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The aim of this study was to evaluated the impact of different fibre levels in alpaca diet on voluntary feed intake and apparent digestibility, and to estimate the digestibility of organic matter (OMD) from the content of crude protein (CP) in feces. The study was carried out with twelve alpacas (36.7 ± 6.4 kg body weight- BW), which were offered 4 treatments with different neutral detergent fiber content (NDF. T1: 40.3%; T2: 62%; T3: 68%; T4: 72%) under a switch back design. Absolute daily dry matter intake (DMI) was higher for T1 (678 g/d) than T4 (312 g/d) (p ≥ 005). NDF intake was similar between treatments when related to BW or MW (on average 1% BW and 22 g/kg MW. p ≥ 0.05). Water intake (L/kg DMI) was higher in T1 compared to the other treatments, with values ranging from 2.9 L/kg DMI(T1) to 2.8 L/kg DMI(T4), respectively (p ≤ 0.05). Digestibility of dry matter, organic matter and CP was higher in T1 than in the other treatments, with average values ranging from 72% for T1 to 32% for T4 (p ≤ 0.05). NDF digestibility was similar among treatments (p ≥ 0.05). The regression equation generated to predict OMD (y) was as follows: y = 0.360 + 0.08294*fecal CP (g/kg OM). Further studies will indicate whether faecal nitrogen can be used to estimate digestibility and hence diet quality in South American camelids., (© 2024. The Author(s).)

All vertebrates possess a daily rhythm, encompassing a comprehensive set of physiological, cognitive, and behavioral patterns that manifest throughout a 24-hour period. The aim of this study was to compare the effect of crude protein (CP) levels in the diet on the daily rhythm of urea in serum (US) and saliva in alpacas and sheep. Ten alpacas and ten sheep, adult, clinically healthy males, were used; they were maintained in natural light conditions and fed ad libitum with two levels of CP (16 and 7%). Blood and saliva samples were taken every 4 h for 48 h. A two-way ANOVA was conducted to analyze the parameters, including adjusted mean rhythm (MESOR), amplitude, and acrophase. It was found that both US and saliva urea exhibited circadian rhythms with a peak during the midday (10:59 - 12:16 h). The MESOR with low CP diets was higher (P < 0.05) in alpacas. The MESOR of urea levels in saliva was greater (P < 0.05) at the highest level of CP in the diet, with no differences between alpacas and sheep (P > 0.05). The amplitude was greater (P < 0.05) in alpacas and at the high level of CP compared to the low level of CP in the diet. Our findings reveal that both serum and saliva urea levels in alpacas and sheep follow a daily rhythm and the MESOR of US was higher in alpacas when they consume food with low CP content, and this difference disappears when CP levels are increased in the diet., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Many habitat-specialist organisms occur in distinct, patchy habitat, yet do not occupy all patches, and an important question is why apparently suitable habitat remains unoccupied. We examined factors influencing patch occupancy in near-threatened, little-known Diademed Plovers (Phegornis mitchellii), arguably the bird most specialized to life in High Andean peatlands. Andean peatlands are well-suited to occupancy modelling because they are discrete patches of humid habitat within a matrix of high-altitude steppe. We hypothesized that Diademed Plovers occupy preferably larger and more humid peatlands, and avoid peatlands used for grazing by llamas and vicuñas, which may trample vegetation and nests. From December 2021 to February 2022 (breeding season), we conducted plover occupancy surveys (2-4) on 40 peatlands at Lagunas de Vilama, a landscape of arid steppe and wetlands above 4,500 m in NW Argentina. We measured peatland size, grazing pressure, topographic and remotely-sensed variables that correlate with humidity, and incorporated these as covariates in occupancy models. Occupancy models showed that more than 50% of the studied peatlands were used by Diademed Plovers and most showed signs of reproduction, highlighting the importance of the Vilama Wetlands for Diademed Plover conservation. Within peatlands, Diademed Plovers were most often associated with headwaters. The top ranked occupancy model included constant detection, random spatial effects, and a single occupancy covariate: mean NDWI (Normalized Difference Water Index, an index correlated with water content and humidity) over the previous three years. Contrary to our prediction, Diademed Plovers preferred less water-saturated peatlands (lower NDWI), possibly to avoid nest flooding. This may be especially important in wet years, like the year when we conducted our surveys. Neither peatland size nor grazing by llamas and vicuñas affected peatland use by Diademed Plovers, suggesting that llama grazing at current levels may be compatible with plover conservation. For organisms that specialize on humid habitats, such as peatlands, factors affecting occupancy may vary temporally with variation in climate, and we recommend follow-up surveys across multi-year timescales to untangle the impact of climate on animals' use of humid habitats., Competing Interests: The authors have declared no competing interests., (Copyright: © 2024 Pietrek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In their natural environment, animals face a variety of ecological and social challenges, which might be linked to the emergence of different cognitive skills. To assess inter-specific variation in cognitive skills, we used ungulates as a study model, testing a total of 26 captive individuals across 5 different species (i.e., dwarf goats, Capra aegagrus hircus, llamas, Lama glama, guanacos, Lama guanicoe, zebras, Equus grevyi, and rhinos, Diceros bicornis michaeli). Across species, we used the same well-established experimental procedures to test individuals' performance in naïve physics tasks, i.e. object permanence, short-term spatial memory, causality, understanding of object properties, and gravity. Our results revealed that study subjects showed object permanence, were able to remember the position of hidden food after up to 60 s, and inferred the position of hidden food from the sound produced or not produced when shaking containers. Moreover, they showed an understanding of basic object properties, being able to locate objects hidden behind occluders based on their size and inclination, and could reliably follow the trajectory of falling objects across different conditions. Finally, inter-specific differences were limited to the understanding of object properties, and suggest that domesticated species as goats might perform better than non-domesticated ones in tasks requiring these skills. These results provide new information on the cognitive skills of a still understudied taxon and confirm ungulates as a promising taxon for the comparative study of cognitive evolution., (© 2024. The Author(s).)

Recent studies document the LH-releasing pathway of nerve growth factor (NGF) in male camelids and that the LH response to seminal NGF is associated with elevated plasma testosterone concentration. Results provide rationale for the hypothesis that NGF in semen is associated with male fertility. In Experiment 1, the association between the amount of NGF in the ejaculate and characteristics of the male reproductive system was examined in alpacas. The concentration of NGF was measured by radioimmunoassay in semen samples collected from male alpacas (n = 47) and correlated with sperm morphology and motility, and measurements of the male reproductive anatomy. Most ejaculates had NGF concentrations that, based on previous studies, triggered ovulation in female camelids, however, we only found a positive correlation between NGF concentration with sperm concentration, thread formation and total NGF, and a negative correlation with pH. In Experiment 2, a retrospective analysis was carried out to determine if breeding performance during the previous season was related to recent concentrations of seminal NGF in male alpacas (n = 22). Birth rates tended to be correlated with sperm concentration and total amount of NGF in the ejaculate (P = 0.09). Experiment 3 was a prospective study to determine the relationship between seminal NGF (n = 8 male alpacas) and ovulation and pregnancy rates in a breeding trial. No association was detected between seminal NGF concentration and ovulation rate, pregnancy rate, or LH response in the female. We conclude that among the breeding males used in our study, the abundance of seminal NGF was correlated with sperm concentration and thread formation, however, it was not predictive of male fertility in alpacas. Examination of males not previously selected as breeding stock may be expected to include a broader range of seminal NGF and provide a more comprehensive understanding of the relationship between seminal NGF and male fertility., Competing Interests: Declaration of conflict of interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3-4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3-4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species., Competing Interests: Declaration of Competing Interest None of the authors have any conflict of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5'UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5'RACE-PCR we isolated seven ASIP transcripts with alternative 5'UTR, where exons 1A, 1A', 1C, 1D, and a novel non-coding exon 1A" were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5'UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3' splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5'UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Auditory loss has been reported in camelids using brainstem auditory evoked responses (BAER). Differentiation between conductive versus sensorineural dysfunction has not been investigated. Therefore, the objective of the study was to investigate auditory function using BAER and bone conduction (BC). Twenty-four alpacas: 15 females, 9 intact males (2-16 years of age) were included in a randomized clinical trial. BAER and BC were recorded using two derivations (vertex to mastoid and vertex to cranial aspect of second cervical vertebra). All alpacas underwent complete physical examinations and were sedated with xylazine hydrochloride at 0.6 mg/kg IM. Peaks, when present, were identified and latencies, amplitudes, and amplitude ratios were determined. Eleven alpacas had normal responses and 13 had auditory loss based on BAER. The latter consisted of complete absence of peaks bilaterally (n = 3), absence of peaks unilaterally (n = 1), delayed latencies bilaterally (n = 4), and delayed latencies unilaterally (n = 5). Distinct peaks on BC supported conductive auditory loss in 6 alpacas, difficult to interpret due to stimulus artifact and additional undefined peaks in 4, and absent peaks in 3 alpacas. The cause of auditory loss was presumed to be due to otitis in 6, aging in 4 (10-16 years old), and congenital sensorineural (absent peaks on BAER and BC) in 3 alpacas with unpigmented fiber and irises. BAER and BC are useful and non-invasive to perform techniques for the investigation of auditory loss in alpacas, and further characterization as conductive or sensorineural., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The present study examined the effects of different photoperiods and melatonin treatment on plasma prolactin concentrations in guanacos, a South American camelid, in captivity. Fourteen adult female guanacos, not gestating or lactating and isolated from males, were studied. The control group was exposed to natural daylight, during short days (N = 7, 10L:14D) and long days (N = 7, 16L:8D). The treatment group (N = 7, 10L:14D) received melatonin implants every 23 days for 6 weeks during long days. Blood samples were taken at intervals of 1 week for 3 weeks, starting the third week of treatment. Prolactin concentrations were measured using competitive ELISA. Plasma concentrations of prolactin in non-lactating female guanacos have seasonal changes, with a higher concentration (p < .001) in short days (3.50 ± 2.24 ng/ml) than long days (1.10 ± 0.91 ng/ml). Melatonin treatment significantly decreases (p < .05) plasma concentrations of prolactin on the 21st day after the treatment. These findings are the first report of an endogenous circannual rhythm of plasma prolactin concentration and the action of melatonin treatment on prolactin secretion in this wild camelid., (© 2020 Wiley-VCH GmbH.)

The objective of this study was to evaluate effects of dehydration on sperm DNA with the aim of eventually using this method for preserving llama spermatozoa. Two experiments were conducted: 1) sperm preservation at 5 °C for 60 days in different hyperosmotic solutions (500, 800, 1000 and 1200 mOsmol/l) (n = 6, replications = 2) and 2) sperm preservation at 5 and -20 °C for 60 days in the same hyperosmotic solutions, with supplementary antibiotics (n = 6, replications = 2). Sperm motility, membrane functional integrity, viability and morphology were evaluated at 0 and 48 h of the preservation period (Experiment 1) and at 30 min and 24 h (Experiment 2). Sperm DNA was evaluated at 0 or 30 min (Experiment 1 and 2, respectively) and on days 7, 14, 21, 30 and 60 of the preservation periods. Motility, membrane functional integrity and viability were less when sperm were dehydrated, while sperm cell morphology was not affected. There was a smaller percentage of sperm with condensed chromatin as duration of the preservation period increased when stored in the different hyperosmotic solutions. There was a markedly smaller (P < 0.05) percentage of sperm with intact DNA in all solutions as the duration of preservation increased, with there being greater values for intact DNA at -20 °C than sperm preserved at 5 °C. Llama sperm chromatin condensation was slightly affected by the process of dehydration. There was a markedly smaller percentage of sperm with intact DNA in the dehydrated semen samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: South American camelids have become popular with an increasing number of breeders in Austria, Germany and Switzerland. The aim of the present online survey was to gain information concerning housing and management of South American camelids, particularly during pregnancy and parturition., Material and Methods: A link to the online survey was sent via email to 8 breeder associations in Austria, Germany and Switzerland. The questionnaire included a total of 24 closed or open questions. The period for participation was May to October 2019., Results: A total of 92 breeders participated and 64 answered all questions. The survey revealed that South American camelids were mainly used for breeding (87 %) and production of fibre (74 %). The median flock size was 27 animals, mainly females. Most breeders (74 %) acquired their knowledge on South American camelids via personal contacts and advanced training courses. The majority (57 %) mated their females with an own sire that is kept separately. Using teaser males was the most popular pregnancy test (91 %), often combined with other techniques, especially ultrasonography. In most mares (88 %) duration of pregnancy was in the normal range of 335-360 days. Repeated problems with loss of pregnancy were reported by 76 % of breeders for less than 5 % of their mares. Postpartum problems occurred sporadically, e. g. lack of milk or mastitis (each < 5 %). Problems with the cria at parturition within the last 3 years were reported by > 50 % of the breeders, but reported reasons for dystocia showed no clear focus. The majority of the participants (69 %) were satisfied with the veterinary care in general and in the fields of pregnancy and obstetrics., Conclusion: The results of this survey provide new insights into the management practices of South American camelids breeders in Austria, Germany and Switzerland. Further studies could compare these practices with those in countries where breeding of South American camelids possesses a longer tradition and different commercial backgrounds., Clinical Relevance: The results provide information to practitioners in preparation for problems and challenges in the veterinary care of South American camelids., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Artificial insemination would be a useful technique for alpaca breeders to use as an aid to breeding to increase fleece quality. The technique, however, is not well developed in alpacas, partly because of the viscous nature of their seminal plasma. Castration conducted for husbandry purposes can provide a source of epididymal spermatozoa to test semen extenders or handling regimens, thus circumventing the problem of the viscous ejaculate. In this experiment, two semen extenders (Andromed and INRA96) developed for other species (bovine and equine, respectively) were tested with alpaca spermatozoa derived from the cauda epididymis. Sperm total motility (mean ± SEM A: 29.1 ± 4.8 % compared with I: 35.4 ± 4.8 %; NS), membrane integrity (A: 58 ± 9% compared with I: 56 ± 9%; NS) and acrosome integrity (A: 65 ± 7% compared with I: 54 ± 7%; NS) were not different between the two extenders. Progressive motility with use of INRA96 was greater after incubating for 30 min than after incubating for 10 min (35 ± 4% vs. 12 ± 4%, respectively; P = 0.03). In conclusion, viable epididymal spermatozoa could be extracted from the castrated organs after overnight transport. There were no differences in sperm quality between the two extenders; therefore, it appears that either extender could be used for alpaca spermatozoa. These results could help in the development of a technique for artificial insemination in alpacas., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of β-nerve growth factor (β-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. β-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both β-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative β-NGF abundance was higher in the UTJ of copulated females (p < .05). β-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the β-NGF/TrKA system and that an increase in β-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas., (© 2020 Wiley Periodicals LLC.)

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E ™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies., (© 2020 Blackwell Verlag GmbH.)

The low fertility and offspring survival indicators in alpacas can be partially due to their particularity seasonal reproduction that reduces the opportunities of the females to become pregnant within a season, with the survival of the offspring concerned by the availability of food and exposure to diseases that depends on the calving date. Optimizing the date of delivery and reducing its variability are shown as eligible criteria that could be used as selection criteria within the genetic improvement programmes in alpacas, the calving date being a much more appropriate trait to measure and optimize fertility unlike of age at first calving and the calving interval, this due to the reproductive seasonality in camelids. For this study, 6,533 birth date records were taken between 2001 and 2018 of Peruvian alpacas, to estimate the genetic parameters. Models assuming heterogeneity in the residuals were fitted besides classical homogeneous models to address, not only the possibility of forwarding or delaying the calving date, but also the trend to have parturitions in similar dates. The heritability and repeatability ranged from 0.07 to 0.20 for a homogeneity model and from 0.08 to 0.23 for a heterogeneity model, and suggest the possibility of advancing or delaying the calving date. It should be taken into account that the gestation length of camelids makes it difficult to adapt many reproductive traits, and trying to centre the calving date could delay it. It was concluded the feasibility to genetically select the calving date, also in the production of camels and dromedaries, which have the same reproductive characteristics as alpacas. This selection can be combined with other traits. The heterogeneity model was shown to provide a better fit., (© 2020 Blackwell Verlag GmbH.)

The ovulation-inducing effect of seminal plasma was first suggested in Bactrian camels over 30 years ago, initiating a long search to identify the 'ovulation-inducing factor' (OIF) present in camelids semen. During the last decade, primarily in llamas and alpacas, this molecule has been intensively studied characterizing its biological and chemical properties and ultimately identifying it as β-Nerve Growth Factor (β-NGF). The high concentration of OIF/β-NGF in seminal plasma of llamas and alpacas, and the striking effects of seminal fluid on ovarian function strongly support the notion of an endocrine mode of action. Also, have challenged the dogma of mating induced ovulation in camelid species, questioning the classical definition of reflex ovulators, which at the light of new evidence should be revised and updated. On the other hand, the presence of OIF/β-NGF and its ovulatory effect in camelids confirm the notion that seminal plasma is not only a transport and survival medium for sperm but also, a signaling agent targeting female tissues after insemination, generating relevant physiological and reproductive consequences. The presence of this molecule, conserved among induced as well as spontaneous ovulating species, clearly suggests that the potential impacts of this reproductive feature extend beyond the camelid species and may have broad implications in mammalian fertility. The aim of the present review is to provide a brief summary of all research efforts undertaken to isolate and identify the ovulation inducing factor present in the seminal plasma of camelids. Also to give an update of the current understanding of the mechanism of action of seminal β-NGF, at central and ovarian level; finally suggesting possible brain targets for this molecule., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Alpacas are being more frequently presented to veterinarians in the UK. It is important to validate whether published normal ocular parameters are consistent with the alpaca population in the UK., Methods: Ophthalmic examinations were performed on healthy alpacas ( Vicugna pacos ) from three farms in East Anglia, UK., Results: On direct ophthalmoscopy of 35 alpacas, there was a 50 per cent prevalence of opacities within the lens in alpacas older than two years old (n=8/16). There was a 36.8 per cent prevalence of persistent hyaloid arteries in alpacas under two years old (n=7/19). The mean Schirmer tear test-1 value was 20.0 ±6 mm/minute (n=40). The mean intraocular pressure measured by rebound tonometry was 17.2 ±5.5 mmHg (n=46), and applanation tonometry resulted in statistically similar values (P=0.30; n=25). There was a significant variation in intraocular pressure throughout a 24-hour period (n=8). Fluorescein dye was not detected at the nostrils of any of the alpacas which underwent a Jones test to assess nasolacrimal duct patency (n=8)., Conclusion: The ophthalmic findings appear largely consistent with previously published values from North America and continental Europe. Variations include the large range of measurements obtained and evidence of diurnal variation in intraocular pressure., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. Re-use permitted under CC BY. Published by BMJ.)

From the camel's toes to the horse's hooves, the diversity in foot morphology among mammals is striking. One distinguishing feature is the presence of fat pads, which may play a role in reducing foot pressures, or may be related to habitat specialization. The camelid family provides a useful paradigm to explore this as within this phylogenetically constrained group we see prominent (camels) and greatly reduced (alpacas) fat pads. We found similar scaling of vertical ground reaction force with body mass, but camels had larger foot contact areas, which increased with velocity, unlike alpacas, meaning camels had relatively lower foot pressures. Further, variation between specific regions under the foot was greater in alpacas than camels. Together, these results provide strong evidence for the role of fat pads in reducing relative peak locomotor foot pressures, suggesting that the fat pad role in habitat specialization remains difficult to disentangle.

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r 2  = .46, p < .0001) and non-pregnant llamas (r 2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p ˂ .05 and p ˂ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration., (© 2019 Blackwell Verlag GmbH.)

Monitoring the cardiopulmonary signal of animals is a challenge for veterinarians in conditions when contact with a conscious animal is inconvenient, difficult, damaging, distressing or dangerous to personnel or the animal subject. In this pilot study, we demonstrate a computer vision-based system and use examples of exotic, untamed species to demonstrate this means to extract the cardiopulmonary signal. Subject animals included the following species: Giant panda (Ailuropoda melanoleuca), African lions (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), koala (Phascolarctos cinereus), red kangaroo (Macropus rufus), alpaca (Vicugna pacos), little blue penguin (Eudyptula minor), Sumatran orangutan (Pongo abelii) and Hamadryas baboon (Papio hamadryas). The study was done without need for restriction, fixation, contact or disruption of the daily routine of the subjects. The pilot system extracts the signal from the abdominal-thoracic region, where cardiopulmonary activity is most likely to be visible using image sequences captured by a digital camera. The results show motion on the body surface of the subjects that is characteristic of cardiopulmonary activity and is likely to be useful to estimate physiological parameters (pulse rate and breathing rate) of animals without any physical contact. The results of the study suggest that a fully controlled study against conventional physiological monitoring equipment is ethically warranted, which may lead to a novel approach to non-contact physiological monitoring and remotely sensed health assessment of animals. The method shows promise for applications in veterinary practice, conservation and game management, animal welfare and zoological and behavioral studies.

The alpaca classic grey phenotype is of particular interest to the industry. Until now, there were only indirect data suggesting that the KIT gene was involved in the classic grey phenotype. All exons of KIT in three black and three classic silvergrey alpacas were sequenced. Five non-synonymous SNPs were observed. There was only one SNP found that was present only in the silvergrey alpacas, and this was also the only SNP predicted to be damaging. This variant results in a change of a glycine (Gly) to an arginine (Arg) at amino acid position 126 (c.376G>A), occurring in the second Ig-like domain of the extracellular domain of KIT. Basic protein modelling predicted that this variant is likely destabilising. Therefore, an additional 488 alpacas were genotyped for this SNP using the tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS-PCR). All classic grey alpacas were observed to be heterozygous, and 99.3% of non-grey dark base colour alpacas were found to be homozygous for the wildtype allele in this position. These results confirm that the classic grey phenotype in alpacas is the result of a c.376G>A (p.Gly126Arg) SNP in exon 3 of KIT. These data also support the hypothesis that the grey phenotype is autosomal dominant and that the mutation is most likely homozygous lethal., (© 2019 Stichting International Foundation for Animal Genetics.)

Group vigilance is a cooperative behaviour in social species that reduces individual risk of predation. Lama guanicoe is a social species of camelid performing cooperative vigilance, but little is known about the vigilance behaviour of different social units. We analysed the vigilance behaviour in different types of social units of L. guanicoe, to better understand the complexity of this behaviour. The best supported models for both the frequency of vigilance and the proportion of time vigilant included the type and size of social units as the most important predictors that affect the vigilance behaviour. Solitary males devoted proportionally more time in vigilance behaviour than family, mixed or bachelor groups, whereas females in female groups spent more time vigilant than guanacos in mixed groups. Frequency of vigilance was higher in family individuals and solitary males than in bachelor or mixed groups. It is likely that that in family and females groups, the presence of offspring would increase the vigilance behaviour to detect possible predators. Topography and habitat characteristics that determine the predation risk, affected vigilance behaviour of mixed groups and solitary males. Our results suggest that vigilance behaviour should not be generalized for this species, since there are differences between the types of social units, probably related to intrinsic characteristics of each one and the perception of risk., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination.

The objective of this study was to evaluate the effects of plasma progesterone (P 4 ) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day -2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P 4 (P 4 group) and (2) no ID (Control group). On Day 5, females were administered PGF 2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P =  0.04) in the control (9.7 ± 4.2) than P 4 -treated (6.7 ± 4.9) group; number of corpora lutea did not differ (5.5 ± 3.1 and 5.2 ± 3.4 respectively). Ovulation rate was greater (P <  0.01) in the P 4 -group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P =  0.07) in the P 4 -group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P 4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality., (Copyright © 2019 Elsevier B.V. All rights reserved.)

South American camelids (SAC) are induced ovulating animals. In unmated females, ovarian follicle development occurs in waves of growth and regression, while mating when there is the presence of a mature follicle leads to ovulation. The capacity to respond to an ovulatory stimulus depends on the stage of the follicular wave development. Treatments to control ovarian follicular development have been performed to synchronize timing of wave emergence and development of the dominant follicle at a predictable time. Thus, synchronization of the time of follicular wave development allows for performing fixed time mating or artificial insemination, and superestimulatory treatments for multiple follicule development. Protocols are based on removal of the suppressive effect of the dominant follicle, that can be achieved by physical ablation or by inducing ovulation (with LH or GnRH) or atresia (with progesterone or progestagens alone or combined with estradiol) of this follicle. Differences between treatments should be taken into consideration when choosing a protocol for fixed time mating or artificial insemination, especially when applying the use these technologies for SAC production by commercial enterprises. Furthermore, the objective of applying synchronization protocols should be considered, because not all of these are effective in inhibiting follicular growth before initiation of a superestimulatory treatment for multiple follicle development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Nerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas., Methods: Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment., Results: Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment., Conclusions: Systemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Changes in digesta dry matter (DM) and mean digesta particle size (MPS) along the gastrointestinal tract are well known in ruminants, but not in camelids. We collected digesta from the dorsal (d) and ventral (v) first forestomach compartment (C1), the second forestomach compartment (C2), three proximal segments and the subsequent glandular part of the third compartment (C3A-D), the caecum and the faeces twelve llamas (Lama glama). DM analysis indicates the presence of digesta stratification in the C1, the presence of fluid in the C2 to facilitate the sorting function of this compartment, the fluid-absorbing function of the proximal parts of the C3, the secretion of enzymes and digestive acids in the C3D, and the water-resorbing function of the lower intestinal tract. These findings illustrate the functional resemblance between the gastrointestinal tract of camelids and cattle-like ruminants (C1 equivalent to the rumen with stratified contents, C2 to the reticulum, C3A/B/C to the omasum and C3D to the abomasum). MPS analysis revealed a progressive reduction in MPS from the C1 to the distal C3. This gradual transition is different from the clear-cut threshold in ruminants between the reticulum and the omasum and had so far only been described in dromedaries (Camelus dromedarius). These findings indicate that regardless of the convergent property of rumination and resemblance of general mechanisms involved in contents stratification and particle sorting, differences between ruminants and camelids exist that could be interpreted as a more efficient functionality of the ruminant forestomach., (© 2019 Blackwell Verlag GmbH.)

The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown., (© 2019 Stichting International Foundation for Animal Genetics.)

Some large herbivores exhibit seasonal adjustments in their energy metabolism. Therefore, our aim was to determine if the llama (one of the most extensively kept livestock breeds) exhibits seasonal adjustment of their energy expenditure, body temperature and locomotion, under its natural high altitude Andean habitat. For this purpose, energy expenditure, body temperature and locomotion were measured in seven non-pregnant llama dams for ten months on the Andean High Plateau (4400 m above sea level). Daily energy expenditure was measured as field metabolic rate using the doubly labelled water method at four different measurement times. Additionally, a telemetry system was used to continuously record activity, body temperature (3 min intervals) as well as the position (hourly) of each animal. The results show that llamas adjusted their body temperature and daily energy expenditure according to environmental conditions. Furthermore, llamas under high altitude Andean climatic conditions exhibited a pronounced daily rhythm in body temperature and activity, with low values at sunrise and increasing values towards sunset. Llamas also had remarkably low energy expenditure compared to other herbivores. Thus, despite the domestication process, llamas have not lost the ability to adjust their body temperature and daily energy expenditure under adverse environmental conditions, similar to some wild herbivores.

Here we report the results of excavation and interdisciplinary study of the largest child and camelid sacrifice known from the New World. Stratigraphy, associated artifacts, and radiocarbon dating indicate that it was a single mass killing of more than 140 children and over 200 camelids directed by the Chimú state, c. AD 1450. Preliminary DNA analysis indicates that both boys and girls were chosen for sacrifice. Variability in forms of cranial modification (head shaping) and stable isotope analysis of carbon and nitrogen suggest that the children were a heterogeneous sample drawn from multiple regions and ethnic groups throughout the Chimú state. The Huanchaquito-Las Llamas mass sacrifice opens a new window on a previously unknown sacrificial ritual from fifteenth century northern coastal Peru. While the motivation for such a massive sacrifice is a subject for further research, there is archaeological evidence that it was associated with a climatic event (heavy rainfall and flooding) that could have impacted the economic, political and ideological stability of one of the most powerful states in the New World during the fifteenth century A.D., Competing Interests: The authors have declared that no competing interests exist.

Semen analysis is a key factor when determining the fertility ability in males. South American camelids, and in particular the alpaca, have been studied very little when compared with other farm animals. The aim of this work was to perform the kinematic characterization of alpaca spermatozoa collected directly from the deferent duct by using CASA-Mot (Computer Assisted Semen Analysis for Motility) technology. Samples were obtained every three days throughout the reproductive season during two periods and with a break of seven days in the middle. During both periods, the quality of the sample's motility and kinematics increased over the first two days and then subsequently decreased. This pattern was similar in all animals. It was concluded that the introduction of resting times can be useful to improve sperm quality for artificial insemination purposes in natural conditions., (© 2018 Blackwell Verlag GmbH.)

The aims of the study were twofold: first, the comparison of the pharmacokinetics parameters of two doses of Progesterone BioRelease ® LA, (BioRelease Technologies, Lexington, KY, USA) one of 300 mg and other of 150 mg and their effects on ovarian dynamics in llamas. Based on the results from the first study, the aim of the second study was to evaluate the effect of the doses of 150 mg of progesterone on follicular activity considering the stage of the largest follicle at the beginning of treatment. The results in Study 1 showed that both doses of the formulation induced plasma progesterone concentrations higher than 1 ng/ml during the first 6 days of treatment in all females, progesterone concentrations steadily decline until Day 5 following by a slowly decrease. The total amount of progesterone released during treatment was higher in Group 300 than in Group 150 (p = 0.045). Mean maximum concentrations were 14.9 ± 2.24 and 14.3 ± 2.16 ng/ml for Group A versus Group B (p = 0.58), and they were registered on Day 1.5 ± 0.22 and 1.7 ± 0.34 days, respectively (p = 0.10). None of the animals of Group A showed progesterone concentration below 1 ng/ml during all studied period. The treatment applied in Study 2 was efficient in inhibiting the ovarian follicular dynamics and to start a superestimulatory treatment. The use of progesterone Biorelease ® LA of 150 mg in comparison with the dose of 300 mg could be more effective in the use of synchronization protocols in llamas for AI or prior to the application of an ovarian superstimulatory treatment., (© 2018 Blackwell Verlag GmbH.)

Background: A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus., Methods: Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH., Results: Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons., Conclusions: Results do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

The management of wild vicuñas can trigger a stress response that may compromise welfare. In Santa Catalina, Jujuy Province, Argentina, indices of short-term stress associated with capture, handling, and shearing were studied in 105 wild vicuñas (Vicugna vicugna). The study included 2 groups (n = 59 and n = 46) of wild vicuñas captured in 2 consecutive days. Independent variables analyzed included sex, restraint time, and groups. Cortisol, creatine kinase, glucose, white blood cells, temperature, heart rate, and respiratory frequency were higher than published values. Respiratory rate increased during handling and correlated with holding time and group size, while heart rate decreased. Packed cell volume was higher in females. Cortisol concentrations differed between restraint groups and sex and inversely correlated with agonistic behavior. The most common behavior was increased vigilance. Sternal recumbency increased over holding time. During handling procedures, frequency of sudden movements like kicking and attempts to stand increased as restraint time increased. Females vocalized more than males. In conclusion, the methods used triggered measurable changes suggestive of short-term stress that appeared to be physiologically tolerated by the vicuñas.

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (<3 mm), medium (3-6 mm) and large (>6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa., (© 2018 Blackwell Verlag GmbH.)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca., (© 2018 Blackwell Verlag GmbH.)

Spatially explicit modelling allows to estimate population abundance and predict species' distribution in relation to environmental factors. Abiotic factors are the main determinants of a herbivore´s response to environmental heterogeneity on large spatiotemporal scales. We assessed the influence of elevation, geographic location and distance to the coast on the seasonal abundance and distribution of guanaco (Lama guanicoe) in central Tierra del Fuego, by means of spatially explicit modelling. The estimated abundance was 23,690 individuals for the non-breeding season and 33,928 individuals for the breeding season. The factors influencing distribution and abundance revealed to be the elevation for the non-breeding season, and the distance to the coast and geographic location for the breeding season. The southwest of the study area presented seasonal abundance variation and the southeast and northeast presented high abundance during both seasons. The elevation would be the driving factor of guanaco distribution, as individuals move to lower areas during the non-breeding season and ascend to high areas during the breeding season. Our results confirm that part of the guanaco population performs seasonal migratory movements and that the main valleys present important wintering habitats for guanacos as well as up-hill zones during summer. This type of study would help to avoid problems of scale mismatch and achieve better results in management actions and is an example of how to assess important seasonal habitats from evaluations of abundance and distribution patterns., Competing Interests: The authors have declared that no competing interests exist.

Ovulation of South American Camelids is induced by mating. After copulation, sperm are stored into the oviduct to be released near ovulation time. To study whether copulation induces matrix metalloproteinase-2 (MMP2) secretion in the oviduct, the occurrence of MMP2 in oviductal tissue and oviductal fluid (OF) from 24 h post-mated was compared with non-mated llama females. There was an incremental increase of MMP2 in the oviductal epithelial cells, and MMP2 activity in OF after copulation. Additionally, MMP2 activator (MMP14), inducer (EMMPRIN) and inhibitor (TIMP2) were present in the oviductal epithelial cells of both non-mated and post-mated females. A post-mating segment-specific regulation occurred because relative abundance of TIMP2 mRNA was greater in the utero tubal-junction which was accompanied with a reduced amount of MMP14 in the ampulla in comparison with the non-mated females. To examine the effect of MMP2 on semen liquefaction and sperm physiology, the effects of addition of recombinant human MMP2 was evaluated. The MMP2 had no effect on semen thread formation and seminal plasma protein profile. Sperm viability and plasma membrane function were not influenced by the enzyme treatment either. In summary, in llamas the oviductal microenvironment changes in response to stimuli induced by copulation, increasing the production and secretion of MMP2., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the effectiveness of a protocol based on GnRH and PGF 2α to synchronize the emergence of a new wave of ovarian follicular development in llamas and, therefore, when a new dominant follicle develops. Llamas (n = 18) were assigned to growing, mature or regressing follicle groups according to the phase of the follicular wave at the beginning of treatment. The protocol was initiated with a GnRH analogue (GnRHa) injection on Day 0 followed 7 days later with a d-cloprostenol injection and a second GnRHa injection on Day 10. Ovulation rate after the first GnRHa treatment, day of new follicle emergence, mean plasma progesterone concentration and percentage of animals with a newly developed dominant follicle ≥ 7 mm on Day 10 were evaluated. Ovulation rate after the first GnRHa was less in the regressing than mature and growing follicle groups and new follicular wave emergence occurred earlier in the regressing follicle group than in the other two groups. Mean plasma progesterone concentration in females that had ovulations after the first GnRHa injection was similar. The percentage of animals that had a new follicle ≥ 7 mm on Day 10 was not different among groups and the overall percentage was 66.6%. The total synchronization rate for development of a new wave of follicular development on Day 10 was greater in females having ovulations after the first GnRHa injection than in those that did not have ovulations. In conclusion, the protocol used in the present study was useful for synchronizing ovarian follicular development in 66% of the llamas regardless of the phase of the follicular wave development at the beginning of treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The objectives of this study were to evaluate the effect over time of different percentages of seminal plasma (SP) on llama sperm characteristics in raw semen and correlate the techniques routinely used to evaluate sperm viability and acrosome status with the Fluorescein Isothiocyanate -Arachis hypogea agglutinin/Propidium Iodide (FITC-PNA/PI). Eighteen ejaculates, obtained from 6 male llamas using electroejaculation, were incubated in 0.1% collagenase in HEPES-TALP (HT), centrifuged and resuspended with SP and HT: 0, 10, 50 and 100% SP. Samples were incubated (37 °C) until evaluation at 0; 1.5 and 3 h. Split plot and factorial designs were used to analyze sperm motility, viability, membrane function and acrosome status and Spearman's test was used for correlation. At 0 h, samples with 100% SP showed oscillatory motility; whereas in samples with 0 and 10% SP, progressive motility was predominant. Viability, membrane function and total motility decreased significantly at 3 h of incubation in samples with 100% SP. Sperm with intact acrosomes were fewer in 0% SP media at all times. FITC-PNA/PI correlated with 6-Carboxyfluorescein Diacetate and Propidium Iodide (CFDA/PI) and with Coomassie Blue (CB) stains (r = 0.8; p = 0.0 and r = 0.5; p = 0.0 respectively)., Conclusions: the motility pattern of llama sperm is influenced by the concentration of SP. The use of SP as the only medium is not able to maintain sperm motility, viability and membrane function for 3 h. A certain percentage of SP is necessary in the medium to avoid spontaneous acrosome reactions. The correlations observed could help to shorten evaluation times and reduce costs in sperm laboratories., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Guanacos (Lama guanicoe) are large ungulates that have been valued by human populations in South America since the Late Pleistocene. Even though they were very abundant until the end of the 19th century (before the high deforestation rate of the last decades), guanacos have nearly disappeared in the Gran Chaco ecoregion, with relicts and isolated populations surviving in some areas, such as the shrubland area near the saline depressions of Córdoba province, Argentina. In this report, we present the first data from a locally endangered guanaco wild population, through the study of skeletal remains recovered in La Providencia ranch. Our results showed that most of the elements belonged to adults aged between 36 and 96 months; sex evaluation showed similar numbers of males and females. Statistical analysis of the body size of modern samples from Córdoba demonstrated that guanacos from the Chaco had large dimensions and presented lower size variability than the modern and archaeological specimens in our database. Moreover, they exhibited dimensions similar to those of modern guanacos from Patagonia and San Juan, and to archaeological specimens from Ongamira and Cerro Colorado, although further genetic studies are needed to corroborate a possible phylogenetic relationship. Finally, we used archaeozoological techniques to provide a first characterization of a relict guanaco population from the Chaco ecoregion, demonstrating its value to the study of modern skeletal remains and species conservation biology.

The aim of this study was to evaluated changes in endometrial vascularization area (EVA) between the left and right uterine horn: a) during the ovarian follicular growth in intact llamas, and b) after exogenous estradiol administration of estradiol benzoate in ovariectomized (OVX) llamas. In experiment 1 follicle wave emergence was synchronized (n = 5 llamas) by follicle ablation (Day 0). Females were examined every other day from Day 1 to Day 27, using B mode ultrasonography to evaluate dominant follicle growth profile. Also, EVA was evaluated in each horn using Color-Power Doppler. Blood samples were taken every other day from Day 1 to Day 27 to measure estradiol (E2) plasma concentration by RIA. In experiment 2 OVX llamas (n = 4 llamas/group) were given a single im administration of: a) 1 mg of estradiol benzoate (EB) or b) 1 mL of saline. Females were subjected to ultrasound examinations every 48 h from Day -4 until treatment (Day 0), every 12 h from Day 0 to Day 4, and again every 48 h from Day 5 to Day 11. Evaluation of EVA in both uterine horns was performed as described for experiment 1. Blood sampling for the measurement of E2 was carried out at the same time points indicated for the ultrasound examinations. Serial data were analyzed by one way ANOVA for repeated measures using the MIXED Procedure in SAS. Also, Pearson's correlation was used to determine the relationship between variables. In intact llamas there was an effect of day on the dominant follicle growing profile (P < 0.01) and estradiol plasma concentration (P < 0.05). Dominant follicle diameter positively correlated (r = 0.4; P < 0.017) with estradiol plasma concentration. Also, EVA of right and left uterine horn did not differ (P = 0.89) during the evaluation period; however, it was affected by time (P < 0.05). In ovariectomized llamas estradiol concentration was significantly (P < 0.001) affected by treatment, time and their interaction. Accordingly, treatment with EB (P < 0.0001), time (P < 0.05) and their interaction (P < 0.01) affected EVA of both uterine horns; however, this variable did not differ between horns (P = 0.98). In conclusion, circulating concentrations of estradiol determined an increase in uterine vascularization, during the phase of follicular growth in intact llamas and after the exogenous administration of EB to ovariectomized females; however, no differential effect in endometrial vascularization area between right and left uterine horn was observed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The synthesis of anti-Müllerian Hormone (AMH) by the Sertoli cells in males is crucial for sexual differentiation. There are no studies on AMH in Camelids. The objectives of this research were to 1) compare AMH serum concentrations in prepubertal and adult male alpacas and 2) determine the effect of castration on these concentrations in adult males to provide a validation of a commercial AMH test in alpacas. Serum samples were obtained from 15 prepubertal animals (5 for each age groups of 6, 7 and 8 months) and from 5 adult males (age 5-9 years), pre- and 24 h post-castration. AMH was determined with a quantitative ELISA according to the manufacture's instructions. There was not significant difference (P < 0.05) in AMH level (pg/ml) between pre-pubertal (549.9 ± 120.8, 789.4 ± 172.3, 597.5 ± 177.3 for ages 4, 7, and 8 months, respectively) and adult alpacas (938.7 ± 175.9). In adult males, AMH concentration decreased significantly following castration (P < 0.05) (938.7 ± 383.5 pg/ml) pre-castration, and 222.1 ± 116.5 pg/ml) after castration). There was a positive correlation between testosterone levels and AMH. In conclusion, the quantitative assay used is a reliable test to determine AMH in alpacas. The AMH level in prepubertal and adult alpacas appear to not differ, contrarily from other mammals, this requires further investigation. The decrease in serum AMH concentrations after castration suggests that measurement of this hormone can be used to diagnose bilateral cryptorchid or hemicastrated unilateral cryptorchid animals in this species., (Published by Elsevier Inc.)

Purpose: To determine the normal reference range for Schirmer tear test-1 (STT-1) values in eyes of healthy alpacas (Vicugña pacos)., Methods: Complete ophthalmic examinations were performed on forty healthy alpacas (80 eyes). STT-1 values were obtained in both eyes of all alpacas using a commercial STT strip of a single lot number. Data were analyzed, using a doubly repeated measures ANOVA design, Student's paired t-test, and Pearson correlation test, with P ≤ 0.05 considered significant., Results: The STT-1 values ± standard deviation (SD) were 20.80 ± 4.01 mm/min OD, 20.00 ± 4.13 mm/min OS, and 20.88 ± 4.04 mm/min OU (range 15.50-30.50 mm/min). No significant differences in STT-1 were found between OD and OS. STT-1 was not significantly affected by breed. Schirmer tear test-1 values were significantly increased by 3.45 mm/min for every 10 year increase in age., Conclusions: This study provides a reference range of STT-1 in the healthy alpaca which can assist veterinarians in diagnosing potential keratoconjunctivitis sicca, tear film abnormalities, as well as ocular surface diseases in alpacas., (© 2017 American College of Veterinary Ophthalmologists.)

Pregnancy maintenance depends on the maternal recognition of pregnancy (MRP), a physiological process by which the lifespan of the corpus luteum is prolonged. This mechanism is not well characterized in camelids. The objectives of the present research were to determine if exogenous oxytocin prolongs the corpus luteum activity in alpacas and to evaluate expression and localization of oxytocin receptors within the endometrium at 9 and 14days post-mating. In the oxytocin studies, plasma progesterone profiles were determined after ovulation in the same alpacas on 2 cycles: one cycle without oxytocin treatment and one cycle with oxytocin treatment. Oxytocin was administered daily by intramuscular injections (IM) at a dose of 20IU (experiment 1, n=6) or 60IU (experiment 2, n=7 from day 3 through day 10 after induction of ovulation with GnRH IM. There was no significant difference in the length of the luteal phase (i.e. corpus luteum lifespan) between the treated and control cycles using either 20 or 60IU of oxytocin. In the final experiment, uteri from open and pregnant alpacas (n=4 per group) at 9 and 14days post-mating were evaluated for expressions of oxytocin receptors by immunohistochemistry. No significant difference (P≤0.05) in the expression of oxytocin receptors was observed between open and pregnant animals in either staining intensity or tissue localization. We conclude that oxytocin is not involved in luteolysis and early MRP in alpacas., (Published by Elsevier B.V.)

The wild South American camelids developed a strategy of seasonal reproduction during spring and summer with singleton birth. The photoperiod is one of the factors that may modulate this seasonality where light would be translated into a hormonal signal. This study evaluated the influence of changes in daily light intensity on melatonin concentration in captive guanacos under a long-day photoperiod (16 hr light/8 hr dark; 33 '28'S). Mean melatonin concentration was 28.3 ± 20.3 pg/ml, with a maximum of 52.14 ± 17.19 pg/ml at 23:30 and minimum of 14.29 ± 6.64 pg/ml at 08:30 (p < .001). There was a negative association between light intensity and melatonin concentration (r = -0.57; p < .001). The results indicate that guanacos respond to variation in daily environmental light with a hormonal response and point to a circannual rhythm as a function of the photoperiod., (© 2017 Blackwell Verlag GmbH.)

The objectives of the study were to determine the effect of seminal plasma β-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 μg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spβ-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1/P450scc and STAR expression by RT-PCR. Ovulation was similar between llamas treated with GnRH or spβ-NGF and CL diameter did not differ between GnRH or spβ-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P < 0.01) in llamas from the spβ-NGF than GnRH-treated group by Day 4 and 8. Plasma progesterone concentration was higher (P < 0.05) in llamas from the spβ-NGF compared to females of GnRH group by Day 4 and 8. The proportion of small and large luteal cells did not differ between GnRH or spβ-NGF groups by Day 8. CYP11A1/P450scc was upregulated 3 folds at day 4 and 10 by spβ-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spβ-NGF. In conclusion, the luteotrophic effect of spβ-NGF could be related to an increase of vascularization and up regulation of CYP11A1/P450scc and STAR transcripts enhancing progesterone secretion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO-PRO-1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post-thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen., (© 2017 Blackwell Verlag GmbH.)