SCD and 22.8 ± 6.9 % for TUNEL using fluorescence microscopy),
however these authors observed similar percentages between both techniques in
fertile men (13.6 ± 3.4 % and 12.9 ± 3.0 % for SCD and TUNEL, respectively).
Chohan et al. (2006) observed similar values of fragmentation using TUNEL by
fluorescence microscopy and SCD, both for infertile and fertile men (19.5 ± 1.3 %
and 20.4 ± 1.3 % for SCD and 11.1 ± 0.9 % and 10.8 ± 1.1 % for TUNEL, re-
spectively).

The TUNEL assay quantifies the percentage of sperm with fragmented DNA by labeling the strand breaks with TdT, whereas the SCD test involves acid denaturation, which generates single-stranded DNA motifs from DNA breaks and further deproteinization of nuclear proteins suppresses the formation of a halo. Though these assays use different principles and protocols, and despite the greater fragmentation values in SCD compared to TUNEL, nevertheless the highly positive correlation observed between the techniques in raw llama semen (r= 0.80; p= 0.0002) was encouraging and further validates the use of the simpler SCD for DNA evaluation in llama sperm.

Conclusion

These results indicate that DNA evaluation with SCD correlates with the TUNEL
assay and so could be used in andrology laboratories that don't possess expensive
equipment and could also be used in high altitude labs, such as those in the Puna
region, making DNA evaluation more readily available.

References

Aller, J.F.; Rebuffi, G.E.; Cancino, A.K.; Alberio, R.H. 2003, Influencia de la criopreservación sobre la motilidad, viabilidad y fertilidad de espermatozoides de llama (Lama glama) (Influence of cryopreservation on the motility, viability and fertility of llama spermatozoa (Lama glama). Archivos de zootecnia, 52:15-23.

Allera, C.; Comercio, E.; Gonzáles Vera, J.; Miragaya, M.; Carretero, M.I. 2016, Evaluación del ADN espermático felino mediante la tinción de azul de toluidina. Invet, 18(1): 58.
Apichela, S.; Argañaraz, M.E.; Giuliano, S.; Zampini, R.; Carretero, I.; Miragaya, M.; Miceli, D.C. 2014, Llama oviductal sperm reservoirs: involvement of bulbourethral glands. *Andrologia* 46(3):290-295.

Bravo, P.W.; Skidmore, J.A.; Zhao, X.X. 2000, Reproductive aspects and storage of semen in Camelidae. *Animal Reproduction Science* (62):173-193.

Carretero, M.I.; Giuliano, S.M.; Casaretto, C.I.; Gambarotta, M.C.; Neild, D.M. 2009, Evaluación del ADN espermático de llamas utilizando azul de toluidina. *InVet, 11*(1):55-63.

Carretero, M.I.; Giuliano, S.; Agüero A.; Pinto, M.; Miragaya, M.; Trasorras, V.; Egey, J.; Von Thungen J., Neild, D. 2010a Guanaco sperm chromatin evaluation using Toluidine Blue. *Reproduction Fertility and Development*, 22(1):310-310.

Carretero, M.I.; Lombardo, D.; Arraztoa, C.C.; Giuliano, S.M.; Gambarotta, M.C.; Neild, D.M. 2012, Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test. *Animal Reproduction Science*, 131 (1-2):63-71.

Carretero, M.I.; Arraztoa, C.C.; Casaretto, C.I.; Huanca, W.; Neild, D.M.; Giuliano, S.M. 2010b, Alpaca sperm chromatin evaluation using Toluidine Blue. In: Fibre production in South American camelids and other fibre animals. Lieke Boersma (Eds), Wageningen Academic Publisherspp. 141-144.

Carretero, M.I.; Arrazto, C.C.; Caldevilla, M.; Ferrante, A.; Lombardo, D.; Neild, D. 2010c, Chromatin Dispersion test in equine spermatozoa. *InVet* 12(2): 249.

Carretero, M.I.; Fumuso, F.G.; Giuliano, S.M.; Neild, D.M.; Cetica, P.; Miragaya, M.H. 2015, Evaluación del ADN en espermatozoides de llama mediante la técnica de TUNEL utilizando citometría de flujo. Resultados preliminares. Primer Congreso de la Sociedad Latinoamericana de Reproducción Animal. ISSN 978-9874586209, pág., 375-378.

Casaretto, C.; Martínez Sarrasague, M.; Giuliano, S.; Rubin De Celis, E.; Gambarotta, M.; Carretero, M.I.; Miragaya, M. 2012, Evaluation of Lama glama semen viscosity with a cone-plate rotational viscometer. *Andrologia*, 44:335-341.

Chohan, K.R.; Griffin, J.T.; Lafromboise, M.; De Jonge C.J.; Carrell, D.T. 2006, Comparison of Chromatin Assays for DNA Fragmentation Evaluation in Human Sperm. *Journal of Andrology*, 27(1):53-59.

Cortés-Gutiérrez, E.I.; Dávila-Rodríguez, M.I.; López-Fernández, C.; Fernández, J.L.; Gosálvez, J. 2007, Evaluación del daño del DNA espermático. *Actas Urológicas España*, 31(2): 120-131.

Director, A.; Giuliano, S.; Trasorras, V.; Carretero, M.I.; Pinto, M.; Miragaya, M. 2007, Electroejaculation in llama (Lama glama). *Journal of Camel Practice and Research*, 14(2):203-206.

Feijó, C.M.; Esteves, S.C.; in press., Diagnostic accuracy of sperm chromatin dispersion test to evaluate sperm deoxyribonucleic acid damage in men with unexplained infertility. *Andrology*, http://dx.doi.org/10.1016/j.fertnstert.2013.09.002.

Fernández, J.L.; Muriel, L.; Rivero, M.T.; Goyanes, V.; Vázquez, R.; Alvarez, J.G. 2003, The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation. *Journal of Andrology*, 24(1):59-66.

Fernández, J.L.; Muriel, L.; Goyanes, V.; Segrelles, E.; Gosálvez, J.; Enciso, M.; Lafromboise, M.; De Jonge, C. 2005, Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. *Fertility and Sterility*, 84(4):833-842.
Giuliano, S.; Director, A.; Gambarotta, M.; Trasorras, V.; Miragaya, M. 2008, Collection method, season and individual variation on seminal characteristics in the llama (Lama glama). Animal Reproduction Science, 104:359–369.

Giuliano, S.M.; Carretero, M.I.; Gambarotta, M.C.; Neild, D.M.; Trasorras, V.L.; Pinto, M.; Miragaya, M.H. 2010, Improvement of llama (Lama glama) seminal characteristics using collagenase. Animal Reproduction Science, 118 (1):98-102.

Henkel, R.; Hajimohammad, M.; Stalf, T.; Hoogendijk, C.; Mehnert, C.; Menkveld, R.; Gips, H.; Schill, W.B.; Kruger, T.F. 2004, Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertility and Sterility, 81:965–72.

Monachesi, N.; Carretero, M.I. 2014, DNA evaluation of raw dog semen using toluidine blue. Preliminary results. Invet, 16:166.

Sardoy, M.C.; Carretero, M.I.; Neild, D. 2008, Evaluation of stallion sperm DNA alterations during cryopreservation using toluidine blue. Animal Reproduction Science, 107:349-350.

Zhang, L.; Qiu, Y.; Wang, K.; Wang, Q.; Tao, G.; Wang, L. 2010, Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labeling assay. Fertility and Sterility, 94(3): 1027-1032.
# Teeth in Camelids: Myths, Facts and Problems

I. Gunsser

Aelas e.V., Römerstr.23, D-80801 Munich;
Llama & Alpaca Registries Europe (LAREU), P.O. Box 666, CH-3900 Brig-Glis
E-Mail: Ilona.gunsser@t-online.de

**Abstract.** Most owners of alpacas and llamas in Europe have small groups of these animals and most of them have no education in keeping and breeding agricultural animals. In addition, it is usually difficult for them to find correct information about alpacas and llamas in their native language. There are different tooth problems seen in camelids like prognatism, sharp fighting teeth, root infections and real or assumed problems with cheek teeth. In this paper “myths” about the reasons of these problems are presented and, in contrast, the real reasons for these problems are explained and some therapeutic proposals are added.

**Resumen.** La mayoría de los propietarios de alpacas y llamas de Europa poseen pocos animales y carecen de formación para mantenerlos y reproducirlos. Además, es difícil en-contrar información correcta sobre alpacas y llamas en su lengua materna. Se observan diversos problemas dentales en camélidos, como prognatismo, dientes filosos, infecciones de las raíces y problemas reales o no en los carrillos. En este trabajo se presentan "mitos" sobre las razones de estos problemas mostrando de manera contrasada la verdadera natura-leza de estos problemas, sugiriendo propuestas terapéuticas.

**Keywords:** camelid teeth, anatomy, function, myths

## Introduction

Alpacas and llamas are kept in Europe since more than 20 years, but up to now breeding these species is a niche. Most owners have only small groups of these animals, and most of them have no education in keeping and breeding agricultural animals. In addition, it is usually difficult for the owners to find correct information about alpacas and llamas in their native language. The nutrition of camelids consists of grass and hay and they can ruminate. This eating technique is very efficient but it requires correct and healthy teeth. There are different tooth problems seen in camelids and one can find various “explanations”, better “myths”, for these tooth problems.
## Eating Facts

Camelids eat mostly grass, hey and minerals. They cut the grass with their incisors and use their lips to transport the grass (or hey) into the mouth. Then they chew the grass (hey) with the molar teeth as long as the “balls” reach a size which is the correct size to be swallowed. Later they ruminate the content of C1, chew it and swallow it again.

## Anatomy of Camelid Teeth

Alpacas and Llamas have incisor (I), canine (C) and cheek (P/M) teeth. The third incisor and the canine tooth in the maxilla and the canine tooth of the mandibular of each side are considered as fighting teeth.

The dental formula of each side in deciduous teeth is:

1-2 Jd, 1 Cd, 3 Pd

3 Jd, 1 Cd, 2 Pd

The dental formula of each side in permanent teeth is:

1 J, 1 C, 2 P, 3 M

3 J, 1 C, 1 P, 3 M

<figure><img src="image_63.png" /><figcaption>**Figure 1:** Skull of an adult male llama. Photo: Gunsser</figcaption></figure>

Figure 1 shows the skull of a male adult llama with correct incisors and cheek teeth. In intact male alpacas and llamas the fighting teeth are more prominent than in females or geldings castrated before the influence of testosterone (1 – 1.5 years of age).
The incisors of alpacas are relatively long compared to the incisors of llamas. In llama incisors, the whole tooth is covered by enamel. In alpacas only the labial part of the incisors is covered by hard enamel. The lingual part of the incisors consists of softer dentin.

Figure 2 explains the differences seen in llama and alpaca incisors. Because of the softer dentin on the lingual part of the alpaca incisor the teeth are more easily worn down. For that reason incisors of alpacas are longer than llama incisors because they move up more to restore the contact with the dental pad.

<figure><img src="image_64.png" /><figcaption>**Figure 2:** Left incisor llama, right incisor alpaca. Photo: Gunsser</figcaption></figure>

*Myth: "Incisors grow a life long"*

One such myth concerns the prognatism of incisors in some alpacas, stating that the incisors are growing during the whole life of the animal and that they protrude from the mouth because the grass and hay in Europe is not hard enough to keep them in the correct length. Fact is that, if used, the very long incisors move up during the lifetime of the animal to restore the contact with the dental pad (like in other species), but they do not grow permanently. The incorrect position of the incisors prevents contact with the dental pad and the dentin is not sufficiently worn down. Prognatism is an inherited problem. Too long prognate incisors impede the movement of the lips (Fig. 3) and make the cutting of grass and the transport of hay into the mouth difficult.
<figure><img src="image_65.png" /><figcaption>**Figure 3:** Long incisors impede movement of lips. Photo: Gunsser</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>**Figure 4:** Incisor before (left) and after (right) cutting to restore a correct bite. Photo: Gunsser</figcaption></figure>

Therefore cutting the incisors and restoring the correct bite (Fig.4) may in some cases help for a limited time. But one has to consider that there is a limited length of the incisors. Controlled breeding selection for anatomical correct teeth would be the better solution.

*Myth: "Cutting fighting teeth without anaesthesia does no harm to the animal"*

Another myth is that cutting fighting teeth without anesthesia is admissible. Fact is that fighting teeth have a pulpa with nerves, but flight animals do not express pain by screaming when the tooth is cut. Cutting teeth without anesthesia is not compatible with animal welfare. In Fig. 5 an example of a fighting tooth is cut
open to make visible the pulp canal. The pulp canal is usually filled with blood vessels and nerves.

<figure><img src="image_67.png" /><figcaption>**Figure 5:** Opened fighting tooth to show pulp canal. Photo: Gunsser</figcaption></figure>

*Myth: "Sharp points of cheek teeth are the reason for not eating"*

Alpacas and llamas cannot open their mouths very wide and therefore the control of premolar and molar teeth by the animal owner or by the veterinarian is not easy. But the control is necessary, for example, when the animals do not want to eat. Young and middle age animals have cheek teeth with an oblique, very sharply pointed surface, necessary to chew the hay and grass. Fig. 6 shows a prepared skull with cheek teeth of the upper and lower jaw. The teeth in the mandibular are smaller than the teeth in the maxilla. While chewing, the mandibular teeth move oblique along the maxilla teeth and form the grass/hey to balls. With increasing age of the animal the surface of the cheek teeth will flatten out in the middle of the surface, the lingual edge (mandibular) and the labial edge (maxilla) will stay pointed for longer time. Some veterinarians (mostly horse practitioners) believe that these points may be a problem for the animal and will want to grind them off. But grinded teeth (like old used teeth) inhibit correct chewing. Insufficiently chewed
balls cannot be swallowed. Grinding of a cheek tooth is only necessary if the position of the tooth has changed and is going to block the movement of chewing.

<figure><img src="image_68.png" /><figcaption>**Figure 6:** Cheek teeth with sharp points. Photo: Gunsser</figcaption></figure>

*No Myth but problems: Root infections*

The most important cheek teeth are the three molar teeth in the maxilla and mandibula of each side. Infections of the roots are a big problem for the camelids. Fig. 7 shows as example an x-ray of a young alpaca with infection in the region of the roots of pd1 and pd2 of the mandibula. Since root infections are seen mostly in more than one tooth, extractions may lead to chewing problems, because the teeth in the counterparts are missing. So regular tooth and mandibular/maxilla control and, if necessary, early conservative therapy should be preferred to conserve the functioning of the teeth as long as possible.

In old animals worn and lost teeth are the main reason for nutrition problems. Alpacas and llamas use the same eating techniques as ruminants, they chew everything twice. For this reason functioning teeth are very important. Most old animals die of starvation because they have lost cheek teeth or their teeth do not have sharp points to chop the food. Fig. 8 shows a mandibular of an old llama with missing cheek teeth and missing incisors.
<figure><img src="image_69.png" /><figcaption>**Figure 7:** X-ray of alpaca cheek teeth with infection of the roots. Photo: Gunsser</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>**Figure 8:** Mandibula old llama, 2 molars and all incisors lost. Photo: Gunsser</figcaption></figure>

## Conclusion

Since alpacas and llamas are plant eaters and ruminate, correct and healthy teeth are very important for these species. The lifetime of alpacas and llamas depends on the quality of their teeth. In view of animal welfare correct conformation with correct teeth should therefore be a breeding goal. For that reason owners should have correct information and no “myths” about the anatomy and functioning of the teeth, and should be aware of potential tooth problems.
<figure><img src="image_71.png" /></figure>
Nutrition

<figure><img src="image_72.png" /></figure>
<figure><img src="image_73.png" /></figure>
# Advances in Nutrition on Chinese Cashmere Goat: A Review

Sun Haizhoua, Li Shenglia, Zhang Chongzhia, Jin Lua, Sang Dana and Zhang Chunhuua

Institute of Animal Nutrition and Feed Research, Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Hohhot, 010018, China

E-Mail: sunhaizhou@china.com

**Abstract.** China has the world's largest number of cashmere goat populations, and produces over 50% the world's total cashmere. The quality of the cashmere is best, and output quantity is highest in the world. The Inner Mongolian, Liaoning and Hanshan cashmere goats are the most popular of the Chinese cashmere goats. Inner Mongolian cashmere goats are one of the best Cashmere goat breeds in China and the world. This paper reviews the progress in Vhinese cashmere goat nutrition research.

**Resumen.** China tiene la población más numerosa de cabra cashmere del mundo, y produce más del 50 % de la lana cashmere total del mundo. La calidad del cashmere es la mejor, y el volume de producción es el más alto del mundo. Las cabras cashmere de Mongolia Interior, Liaoning y Hanshan son las más populares en China. Las cabras cashmere de Mongolia Interior están entre las mejores razas de China y del mundo. En este trabajo se revisa el progreso en investigación nutricional de la cabra cashmere de China.

**Keywords:** cashmere goat, nutrition, hair follicle, diets evaluation

## Introduction

Inner Mongolian cashmere goats are one of the best Cashmere goat breeds in the world. Cashmere goat industry has made a great contribution in promoting local economic development and improving farmers' living standard through providing excellent cashmere raw material. For years, the cashmere goats' grazing and protecting the ecological environment of grassland have been in intense conflict due to overgrazing. The best choice to solve this problem is to carry out a confinement and semi confinement feeding instead of year-round grazing system only. But after the extension of the confinement feeding system, a considerable problem has been found: Feeding costs are increased and the economic returns are decreased compared with year-around grazing systems. Nevertheless, a profitable confinement system for cashmere goats can be established through manipulations
of nutrient partitioning. One is to develop techniques for using light duration or melatonin treatment, the other is to develop a basic technique of manipulating dietary nutrient balance. The purpose of this paper is to identify the nutritional factors responsible for increasing fiber production and economic returns.

## Dietary Nutrient Balance Technique

Feeding total mixed ration (TMR) is a widely-applied feeding strategy particularly on medium and large goat farms in China. The classic methods for evaluating TMR are in vivo techniques, but there are a lot of limitations because they are time-consuming, laborious and of poor accuracy. Now, it is popular using in vitro gas methods (Makkar et al., 2005) which allow to maintain experimental conditions more precisely than in vivo trials. It is a novel laboratory method utilizing a batch-culture, rumen-fluid, gas-fermentation system combined with mathematical curve-peeling techniques, allowing for the differentiation of rapid and slowly fermenting carbohydrate pools in TMR samples. Moreover, when animals are fed with TMR, digestive interactions can occur between single ingredients. These interactions, called associative effects, can modify the fermentation processes in the rumen in such a way that the response of cashmere goats to a combination of feed ingredients may differ from the components when considered individually.

A series of studies was undertaken to evaluate the associative effect on in vitro gas production from total mixed ration (TMR), based on alfalfa and corn-soybean concentrate, and separate TMR components (Table 1). These experiments showed that associative effects on gas production were much higher in early hours of incubation, and declined as time of incubation progressed, which is similar to results reported by Liu et al. (2002) in an in vitro system with sufficient N to support microbial growth. Associative effects commonly occur, when the digestion of one feedstuff is not independent of another (Niderkorn et al. 2009). Because microbial fermentation of individual and combined feedstuffs may diverge, the velocity of fermentation may be different, which may lead to a misjudgment of the risk for rumen disorders in cashmere goats.

In addition, the amount (ml) and relative proportions of gas produced by each pool (fast and slow pool, gases derived from the degradation of the rapid or slow digestion soluble fraction, respectively) can help characterize the fermentation (Johnston, 2009). Field experience with hundreds of herds experiencing production challenges indicates, that the vast majority of problem herds are fed a TMR with an excessively fast "fast pool" and a relatively slow "slow pool" (Figure 1). The results also showed the extremely high gas production in the TMR, indicating that the cause of the excessively fast "fast pool" was primarily due to the B2 (soluble fiber) pool producing lots of methane and carbon dioxide gas (along with acetate) from rapidly digested soluble fiber rather than from excessively fermentable starch (B1).
<table><caption>**Table 1:** Associative effects of gas production of three TMR diets</caption><thead><tr><th>NFC/NDF</th><th>Time (h)</th><th>Ratio TMR</th><th>Ratio Alfalfa</th><th>Ratio Concentrate</th><th>Calculated value</th><th>Associative effect (%)</th></tr></thead><tbody><tr><td rowspan="8">2.00</td><td>2</td><td>27.33</td><td>22.89</td><td>30.95</td><td>29.33</td><td>- 6.80</td></tr><tr><td>4</td><td>54.51</td><td>34.15</td><td>59.60</td><td>54.47</td><td>0.07</td></tr><tr><td>8</td><td>96.46</td><td>49.59</td><td>113.95</td><td>100.98</td><td>- 4.48</td></tr><tr><td>12</td><td>124.92</td><td>58.13</td><td>146.10</td><td>128.38</td><td>- 2.69</td></tr><tr><td>24</td><td>178.14</td><td>72.98</td><td>211.11</td><td>183.28</td><td>- 2.81</td></tr><tr><td>30</td><td>185.58</td><td>77.12</td><td>215.93</td><td>187.96</td><td>- 1.26</td></tr><tr><td>48</td><td>191.18</td><td>89.05</td><td>221.99</td><td>195.20</td><td>- 2.06</td></tr><tr><td rowspan="8">2.35</td><td>2</td><td>43.40</td><td>22.89</td><td>39.68</td><td>36.97</td><td>17.39</td></tr><tr><td>4</td><td>79.60</td><td>34.15</td><td>74.23</td><td>67.76</td><td>17.47</td></tr><tr><td>8</td><td>132.31</td><td>49.59</td><td>136.18</td><td>122.22</td><td>8.26</td></tr><tr><td>12</td><td>153.14</td><td>58.13</td><td>164.41</td><td>147.27</td><td>3.98</td></tr><tr><td>24</td><td>184.45</td><td>72.98</td><td>210.41</td><td>188.24</td><td>- 2.02</td></tr><tr><td>30</td><td>191.86</td><td>77.12</td><td>222.63</td><td>199.16</td><td>- 3.66</td></tr><tr><td>48</td><td>199.74</td><td>89.05</td><td>230.21</td><td>207.44</td><td>- 3.72</td></tr><tr><td rowspan="8">3.00</td><td>2</td><td>33.36</td><td>22.89</td><td>31.06</td><td>29.83</td><td>11.85</td></tr><tr><td>4</td><td>64.31</td><td>34.15</td><td>59.60</td><td>55.75</td><td>15.35</td></tr><tr><td>8</td><td>116.21</td><td>49.59</td><td>117.41</td><td>107.16</td><td>8.44</td></tr><tr><td>12</td><td>153.56</td><td>58.13</td><td>163.32</td><td>147.42</td><td>4.17</td></tr><tr><td>24</td><td>219.17</td><td>72.98</td><td>232.67</td><td>208.52</td><td>5.11</td></tr><tr><td>30</td><td>222.14</td><td>77.12</td><td>241.32</td><td>216.49</td><td>2.61</td></tr><tr><td>48</td><td>228.25</td><td>89.05</td><td>246.80</td><td>222.95</td><td>2.38</td></tr></tbody></table>
<figure><img src="image_74.png" /></figure>

<table><thead><tr><th colspan="3">Relative pool contributions (NFC/NDF=2.00)</th></tr><tr><th></th><th>Gas(ml)</th><th>% of total</th></tr></thead><tbody><tr><td>Fast pool</td><td>23.03</td><td>30.50</td></tr><tr><td>Slow pool</td><td>52.48</td><td>69.50</td></tr><tr><td>2-pool total</td><td>75.51</td><td></td></tr></tbody></table>

<figure><img src="image_75.png" /></figure>

<table><caption>**Figure 1:** Application of a logistic-dual pool model to divide the cumulative gas production of three TMR diets during the incubation period</caption><thead><tr><th colspan="3">Relative pool contributions (NFC/NDF=2.35)</th></tr><tr><th></th><th>Gas(ml)</th><th>% of total</th></tr></thead><tbody><tr><td>Fast pool</td><td>43.03</td><td>54.43</td></tr><tr><td>Slow pool</td><td>36.02</td><td>45.57</td></tr><tr><td>2-pool total</td><td>79.04</td><td></td></tr></tbody></table>
<figure><img src="image_76.png" /></figure>

<table><thead><tr><th colspan="3">Relative pool contributions (NFC/NDF=3.00)</th></tr><tr><th></th><th>Gas(ml)</th><th>% of total</th></tr></thead><tbody><tr><td>Fast pool</td><td>25.32</td><td>28.02</td></tr><tr><td>Slow pool</td><td>65.03</td><td>71.98</td></tr><tr><td>2-pool total</td><td>90.35</td><td></td></tr></tbody></table>

**Figure 1 (continued):** Application of a logistic-dual pool model to divide the cumulative gas production of three TMR diets during the incubation period

## Regulation Technology of Cashmere Growth

The growth progress of cashmere in goat which is affected by photoperiod, nutrition, management, environment and genetic factors (Sun et al., 1998; Wang, 2004; Zhang et al., 2016), exhibits a seasonal pattern arising from circannual changes in the natural photoperiod, it also has been confirmed that the number, function and activity of the hair follicle cells in the skin influence cashmere growth (Sun et al., 1998; Duan et al., 2015; Zhang et al., 2017). However, the hair follicles in the skin undergo recurrent cycling of controlled anagen, catagen and telogen with a defined periodicity. The short duration of sunshine accelerates the development of hair follicles while the long duration of sunshine retards the development of hair follicles until it stops. For many years, a lot of techniques and methods have been explored to improve cashmere production. Until now, photoperiod alteration and melatonin (MT) implantation have been the best methods used. It was found that altering the photoperiod and implanting melatonin had potential use in cashmere goat feeding (Wang, 2004). More melatonin which was secreted by the pineal gland in short daily photoperiod might be the key factor for increased activity of secondary hair follicles (Wang, 2004; Wang et al., 2007; Hardeland et al., 2012; Liu et al., 2017). The study inferred that the secondary hair follicle activity of cashmere goats was increased under short daily photoperiod conditions, which might contribute to the pineal gland secreting
more melatonin (Zhang et al., 2017). In recent years, the skin has emerged as a model for studying the circadian clock regulation of the hair follicle cycle process (Tanioka et al., 2009; Lin et al., 2009; Plikus et al., 2015; Hardman et al., 2015). Previous studies of Liaoning cashmere goat have emphasized that not only Bmal1, Clock, Cry1, Dbp, Nr1d1 were expressed in the skin, but also Clock genes expressions modified the secondary follicle cycle process (Shu, 2014). As these mitotic rhythms partly persist even in constant darkness, they might be under control of an endogenous clock. Recently, in liver (Eckel-Mahan et al., 2013; Jacobi et al., 2015; Dang et al., 2016), the circadian clock genes were shown to control the nutrients digestibility in goats (articles in press). Consequently, from my own perspective, providing a novel strategy for differential “peripheral clock” to research cashmere growth is a new development direction. The progression in photoperiod alteration, melatonin (MT) implantation and nutrition level used in cashmere goat feeding in 1998-2007 is collected in Table 2.

## Regulation Technology of Functional Amino Acids

Amino acids have enormous physiological importance, serving as building blocks for proteins and substrates for synthesis of low-molecular-weight substances (Wu et al., 2014). Especially, amino acids content in the diet during pregnancy are one important modifiable factor that can have a substantial influence on the viability and body composition of the newborn (Symonds et al., 2010). Furthermore, amino acids in histotroph of goat can activate mTOR cells signaling that stimulates migration, hypertrophy and hyperplasia of cells of the conceptus (Bazer et al., 2015).

The study indicated that the diet supplemental rumen-protected leucine (RPLeu) and HMB-Ca increased nitrogen intake, retained nitrogen and daily gain, and tended to decrease urine urea nitrogen excretion. RPLeu and HMB-Ca not only induced protein synthesis in skeletal muscle (Guo et al., 2009), but also played an important role in enhancing the body non-specific immune function (Guo et al., 2010). Gu et al. (2012) demonstrated that dietary N-Carbamylglutamate (NCG) supplementation increased meat rate of carcass and intramuscular fat, while dietary HMB-Ca supplementation increased the muscle fatty acids and decreased intramuscular fat in goats (Gu et al., 2012; Gu, 2012). Moreover, Wu et al. (2017) demonstrated that dietary arginine supplementation could help fetal growth and development, increase amino acid concentration in the blood, and induce the genes expression (OPN, VEGF, SLC7A and so on) of related conceptus development in the goat (Wu et al., 2017).
<table><caption>**Table 2:** The progress in photoperiod alteration, melatonin (MT) implantation and nutrition level using in cashmere goat feeding.</caption><thead><tr><td>Target tissue</td><td>Test result</td><td>Author</td></tr></thead><tbody><tr><td>Skin</td><td>The cashmere production and fiber length in Inner Mongolia Arbas white cashmere goat were significantly affected by protein and energy levels in the diet.</td><td>Sun et al., 1998</td></tr><tr><td>Skin</td><td>Study on the growth and activity changes of hair follicle in skin of Inner Mongolia Arbas white cashmere goat.</td><td>Sun et al., 1998</td></tr><tr><td>Tissue and organs</td><td>Experimental design to study the effects of different dietary MG levels on nutrient partitioning among tissue and organs in Cashmere goats.</td><td>Su, 2003</td></tr><tr><td>Gastrointestinal tract</td><td>The nitrogenous compounds partitioned more to cashmere growth when goats were treated by short daily photoperiod or melatonin implanted goats.</td><td>Wang, 2004</td></tr><tr><td>Gastrointestinal tract</td><td>The nitrogenous compounds partitioned more to body deposition when goats were treated by long daily photoperiod.</td><td>Wang, 2004</td></tr><tr><td>Skin and gastrointestinal tract</td><td>Not only the yield, diameter and length of cashmere fibres and nutrients digestibility of goat were changed significantly with short daily photoperiod, but also the cashmere growth performance and nutrients digestibility were improved significantly in high energy concentration diet.</td><td>Wang et al., 2007; Li et al., 2014; Kong et al., 2014; Zhang et al., 2017</td></tr><tr><td>Skin</td><td>Liaoning Cashmere goats have emphasized that not only Bmal1, Clock, Cry1, Dbp, Nr1d1 were expressed in skin, but also Clock genes expressions modified secondary follicle cycle process.</td><td>Shu, 2014</td></tr><tr><td>Blood</td><td>Analysis of metabolic pathways revealed that the energy levels affected valine, leucine, isoleucine biosynthesis, citrate cycle, glycolysis or gluconeogenesis, lactose metabolism, tyrosine metabolism, arginine and praline metabolism, cysteine and methionine metabolism, and the photoperiod influences oxidation of fatty acids, tyrosine metabolism, β-alanine metabolism, pyruvate metabolism.</td><td>Zhang et al., 2016</td></tr><tr><td>Skin</td><td>The intrinsic oscillating molecular clock (Bmal1, Clock, Cry1, Per1 and Rev-erbα five core clock genes) exists in skin hair follicles, which contributes to the regulation mechanisms of hair follicles cell proliferation.</td><td>Zhang et al., 2017</td></tr></tbody></table>
Nutrition of the Grazing Goat

Dry matter intake and botanical pasture composition are the keys to assess the nutritional status of theInner Mongolian Cashmere goats, so a series of studies were undertaken in different phases of pastures (sprout, growing and senescing period) (Sun, 1995; Bian, 1996; Guo, 2008). The results showed the seasonal variation of food intake and food composition of cashmere goats under natural grazing conditions using Alkane technique (Guo, 2008). The dry matter intakes (DMI) at different stages were 0.779, 0.845 and 1.632 kg/d respectively. Kenney et al. (1984) showed that the rate of intake of fresh forage increased as its water content increased, but the rate of dry matter intake declined once the dry matter content of the forage fell below 40 %. The feed intake of goats in the senescing period was higher than that of the sprout and growing period, which was also found in Liaoning Cashmere goats (Bian, 1996).

Pasture botanical composition were *Carex duriusula* 13.42 %, *Artemisia frigida Willd* 6.43 %, *Stipa breviflora* 71.85 %, *Iris bungei maxim* 3.93 % and *Peganum harmala* L 4.35 % in sprout period pasture and *Puncturevine Caltrop* Fruit 7.9 %, *Peganum harmala* L 42.28 % *Erodium stephanianum Willd* 8.78 %, *Allium victorialis* L 12.1 %, *Green Bristlegrass Herb* 8.56 % and *Poa alpigena* 20.4 % in growing period pasture, and *Sweet Wormwood* Herb 68.9 %, *Artemisia annua* L 13.3 % and *Carex duriusula* 17.82% in senescing period pasture, respectively (Table 3).

The nutritional values and dry matter intake of cashmere goats were affected greatly by the change of season (Figure 2), also for the nutrient digestibility of the herbage (Sun, 1995). When the nutrient digestibility of the herbage reached its peak, the grazing cashmere goats were fast growing.With the growth of the grass, the digestibility of various nutrients decreased and reached the lowest point in December or February respectively (Figure 3).
<table><caption>**Table 3:** Comparison of body weight, feed intake and pasture botanic composition in different pastures growing periods (means±SD).</caption><thead><tr><th>Item</th><th>Sprout</th><th>Growing</th><th>Senescing</th></tr></thead><tbody><tr><td>Body Weight (kg)</td><td>25.7±1.3<sup>a</sup></td><td>34.0±2.53<sup>b</sup></td><td>41.7±3.8<sup>c</sup></td></tr><tr><td>Feed Intake (kg DM/d)</td><td>0.779±0.23<sup>a</sup></td><td>0.845±0.31<sup>a</sup></td><td>1.632±0.21<sup>c</sup></td></tr><tr><td>Carex duriusula</td><td>13.42</td><td>—</td><td>17.82</td></tr><tr><td>Artemisia frigida Willd</td><td>6.43</td><td>—</td><td>—</td></tr><tr><td>Stipa breviflora</td><td>71.85</td><td>—</td><td>—</td></tr><tr><td>Iris bungei maxim</td><td>3.93</td><td>—</td><td>—</td></tr><tr><td>Peganum harmala</td><td>4.35</td><td>42.28</td><td>—</td></tr><tr><td>Puncturevine Caltrop Fruit</td><td>—</td><td>7.90</td><td>—</td></tr><tr><td>Erodium stephanianum Willd</td><td>—</td><td>8.78</td><td>—</td></tr><tr><td>Allium victorialis L</td><td>—</td><td>12.10</td><td>—</td></tr><tr><td>Green Bristlegrass Herb</td><td>—</td><td>8.56</td><td>—</td></tr><tr><td>Poa alpigena</td><td>—</td><td>20.40</td><td>—</td></tr><tr><td>Sweet Wormwood Herb</td><td>—</td><td>—</td><td>68.9</td></tr><tr><td>Artemisia annua L</td><td>—</td><td>—</td><td>13.3</td></tr></tbody></table>

The crude protein and energy intake of the grazing cashmere goat in each period can be calculated from the feed intake and nutrient content. The nutrient requirements of protein and energy of angora goats are shown in Figure 4 and Figure 5 (Sun, 1995). It can be seen, that in winter and spring season, the protein and energy intakes of grazing cashmere goats from the pasture were less than their protein and energy requirements, even though supplementary feeding (250 g/d) was provided.
<figure><img src="image_77.png" /><figcaption>**Figure 2:** Food intake of goats in different pastures growing periods</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>**Figure 3:** The apparent digestibility of the herbage in different pastures growing periods</figcaption></figure>
<figure><img src="image_79.png" /><figcaption>**Figure 4:** Daily crude protein intake of goats in different season</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>**Figure 5:** Daily energy intake of goats in different season</figcaption></figure>
## Rumen Fermentation Regulation

As the forage ratio increased, the pH value of rumen improved (Shi, 2008; Ma, 2008), and the nutrient passage rates showed that rumen Kp of feeding a 3:7 (concentrate to roughage ratio) diet was lower than that of feeding a 2:8 diet. After adding malate, Kp in the rumen decreased (Shi, 2008). With the increasing of the forage in the diets, the *Entodinium* decreased; however, the *Fibrobacter succinogenes*, *Ruminococcus flavefaciens* and *Ruminococcus albus* increased (Ma, 2008). Adding malate in the diets decreased rumen NH3-N concentration, lactate concentration and A/P ratio. The number of ruminal total bacteria, cellulolytic bacteria, and S. Ruminantium was higher than in the control group. Meanwhile, adding malate also could improve total flowing of DM, OM, NDF and ADF in the rumen, while the flowing of those in the duodenum was decreased. In other words, the amount of disappearance of NDF and ADF was increased in the gastric area. At the same time, daily gain and cashmere production of Cashmere goats was increased by adding malate in the diet (Shi, 2008). The gas production and methane emission of rumen were influenced strongly by addition of different doses of essential oil and antibiotics, and methane emission was decreased while gas production was increased (Xue, 2010).

## Nitrogen Digestion and Metabolism

The dietary nitrogen levels and partial ruminal defaunation have important effects on nitrogen metabolism, microbial protein synthesis and urea nitrogen recycling in the Inner Mongolia White Cashmere goats. Studies by Du (2010) have shown that the nitrogen intake, degradable nitrogen, retained nitrogen and retained nitrogen/metabolic body weight were increasing with the dietary crude protein increasing.

The ratios of retained nitrogen/metabolic body weight were significantly raised, but the plasma ammonia nitrogen was markedly reduced by feeding swelling corn compared to cracked corn. For the urea transporter-B (UT-B) expression in the gastrointestinal tracts, the high crude protein level of 13.5% in the diet could enhance the expression of UT-B protein in fore-stomach, ileum, cecum, caecum, and kidney, while inhibiting the expression of the protein in duodenum. However, the parotid gland, the abomasum and the liver were less affected. Fan (2014) reported that the appropriate reduction of diet N was a benefit to improve the utilization of N when the microbial protein met the animals' demand. Partial defaunation improves the utilization of N in goats fed a low nitrogen diet while feeding oscillating dietary CP benefits the utilization of N and microbial protein synthesis.

## Conclusion

There is presently an export demand for both goat cashmere and meat, and Chinese scientists and producers face the exciting prospect being able to produce more and better cashmere at less cost. There is an increase in knowledge on the various factors that limit the performances of cashmere goats. Each of the
improvements mentioned in the paper, including dietary nutrient balance technique, cashmere growth regulation technology, functional amino acids regulation technology, nutrition of the grazing goat, rumen fermentation regulation and nitrogen digestion and metabolism will improve the animal performance greatly by integrating the fundamental knowledge about cashmere goat nutrition and physiology.

## References

* Bazer FW, Wang XQ, Johnson GA & Wu GY, 2015, Select nutrients and their effects on conceptus development in mammals. Anim Nutr 1, 85-95.

* Bian DX, Meng ZY, Sun HR, 1996, A Study on inter month dynamics of grazing intake at the ewes of lioaning cashmere goat. Acta agrestia since 4, 173-179

* Dang F, Sun X, Ma X, Wu R, Zhang D, Chen Y, Xu Q, Wu Y & Liu Y, 2016, Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock. Nat Commun 7, 12696.

* Du JW, 2010, Study on Effects of various dietary on gastrointestinal tract UT-B expression of the inner mongolia white cashmere goat. Hohhot: Inner Mongolia Agricultural University.

* Duan C, Xu J, Sun C, Jia Z & Zhang W, 2015, Effects of melatonin implantation on cashmere yield, fiber characteristics, duration of cashmere growth as well as growth and reproductive performance of Inner Mongolian cashmere goats. J Anim Sci Biotechnol 6, 22.

* Eckel-Mahan KL, Patel VR, de Mateo S, Orozco-Solis R, Ceglia NJ, Sahar SS, Dilag-Penilla SA, Dyar KA, Baldi P & Sassone-Corsi P, 2013, Reprogramming of the circadian clock by nutritional challenge. Cell 155, 1464-1478.

* Fan YH, 2014, Effects of diet and rumen regulation on nitrogen metabolism, urea nitrogen recycling and microbial protein synthesis in growing white cashmere goats. Hohhot: Inner Mongolia Agricultural University.

* Gu Y, 2012, Effect of NCG and HMB on growing performance and meat quality of white cashmere goats and sheep. Hohhot: Inner Mongolia Agricultural University.

* Gu Y, Sun HZ, Zhao CF, Sang D, Zhang CH, Li SL, Shan D, Ren XP, Ling SL & Na R, 2012, Effect of HMB on growing performance and meat quality of white cashmere goats. 9th Chinese Sheep Development Conference.

* Guo JQ, Sun HZ, Sang D, Li JX & Zhao CF, 2010, Effects of rumen-protected leucine and β-hydroxy β-methylbutyrate-Ca on immune function of Inner Mongolian white cashmere goats. Chinese J Anim Nutr 22, 1762-1767.

* Guo JQ, 2009, Study on effects of leucine and HMB-Ca on immunity function and performance of Inner Mongolia white cashmere goats. Hohhot: Inner Mongolia Agricultural University.

* Guo XF, 2008, Studies on establishing of methane production prediction model and influencing factors on methane production of grazing in inner mongolian cashmere goats. Hohhot: Inner Mongolia Agricultural University.

* Hardeland R, Madrid JA, Tan DX & Reiter RJ, 2012, Melatonin, the circadian multioscillator system and health: the need for detailed analyses of peripheral melatonin signaling. J Pineal Res 52, 139-166.
Hardman JA, Haslam IS, Farjo N, Farjo B & Paus R, 2015, Thyroxine differentially modulates the peripheral clock: lessons from the human hair follicle. Plos One 10, 1-15.

Jacobi D, Liu S, Burkewitz K, Kory N, Knudsen NH, Alexander RK, Unluturk U, Li X, Kong X, Hyde AL, Gangl MR, Mair WB & Lee CH, 2015, Hepatic Bmal1 regulates rhythmic mitochondrial dynamics and promotes metabolic fitness. Cell Metab 22, 709-720.

Kenney PA & Black JL, 1984, Factors affecting diet selection by sheep I. Potential intake rate and acceptability of feed. Aust J Agric Res 35, 551-563.

Kearl L C, 1982, Nutrient requirements of ruminants in developing countries. Pp. 381. International Feedstuffs Institute. Utah Agricultural Experimental Station. Utah State University Logan 84 322, USA.

Kong XT, 2014, Effects of diet energy level on growth performance, nutrient digestibility and Methane yield in Shanbei white cashmere goats. Yangling: Northwest A & F University.

Li SL, Sun HZ, Zhao CF, Hao BYSHL, Gu Y, Sang D, Ling SL, Shan D & Hua L, 2014, Effects of photoperiod on nutrient digestibility of Inner Mongolia white cashmere goat. Acta Ecologiae Animalis Domastici 35, 28-32.

Lin KK, Kumar V, Geyfman M, Chudova D, Ihler AT, Smyth P, Paus R, Takahashi JS & Andersen B, 2009, Circadian clock genes contribute to the regulation of hair follicle cycling. PLoS Genet 5, 10573.

Liu Z, Gan L, Luo D & Sun C, 2017, Melatonin promotes circadian rhythm-induced proliferation through interaction of Clock/HDAC3/c-Myc in mice adipose tissue. J Pineal Res 62, 12383.

Liu JX, Susenbeth A & Sudekum KH, 2002, In vitro gas production measurements to evaluate interactions between untreated and chemically treated rice straws, grass hay, and mulberry leaves. J Anim Sci 80, 517-524.

Johnston JD & Tricarico JM, 2007, Practical implications of fiber in dairy rations: Making use of forage fiber. Proceedings of the 22nd Annual Southwest Nutrition & Management Conference. Feb. 22-23. Tempe, Ariz.

Makkar HPS, 2005, In vitro gas methods for evaluation of feeds containing phytochemicals. Animl Feed Sci and Technology 123, 291-302.

Ma HZ, 2008, The Effect of Different Forage to Concentrate Ratio on Rumen Fermentation and Microflora of Inner Mongolia Cashmere Goat Hohhot: Inner Mongolia Agricultural University.

Niderkorn V & Baumont R, 2009, Associative effects between forages on feed intake and digestion in ruminants. Animal 3, 951-960.

Plikus MV, Van Spvk EN, Pham K, Geyfman M, Kumar V, Takahashi JS & Andersen B, 2015, The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity. J Biol Rhythms 30, 163-182.

Shi LL, 2008, Study on Effects of Malate on Rumen Fermentation and Performance of Inner Mongolia White Cashmere Goats Hohhot: Inner Mongolia Agricultural University.

Shu G T, 2014, Study on secondary hair follicles annual cycle changes and the related circadian clock genes of Liaoning cashmere goat. Chinese Academy of Agricultural Sciences.
Su PC, 2003, Study on partitioning of proteins (amino acids) in white cashmere goats fed diets with different dietary metabolizable glucose Levels. Hohhot: Inner Mongolia Agricultural University.

Sun HZ, 1995, Studies on the Fleece Growth Patterns and Post-natal Follicle Development in AERBAS Cashmere Goats Under Natural Grazing Conditions, Hohhot: Inner Mongolia Agricultural University.

Sun HZ, Hou XZ, Yu ZH, Zhao ZG & Hong M, 1998, Effects of protein and energy level in diet on cashmere production performance of Arbas white cashmere goat. Chinese J Anim Sci and Production 19, 5-7.

Sun HZ, Hou XZ & Hong M, 1998, Study on the growth and activity changes of hair follicle in skin on Inner Mongolia Arbas white cashmere goat. Inner Mongolia Journal of Animal Science and Production 19, 3-6.

Symonds ME, Sebert SP & Budge H, 2010, Nutritional regulation of fetal growth and implications for productive life in ruminants. Animal 4, 1075-1083.

Tanioka M, Yamada H, Doi M, Bando H, Yamaguchi Y, Nishigori C & Okamura H, 2009, Molecular clocks in mouse skin. J Invest Dermatol 129, 1225-1231.

Wang LF, 2004, Effects of photoperiod and implanted melatonin on nitrogenous compounds partitioning, cashmere growth and its manipulation in Inner Mongolia white cashmere goats. Hohhot: Inner Mongolia Agricultural University.

Wang LF, Lu DX & Sun HZ, 2007, Effects of photoperiod and melatonin on nitrogen partitioning and body composition in Inner Mongolia white cashmere goats. J Anim Feed Sci 16, 576-580.

Wu BS, Sun HZ, Zhang CZ, Li SL, Sang D, Zhang CH, Jin L & Zhao CF, 2017, Effects of supplement arginine on conceptues growth and development in Inner Mongolian white cashmere goats. Chinese Journal of Animal Nutrition (in press).

Wu GY, Bazer FW, Dai ZL, Li DF, Wang JJ & Wu ZL, 2014, Amino Acid Nutrition in Animals: Protein Synthesis and Beyond. Annu Rev Anim Biosci 2, 387-417.

Xue XM, 2010, Study on Effects of Different Essential oils and Monensin On Rumen Fermentation of Inner Mongolia Cashmere Goat Hohhot: Inner Mongolia Agricultural University.

Zhang CZ, Sun HZ, Li SL, Sang D, Zhang CH & Jin L, 2016, The role of the circadian clock system in animal nutrition and metabolism. Acta Ecologiae Animalis Domastici 37, 1-8.

Zhang CZ, Sun HZ, Li SL, Sang D, Zhang CH, Jin L & Zhao CF, 2016, Effects of photoperiod and diet energy concentration upon hair follicle activity and blood metabolomics in Inner Mongolia white cashmere goats. IGA 2016 12th International conference on goats 2016,7.

Zhang CZ, Sun HZ, Li SL, Sang D, Zhang CH & Jin L, 2017, Effects of photoperiod and diet energy intervention on nutrient digestibility, hair follicles activity, cashmere production for cashmere goats. Acta Ecologiae Animalis Domastici 38, 19-24.

Zhang CZ, Sun HZ, Li SL, Sang D, Zhang CH, Jin L, Antonini M & Zhao CF, 2017, Effects of photoperiod and diet energy concentration upon nutrients digestibility, hair follicle activity and circadian clock genes contributed the regulation of hair follicle cycling in Inner Mongolia white cashmere goats. Livestock (in press).
<figure><img src="image_81.png" /></figure>
# Alfalfa Hay Supplementation to Improve Llama Meat Production for Smallholders in Pasco Region, Peru

G. Gutierrez<fnref n="1" />, A. Corredor<fnref n="1" />, R. Robles<fnref n="1" />, J. Mendoza<fnref n="1" />, V. Hidalgo<fnref n="1" /> and M. Wurzinger<fnref n="2" />

E-Mail: gustavogr@lamolina.edu.pe

E-Mail: maria.wurzinger@boku.ac.at

**Abstract.** This study was carried out in Ninacaca, Pasco, Peru at an altitude of 4,350 meters above sea level. The scope of the study was to evaluate the effect of two feeding systems: (i) grazing on native pasture and (ii) grazing on native pasture plus alfalfa hay supplementation, on live weight, weight gain, biometric measurements and carcass dressing. Live body weight and biometric measurements were recorded every 15 days from 32 male llamas (8-12 months old). At 100 days of the experiment llamas were slaughtered, thus cold and hot carcass weight were recorded. Models used for analyzing final weight, total weight gain and weight gain by period included the feeding system as fixed factor and initial weight as covariate. Significant differences (p<0.01) were found due to the effect of alfalfa hay supplementation on final weight and total weight gain. Also, significant statistical differences were found between live weights per period of different feeding strategies. Supplementation with alfalfa hay showed better weight gain response at 13, 27 and 41 days after the beginning of the experiment. Supplementation with alfalfa hay improves the growing performance of young llamas kept on natural pasture.

**Resumen.** Este estudio se realizó en Ninacaca, Pasco, Perú a una altitud de 4.350 metros sobre el nivel del mar. El objetivo del estudio fue evaluar el efecto de dos sistemas de alimentación sobre el peso corporal, la ganancia de peso, medidas biométricas y rendimiento a la canal: (i) pastoreo en pasto nativo y (ii) pastoreo en pasto nativo más suplementación con heno de alfalfa. El peso corporal y las medidas biométricas de 32 llamas machos (8-12 meses de edad), fueron medidos cada 15 días. Las llamas se sacrificaron a los 100 días del experimento y se midió el peso de carcasa fría y caliente. Los modelos utilizados para analizar el peso vivo final, ganancia de peso total y la ganancia de peso por periodos incluyeron al sistema de alimentación como efecto fijo y el peso vivo inicial como covariable. Se encontraron diferencias significativas (p<0,01) debidas al efecto de la suplementación con heno de alfalfa sobre el peso vivo final y ganancia de peso total. También se

<footnote n="1">¹Universidad Nacional Agraria La Molina – UNALM. Av. La Molina S/N. La Molina, Lima 12, Peru.</footnote>

<footnote n="2">²Division of Livestock Sciences, Department of Sustainable Agricultural Systems, University of Natural Resources and Life Sciences – BOKU. Gregor-Mendel-Str. 33, A-1180 Vienna, Austria.</footnote>
hallaron diferencias significativas entre los sistemas de alimentación evaluados para el peso vivo entre los períodos del experimento. La suplementación con heno de alfalfa tuvo mejores ganancias de peso a los 13, 27 y 41 días después del inicio del experimento. La suplementación con heno de alfalfa mejoró el crecimiento de llamas jóvenes críadas en pastos nativos.

**Keywords:** llama, nutrition, meat production, morphometric measurements, supplementation

## Introduction

In Peru, llama production is mainly located in the Center and South Andes at an altitude ranging from 3,800 to over 5,000 meters above sea level (Leyva, 1991; Westreicher, 2007). The production system faces different problems such as high mortality of offspring and poor meat quality due to the presence of sarcocystes. In addition, the impoverishment of grasslands of natural pastures due to overgrazing is another factor leading to low production (Buttolph and Coppock, 2004; Flores, 2012). All these limitations lead to low meat production and as a consequence the profitability for smallholders is also low (FAO, 2005).

Previous studies showed that smallholders in the Pasco Region in the Central Andes of Peru keep llamas mainly for meat production (Alvarez, 2001; Cano, 2009; Mendoza, 2013; Mendoza, 2015). Farmers usually sell older animals, which often demonstrate a high incidence of sarcocystis (Castro et al., 2004; Martin et al., 2016) and show low body condition. These factors reduce the value of sales and consequently the profits of the producer (Alva et al., 1980). Therefore, one main constraint for improving meat production in llamas is the availability of appropriate amount and quality of feed, mainly at the beginning of the dry season when most llamas are sold to the meat market. The aim of this study was to evaluate the effect of supplementation with alfalfa hay on body weight gain, biometric measurements and carcass weight in young male llamas selected for meat market.

## Materials and Methods

### *Study site and treatments*

The study was carried out on a smallholder community farm, located at 4,350 meters above sea level (latitude 10°45.524"S and longitude 76°3.338"W), in the district of Ninacaca, Province of Pasco, Peru. The annual average temperature is 4°C and the annual precipitation fluctuates between 650 and 900mm with a rainy season from November to March.

A total of 32 male llamas were randomly sampled from individual breeders in Pasco Province. Age of llamas ranged from 8 to 12 months. Llamas were identified with numbered ear tags and they were assigned randomly to the two treatments
(16 llamas per treatment). In treatment 1 only native pasture (NP) was provided and in treatment 2 native pasture was supplemented with alfalfa hay. The daily amount of alfalfa hay that each llama received was 30 % of dry matter requirements. The amount was calculated from the ME requirements for gaining about 200 g/day (NRC, 2007).

The llamas of the two treatments grazed together 9 hours per day (08:00 to 17:00) on the same native pasture. Llamas from treatment 2 were gathered in pens and alfalfa hay was offered daily from 17:00 onwards individually for each llama. Alfalfa hay intake was recorded daily. The experiment was carried out over a period of 100 days.

## Pasture characteristics

The native pasture area covered 5 ha and it was divided into three plots and fenced with eucalyptus poles of 2.20 m height and a barbed wire fence. Each plot was grazed for 15 days with a resting period of 30 days. The stocking rate was 5.2 llamas/ha based on the calculation according to Flórez and Malpartida (1987). Native pasture was mainly composed of Festucadolichophylla, Stipaichu, Muhlenbergia spp., Bro-musunioloides, Calamagrostis spp., Festucaorthophylla. Llamas had permanent access to fresh water.

## Body weight and biometric measurements

The body weight and body measurements were recorded at the beginning of the study, and thereafter every 15 days. All animals were weighed in the mornings (07:00) by an electronic weight scale (2,000 kg capacity, 1 kg precision). The daily weight gain was estimated from the difference of the initial weight and the final weight, divided by the number of days each test phase lasted. Different body measurements were recorded (Table 1).

## Carcass traits

At the end of the trial, all llamas were weighed and transported together to a slaughterhouse, where they were fasted. Immediately after slaughter, the carcasses were weighed (hot carcass weight, HCW), and the dressing percentage was calculated as the ratio of hot carcass weight to live weight on farm (dressing percentage = HCW × 100/slaughter weight). Cold carcass was weighed (CCW) and chilling loss calculated as the ratio of cold carcass weight to hot carcass weight ([HCW − CCW]/HCW × 100).
<table><caption>**Table 1:** Description of biometric measurements (adapted from Mendoza, 2013)</caption><thead><tr><td>Biometric measurement</td><td>Anatomical reference</td><td>Measuring instrument</td></tr></thead><tbody><tr><td>Height at head (cm)</td><td>Distance between the sagittal suture and the floor (perpendicular)</td><td>Ruler with square</td></tr><tr><td>Height at withers (cm)</td><td>Distance between the meeting of shoulder blades (thorny thoracic process) to the ground (perpendicular)</td><td>Ruler with square</td></tr><tr><td>Height at rump (cm)</td><td>Distance between the highest point of the sacral bone to the ground level (perpendicular)</td><td>Ruler with square</td></tr><tr><td>Thoracic perimeter (cm)</td><td>Circumference at the level of the anterior part of the thorax (immediately behind the axillary area) and the cross (encounter of the shoulder blades)</td><td>Measuring tape</td></tr><tr><td>Body length (cm)</td><td>Distance between the tip of the medial ischium to the tuberosity humeral scapula.</td><td>Measuring tape</td></tr><tr><td>Cane perimeter (cm)</td><td>Circumference at the height of the metacarpus (cane) anterior left</td><td>Measuring tape</td></tr><tr><td>Width of breast (cm)</td><td>Distance between the two scapula humeral joints</td><td>Measuring tape</td></tr><tr><td>Ear length (cm)</td><td>Measure taken from the base to the apex of the ear</td><td>Measuring tape</td></tr></tbody></table>

## Data analysis

The experiment was a completely randomized design with 2 treatments. Data were analyzed using SAS version 9.2 for Windows (SAS, 1996). Two models were used; both models included the feeding systems as fixed effects and initial body weight as covariate. Model 1: a fixed model fitted for final weight and total weight gain. Model II: a linear random coefficient regression model fitted for live weight over period. Differences between fattening periods were determined using the least significant difference (LSD) test.
## Results and Discussion

### Pasture characteristics

During the trial, dry matter availability on native pasture varied from 6,064.86 kg/ha initially and 4,807.73 kg/ha at the end of the experiment. When analyzing the chemical composition (Table 2), the native pasture showed lower crude protein content (7.48 %) and the higher crude fiber content (28.68 %) than alfalfa hay.

<table><caption>**Table 2:** Mean chemical composition of native pasture and alfalfa hay at initial and final stages of the trial</caption><thead><tr><th></th><th>Native pasture (initial)</th><th>Native pasture (final)</th><th>Alfalfa hay (initial)</th><th>Alfalfa hay (final)</th></tr></thead><tbody><tr><td>Dry matter (%)</td><td>91.38</td><td>94.70</td><td>89.23</td><td>89.55</td></tr><tr><td>Crude protein (%)</td><td>7.48</td><td>3.58</td><td>15.97</td><td>15.80</td></tr><tr><td>Lipid (Fat) (%)</td><td>1.66</td><td>1.54</td><td>1.74</td><td>1.70</td></tr><tr><td>Crude fiber (%)</td><td>28.68</td><td>40.20</td><td>24.77</td><td>25.85</td></tr><tr><td>Ash content (%)</td><td>3.19</td><td>4.29</td><td>7.25</td><td>7.80</td></tr><tr><td>Carbohydrate (NFE) (%)</td><td>50.37</td><td>45.09</td><td>39.50</td><td>38.40</td></tr></tbody></table>

NFE=Nitrogen Free Extract

The chemical analysis of the natural pasture and the alfalfa hay showed differences in nutritional value. Natural pasture and alfalfa hay showed a protein composition of 7.48 % and 15.97 % at the initial stage of the trial, respectively. The higher crude protein content of the native pasture at the initial stage is due to the fact that plants are in a vegetative state of growth and flowering. While at the final stage of the trial the native plants are in a vegetative period of ripening and dormancy (Florez and Malpartida, 1987; San Martín, 1989; Farfán and Durán, 1998). The nutrients provided by natural pasture are insufficient to meet the requirements of llamas the dry season period.

### Body weight, total gain and biometric measurements

The average body weight of each treatment is shown in Table 3. The final body weight in alfalfa hay treatment showed a significant difference compared to the pasture-only feeding system (p<0.05). Thus, the total gain and daily weight gain of animals fed with supplementation was higher. Therefore, the percentage of weight gain for animals fed by native pasture was 15.75 % and 21.87 % for those fed native pasture plus alfalfa hay, respectively. Llamas that received alfalfa as a supplement had higher weight gains than llamas only fed on pasture (p<0.01).
Llamas fed with alfalfa hay had better performance, a higher final body weight, total weight and daily weight gain. This response is explained by the higher supply of nutrients in alfalfa hay which allowed covering the maintenance requirements in llamas and the additional requirements for growth (San Martin, 1989; Davies et al., 2007; Liu et al., 2009).

These results are in agreement with similar trials with llamas (Mamani-Linares, 2013; Pérez et al., 2000) and with alpacas (Smith et al., 2017). Assuming that there were no differences in pasture intake, the differences in final body weights found in this study (Tables 3) may be related to the greater input of nutrients from the alfalfa hay supplement, which resulted in a better performance.

<table><caption>**Table 3:** Body weights, total and daily weight gain (means ± SD)</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">Native Pasture (NP)</th><th colspan="2">NP + Alfalfa Hay</th><th rowspan="2">Significance of differences</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Initial live weight (kg)</td><td>56.39</td><td>12.88</td><td>55.79</td><td>12.12</td><td>***</td></tr><tr><td>Final live weight (kg)</td><td>65.27</td><td>12.94</td><td>67.99</td><td>15.13</td><td>***</td></tr><tr><td>Total weight gain (kg)</td><td>8.88</td><td>3.15</td><td>12.20</td><td>3.79</td><td>***</td></tr><tr><td>Daily weight gain (g)</td><td>89.65</td><td>31.85</td><td>123.23</td><td>38.29</td><td>***</td></tr></tbody></table>

The data from biometric measurements at initial and final stages are shown in Table 4. The thoracic perimeter was the only biometric measurement that was significantly different between the two treatments (p<0.01). This would indicate that the size of the thoracic perimeter is effectively affected by the feeding system and would be associated with the gain of weight in llamas. The thoracic perimeter increased with the weight of the animal as also reported by Mendoza (2013), Condori et al. (2003), Llacsa et al. (2007), Zea et al. (2007). However, body length and height at rump did not show significant differences with the feeding system, contrasting with the results of significant increases presented by Llacsa et al. (2007) and Zea et al. (2007). But these measures were reported for young animals of 1 to 8 months of age (Llacsa et al., 2007) and of 2 to 15 months (Zea et al., 2007). Probably, the time range (3 months) considered in the development of the animals in the present study was not sufficient to show the variability needed to find significant differences.

<footnote>*** p<0.01</footnote>
<table><caption>**Table 4:** Average size of biometric measurements at initial and final stages (means ± SD)</caption><thead><tr><th rowspan="2">Biometric measurements</th><th colspan="2">Initial</th><th colspan="2">Final</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Height at head (cm)</td><td>147.69</td><td>10.77</td><td>156.31</td><td>9.98</td></tr><tr><td>Height at withers (cm)</td><td>92.09</td><td>4.18</td><td>96.06</td><td>4.95</td></tr><tr><td>Height at rump (cm)</td><td>92.09</td><td>4.18</td><td>96.06</td><td>4.95</td></tr><tr><td>Thoracic perimeter (cm)</td><td>90.78</td><td>6.82</td><td>96.38</td><td>6.22</td></tr><tr><td>Body length (cm)</td><td>83.19</td><td>5.95</td><td>88.22</td><td>6.40</td></tr><tr><td>Cane perimeter (cm)</td><td>10.56</td><td>0.88</td><td>11.00</td><td>0.88</td></tr><tr><td>Width of breast (cm)</td><td>18.53</td><td>1.83</td><td>19.78</td><td>1.75</td></tr><tr><td>Ear length (cm)</td><td>16.06</td><td>1.22</td><td>17.13</td><td>1.13</td></tr></tbody></table>

## Carcass traits

Table 5 showed the values of hot and cold carcass weight, dressing percentage and chilling loss. Significant differences were found due to the effect of feeding systems (p<0.01). Alfalfa supplementation led to higher carcass weights and to higher dressing percentage (p<0.05). The carcass weights obtained in both feeding systems are similar to those reported in alpacas and llamas (Pérez et al., 2000; Cristofanelli et al., 2004).

<table><caption>**Table 5:** Hot and cold carcass weight, dressing percentage and chilling loss (means ± SD)</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">Native Pasture (NP)</th><th colspan="2">NP + Alfalfa Hay</th><th rowspan="2">Significance of differences</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Hot carcass weight (kg)</td><td>34.65</td><td>7.71</td><td>36.26</td><td>8.15</td><td>**</td></tr><tr><td>Cold carcass weight (kg)</td><td>33.37</td><td>7.94</td><td>35.19</td><td>8.18</td><td>**</td></tr><tr><td>Dressing percentage (%)</td><td>52.92</td><td>3.00</td><td>53.38</td><td>2.69</td><td>*</td></tr><tr><td>Chilling loss (%)</td><td>50.81</td><td>3.31</td><td>51.71</td><td>2.68</td><td>**</td></tr></tbody></table>

<footnote>** p<0.01, * p<0.05</footnote>
*Effect of supplementation in different periods of the experiment*

Also, significant statistical differences were found between live weights per period of different feeding strategies (Figure 1). Supplementation with alfalfa hay showed better weight gain response. The first three fattening periods (at 13, 27 and 41 days) show significant differences (p<0.01) respect to other periods. Supplementation with alfalfa hay improves the performance of young llamas kept on natural pasture.

<figure><img src="image_82.png" /><figcaption>**Figure 1:** Average growth (kg) curves per treatment (1 = Native pasture, 2 = Supplementation with alfalfa hay)</figcaption></figure>

These results are in agreement with other studies conducted in llamas and alpacas (San Martin, 1996; Turin et al., 1999; Garcia et al., 2002). The differences among the first three fattening periods respect to the other periods could be due to the availability of natural pastures with a good nutritional value at the beginning of the experiment from March to April, which is the end of the rainy season (Florez and Malpartida, 1987; San Martin, 1996). In addition, llamas used in the present study came from places where the natural pastures were in poor condition. Therefore, the increase in body weight could be due to compensatory growth effect.
**Conclusion**

This study showed that supplementation with alfalfa hay of young llamas grazing native pastures led to a higher live weight, total and daily weight gain and carcass weight. Feeding with natural pastures of good condition and supplementation with alfalfa hay results in a final live weight of 23 % more compared to the initial body weight in a period of 100 days. Highest gain of body weight occurred until 41 days of the beginning of the experiment for all the treatments.

**Acknowledgments.** The authors thank KEF-Commission for Development Research in Austria and Innovate Peru Program for the financial support of this study. In addition, all farmers, who were willing to support the experiment by providing animals are also acknowledged.

**References**

Alva, J., Rojas, M., Nunez, A. 1980, Decomisos por parasitosis y su importancia económica en alpacas (Lama pacos). Confiscation due to parasitic problems and its economic importance for alpacas (lama pacos). Revista de Investigaciones Veterinarias del Perú 5: 61-63.

Álvarez, G. 2001, Morfometría y sus relaciones en llamas Ccara de la empresa Comunal los Andes Palcán - Pasco. Morfometry and its relations in llamas Ccara of the communal enterprise Los Andes Palcán - Pasco. Tesis Ingeniero Zootecnista. Universidad Nacional del Centro del Perú. Huancayo-Perú. 82 p.

Buttolph, L., Coppock, D. 2004, Influence of Deferred Grazing on Vegetation Dynamics and Livestock Productivity in an Andean Pastoral System. Journal of Applied Ecology,41(4): 664-674.

Cano, L. 2009, Estudio fenotípico y genotípico de una población de llamas de Marcapomacocha caracterizada por "coloración ancestral" Guanaco. Phenotypic and genetic study of a llama population in Marcapomacocha, characterized by "ancestral coloring" "Guanaco". Tesis Médico Veterinario. Facultad de Medicina Veterinaria, Univ. Nacional Mayor de San Marcos. Lima-Perú. 62 p.

Castro, E., Sam, R., Lopez, T., Gonzalez, A., Silva, M. 2004, Evaluación de la edad como factor de riesgo de seropositividad a Sarcocystis sp. en alpacas. Evaluation of age as a risk factor of seropositive in Sarcocystis sp in alpacas. Revista de Investigaciones Veterinarias del Perú 15(1): 83-86.

Condori, G., Ayala, C., Renieri, C., Rodriguez, T., Martinez, Z. 2003, Alometría de cortes comerciales en carcasa de llama en dos fases de crecimiento. Allometry of commercial cuts of llama carcass in two growth phases. En: Memoria del III Congreso Mundial sobre Camélidos Sudamericanos. Potosí- Bolivia. 100 p.

Cristofanelli, S., Antonini, A., Torres, D., Polidori, P., Renieri, C. 2005, Carcass characteristics of peruvian llama (Lama glama) and alpaca(Lama pacos) reared in the Andean highlands. Small Ruminant Research 58 (3): 219-222.

Cristofanelli, S., Antonini, A., Torres, D., Polidori, P., Renieri, C. 2004, Meat and carcass quality from Peruvian llama (Lama glama) andalpaca (Lama pacos). Meat Science 66 (3): 589-593.
Davies, H.L., Robinson, T. F., Roeder, B.L., Sharp, M.E., Johnston, N.P., Christensen, A.C., Schaalje, G.B. 2007, Digestibility, nitrogen balance, and blood metabolites in llama (Lama glama) and alpaca (lama pacos) fed barley or barley alfalfa diets. Small Ruminant Research 73 (1): 1-7.

FAO. 2005. Situación actual de los camélidos sudamericanos en Bolivia. Current situation of South American Camelids in Bolivia. TCP/RLA/2914. Roma, Italia. 56 p.

Farfan, R., Duran, A. 1998, Manejo y técnicas de evaluación de pastizales altoandinos. Manament and tecnics for the evaluation of High-Andes pasture. Pub. Tec. FMV N° 39 Ivita-Maranganí, Perú. p 16-18

Flores, A., Malpartida, E. 1987, Manejo de praderas nativas y pasturas en la región alto andina del Perú. Management of native grassland and pastures of the High Andes of Peru. Tomo II. Banco Agrario. Lima-Perú. p 337 – 651.

Flores, E.R. 2012, Pastores de puna, cambio climático y seguridad alimentaria. Farmes of the Highlands, climate change and food security. In: International Symposium Food Security and Economics of Climate Change. Lima, Peru. 6 – 7 of september 2012.

Garcia, W., San Martin, F., Novoa, C., Franco, E. 2002, Engorde de llamas bajo diferentes regímenes alimenticios. Fattening of llamas under different feeding regimes. Revista de Investigaciones Veterinarias del Perú; 13 (2): 1-9.

Leyva, V. 1991, Informe técnico III fase. Proyecto Camélidos Sudamericanos (IVITA – CIID). p.21.

Liu, Q. Dong, C.S., Li, H.Q., Yang, W.Z., Jiang, J.B., Gao, W. J., Pei, C.X., Liang, Z.Q. 2009, Forestomach fermentation characteristics and diet digestibility in alpacas (Lama pacos) and sheep (Ovis aries) fed two forage diets. Animal Feed Science and Technology 154 (3):151 – 159.

Llacsa, J., Urviola, M. Leyva, V. 2007, Evaluación de indicadores biométricos en llamas (Lama glama) de las variedades Ch'acu y K'ara. Evaluation of biometric indicators of llamas Ch'acu and K'ara. Revista de Investigaciones Veterinarias del Perú 18: 1-10.

Mamani-Linares, L.W., Gallo, C.B. 2013, Effects of supplementary feeding on carcass and meat quality traits of young llamas (Lama glama). Small Ruminant Research 114: 233-239.

Martin, M., Decker, C., Romero, S., Carlatti, T., Schnittger, T., Florin-Christensen, M. 2016, Molecular detection of Sarcocystis aucheniae in the blood of llamas from Argentina. Revista Argentina de Microbiología 48 (3): 200-205.

Mendoza, A. 2015, Situación de la crianza y manejo genético de llamas en las provincias de Pasco y Daniel Alcides Carrión en la Región de Pasco. Situation of the rearing and genetic management of llamas in the provinces of Pasco and Daniel Alcides Carrion in the Pasco Region. Tesis Magister Scientiae. Universidad Nacional Agraria La Molina. Lima, Peru. 105 p.

Mendoza, J. 2013, Medidas corporales en la selección de llamas K'ara e Intermedio (Lama glama) de la Región de Pasco. Body measurements in the selection of llamas K'ara and Intermedio in the Pasco Region. Tesis Magister Scientiae. Universidad Nacional Agraria La Molina. Lima, Peru. 74 p.

Perez, P. Maino, M., Guzman, R., Vaquero, A., Köbrich, C., Pokniak, J. 2000, Carcass characteristics of llamas (Lama glama) reared in Central Chile. Small Ruminant Research 37 (1): 93 – 97.

San Martin, F. 1996, Nutrición en alpacas y llamas. Nutrition of alpacas and llamas. Publicación Científica N° 27. Facultad de Medicina Veterinaria. UNMSM. Lima. 29 p.