Female Alpacas Research Articles

Variación de porcentaje de fibras meduladas y colorimetría en fibra blanca de alpaca Huacaya y Suri

The aim of the study was to evaluate the variability of the percentage of medullated fibres, according to sex and location and to compare the colorimetric characteristics in white fibre from Huacaya and Suri alpaca. The study was carried out in two communities (Juntaya in the Oropesa district and San Juan in the Sabaino district) in the province of Antabamba, Apurímac region (Peru), located between 3950 and 4543 meters above sea level. In total, 451 (Huacaya = 410 and Suri = 41) fibre samples (10 g) were taken from the mid side area. The medullated fibres (%) and the mean fibre diameter (µm) were evaluated using the Fiber Med device. For the colour analysis, a spectrometer coupled to an integrating sphere was used and for the characterization of the chemical bands, an FTIR spectrophotometer. The percentage of total medullation, continuous medullation and strongly medullated fibres were greater in female Huacaya alpacas (p<0.01). The mean diameter of medullated and non-medullated fibres differed depending on the location, while the mean fibre diameter and mean diameter of medullated fibres were higher in males (p<0.01). The colorimetric parameters of luminosity (L*), chromaticity (C*) and tone (Hº) were similar (p>0.05) in the fibres of the two groups of alpacas. In conclusion, in Huacaya alpaca there is variability in the percentage of continuous medullation and mean fibre diameter, depending on sex and location, but the colorimetric characteristics of white fibres are like those of the Suri.

Changes in body weight and condition of female llamas and alpacas throughout the year

The yearly time course of body weight and body condition was evaluated in female llamas and alpacas. One hundred and twenty lactating females, 60 llamas, and 60 alpacas maintained grazing natural pastures were assessed monthly for body weight and body condition. Body weight was registered using a scale to the nearest 1 kg. Body condition was evaluated behind the shoulder, and dorsal protrusion of the thoracic vertebrae was detected. Body weight was analyzed using analysis of variance and body condition using the Chi-square test. Body weight, kg, was different (P≤ 0.05) by species and breeds, 97.6 and 56.2 for llama and alpaca, respectively. Chaku llamas, 94.3; Kcara llamas, 100.9, Huacaya alpacas 56.3, and Suri alpacas 55.9 kg. Body condition varied significantly (P≤ 0.05) between llamas and alpacas. Twelve percent of animals presented good body condition compared to 46% of regular and 42% with poor body condition. More llamas and alpacas were in good body condition, and 82 animals, during the rainy season, December, were also pregnant, more than 15 females with poor body condition and nonpregnant during the same period. Body weight and body condition improved during the rainy than the dry season.

Hematologic parameters in female alpacas during age progression: a retrospective study

Alpacas, like all camelids, have elliptical red blood cells (RBCs) in contrast to other mammals. This particular shape is important for increased osmotic resistance and stability. Age-related changes in the RBC count are known in other species, with alterations in both red and white blood cells being described. In alpacas, there are few data on age-related changes, and only a comparison of crias with adult animals. We characterized age-related hematologic changes in a study of 21 female alpacas from a research herd. A total of 87 records of clinically healthy alpacas of different ages were statistically analyzed retrospectively from the hematologic records over a nine-year period. Significant positive correlations of age with hemoglobin (Hb), HCT, MCV, MCH, neutrophils, platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR) were found as well as significant negative correlations of age with lymphocytes in addition to lymphocyte-to-monocyte ratio (LMR). A paired comparison of eight older animals in the herd at three different ages also showed significant differences in the parameters Hb, HCT, MCV, MCH, MCHC, lymphocytes, eosinophils and neutrophils. Similar changes in hematologic parameters have been reported in other species and should be taken into account when interpreting hematologic results in alpacas.

Intra-horn insemination in the alpaca Vicugna pacos: Copulatory wounding and deep sperm deposition.

Alpacas (Vicugna pacos) are reported to be the rare mammal in which the penis enters the uterus in mating. To date, however, only circumstantial evidence supports this assertion. Using female alpacas culled for meat, we determined that the alpaca penis penetrates to the very tips of the uterine horns, abrading the tract and breaking fine blood vessels. All female alpacas sacrificed one hour or 24 hours after mating showed conspicuous bleeding in the epithelium of some region of their reproductive tract, including the hymen, cervix and the tips of each uterine horn, but typically not in the vagina. Unmated females showed no evidence of conspicuous bleeding. Histological examination of mated females revealed widespread abrasion of the cervical and endometrial epithelium, injuries absent in unmated females. Within one hour of mating, sperm were already present in the oviduct. The male alpaca's cartilaginous penis tip with a hardened urethral process is likely responsible for the copulatory abrasion. The entire female reproductive tract interacts with the penis, functioning like a vagina. Alpacas are induced ovulators, and wounding may hasten delivery of the seminal ovulation-inducing factor beta-NGF into the female's blood stream. There is no evidence of sexual conflict in copulation in alpaca, and thus wounding may also be one of a variety of mechanisms devised by mammals to induce a beneficial, short-term inflammatory response that stimulates blastocyst implantation, the uterine remodeling associated with placental development, and thus the success of early pregnancy.

Serum steroid and thyroid hormone concentrations in healthy domestic male and female alpacas.

Alpacas are high quality fiber producing animals, kept for production purpose and as pets. Endocrine imbalances from adrenal glands, gonads, or thyroid gland may result in coat abnormalities in domestic animals and affect reproduction. Contrary to many domesticated animals, information on hormone concentrations in alpacas is scarce. The purpose of this study was to provide steroid and thyroid hormone values in domestic alpacas. Blood was collected from healthy male (35 intact, 2 castrated) and female (48 non-pregnant, 3 pregnant) alpacas from local farms in Tennessee. Adrenal, reproductive, and thyroid hormones were analyzed. There were no significant differences in median concentrations of progesterone, estradiol, thyroxine (T4), and triiodothyronine (T3) between intact male and female non-pregnant alpacas. Median concentrations of testosterone, androstenedione, 17-hydroxyprogesterone, and cortisol were significantly higher in intact male alpacas compared to female non-pregnant alpacas. This information provides adrenal, gonadal, and thyroid hormone concentrations in alpacas to help with diagnosis of endocrine disorders.

Genome-Wide Association Study of Fiber Diameter in Alpacas.

The aim of this study was the identification of candidate genomic regions associated with fiber diameter in alpacas. DNA samples were collected from 1011 female Huacaya alpacas from two geographical Andean regions in Peru (Pasco and Puno), and three alpaca farms within each region. The samples were genotyped using an Affymetrix Custom Alpaca genotyping array containing 76,508 SNPs. After the quality controls, 960 samples and 51,742 SNPs were retained. Three association study methodologies were performed. The GWAS based on a linear model allowed us to identify 11 and 35 SNPs (-log10(p-values) > 4) using information on all alpacas and alpacas with extreme values of fiber diameter, respectively. The haplotype and marker analysis method allowed us to identify nine haplotypes with standardized haplotype heritability higher than six standard deviations. The selection signatures based on cross-population extended haplotype homozygosity (XP-EHH) allowed us to identify 180 SNPs with XP-EHH values greater than |3|. Four candidate regions with adjacent SNPs identified via two association methods of analysis are located on VPA6, VPA9, VPA29 and one chromosomally unassigned scaffold. This study represents the first analysis of alpaca whole genome association with fiber diameter, using a recently assembled alpaca SNP microarray.

Are alpacas especially protective of lambs? An insight into alpaca guardian behaviour towards sheep using an arena preference test

Alpacas are increasingly used as a guardian species in Australian sheep flocks in an effort to improve lamb survival rates. Yet little is documented about the role and mechanisms of alpaca guarding behaviours within this environment. The first phase of this study, evaluated the temperament of 44 alpacas by assessing their behaviour when individuals were separated from herd mates. Tests included agitation while in an isolation enclosure, flight speed and behaviour in observation arena tests. The second phase of the study evaluated alpaca responses to different auditory, olfactory and visual cues representative of young lambs, adult sheep (ewes) and other novel stimuli. Alpacas (n = 24) which had not previously been exposed to sheep were individually presented with stimuli in a test arena. Behavioural responses were recorded over 2 min and analysed to compare differences between stimuli type (lamb/ewe/other) and stimuli cue form (live animal/auditory only/olfactory only/visual only). In phase one of this study alpacas exhibited little agitation while retained in an isolation enclosure with 67% of animals demonstrating a low agitation score. Females appeared to have a more ‘reactive’ temperament than males, exhibiting significantly faster flight speeds (P < 0.01) and more frequent pacing behaviour (P < 0.01) in the observational pen. In the arena test both male (n = 12) and female (n = 12) alpacas consistently exhibited greater attraction towards the lamb stimuli type (for all cue forms) compared to the ewe and other atypical stimuli (P < 0.05 for all comparisons). Alpacas showed the greatest attraction towards the live lamb compared to visual (P < 0.01), auditory (P < 0.01) and olfactory lamb cues (P < 0.001). Both male and female alpacas demonstrated similar response times and there were no significant differences between alpaca sex in the total time spent reacting to the lamb and ewe stimuli. This study demonstrates that alpacas exhibit a greater attraction towards or interest in young vulnerable animals such as lambs and supports the available anecdotal evidence that the use of alpacas as livestock guardians may be useful in improving lamb survival rates.

Blood selenium and zinc concentration of female alpacas (Vicugna pacos) and their offspring during different physiological conditions.

Minerals are important for animals in many biological functions. There is scarce information, however, about micromineral content in the blood of South American camelids under their prevalent production system. This study aims to determine whole blood selenium (Se) and serum zinc (Zn) concentrations of grazing alpacas at three different physiological states in the Peruvian Andean Highland. Blood samples were collected for measurement of whole blood Se and serum Zn concentrations from 15 female alpacas at late gestation, peripartum, and late lactation, as well as their respective offspring, for them only two times were taken it account (10 days after birth and late lactation). The female alpacas at the beginning had a body weight of 47.5 ± 8 kg and an estimated age of 4.03 ± 0.93 years. Se and Zn content of pastures consumed by alpacas also were determined. The samples were taken during three different months according to the productive calendar of the alpacas. The content of Se and Zn of the diet selected by alpacas met the requirements for the physiological states they go through in comparison with literature references. Serum Zn concentration of female alpacas was higher in peripartum (0.26 µg/ml), compared to late gestation and late lactation (p < 0.05). A similar result was found in the case of their offspring at 10 days after birth (0.23 µg/ml) Zn concentration was higher than for late lactation (p < 0.05). Unlike Zn, in the case of Se, no differences were found between the physiological states studied for alpacas or their offspring. Serum Zn concentration changes through late gestation, peripartum, and late lactation in the female alpacas and their offspring 10 days after birth and late lactation. Further investigation is needed to define the alpaca adequacy of Zn and Se in relation to the blood content of both minerals.

Serum anti-Müllerian hormone concentrations in female alpacas: variations during the reproductive cycle and correlation with ovarian superstimulation response

Earlier, we validated an anti-Müllerian hormone (AMH) enzyme-linked immunosorbent assay kit for male alpacas. First, wecompared the validation data with another kit. There was a high correlation (R2 = 0.94) between these 2 kits. Second, we used thelatter kit to determine serum AMH concentrations during follicular and luteal phases of the reproductive cycle in female alpacas.There were no differences (p = 0.39) in serum AMH concentrations in alpacas (n = 11) between peak follicular and luteal phases(mean ± SEM, 1.33 ± 0.35 versus 1.18 ± 0.34 ng/ml, respectively). Third, we treated female alpacas (n = 13; 5 - 11 years) after 14-day treatment with decreasing doses of porcine follicle-stimulating hormone. There was no effect (p &gt; 0.05) of day of treatmenton serum AMH concentrations. Number of follicles (7 - 10 mm; mean ± SD [as determined via transrectal ultrasonography]) atend of treatment (12.69 ± 5.25; range: 6 - 24) was positively correlated (R2 = 0.7; p &lt; 0.01) with serum AMH concentrations. Toconclude, the kit tested is usable for female alpacas; serum AMH concentrations were not affected by the cycle stage nor by ovariansuperstimulation treatment. Furthermore, a significant correlation between serum AMH serum concentrations and response to superstimulationsuggested that estimation of serum AMH concentrations may be valuable in determining ovarian follicular reserve.

Clinical and Histological Features of Ovarian Hypoplasia/Dysgenesis in Alpacas.

Alpacas have a high incidence of congenital reproductive tract abnormalities, including ovarian hypoplasia/dysgenesis. Diagnosis of this condition is often challenging. The present study describes the clinical, ultrasonographic, and histologic features of ovarian hypoplasia/dysgenesis syndrome in 5 female alpacas. Additionally, serum AMH levels were compared between female alpacas diagnosed with ovarian hypoplasia/dysgenesis and a group of reproductively sound females (n = 11). The syndrome was suspected based on the presence of an infantile uterus and lack of ovaries by ultrasonography and laparoscopy. All females had normal female karyotype (n = 74 XX), but one presented a minute chromosome. The ovaries from these cases showed 3 main histological classifications: hypoplasia (n = 2), dysgenesis (n = 2), and dysplasia (n = 1). Serum AMH levels in affected females were significantly lower (P < 0.05) than those of reproductively sound control females. In conclusion, Serum AMH level may be helpful in the rapid diagnosis of ovarian hypoplasia/dysgenesis syndrome in alpacas. Furthermore, this syndrome in alpacas presents a variety of histological features. Different mechanisms may be involved in the derangement of ovarian differentiation. Further studies are needed to elucidate the causes of the syndrome.

Camelids Orgling Sounds Help Female Alpacas to Ovulate

Camelids Orgling Sounds Help Female Alpacas to Ovulate

First report of Eimeria and Entamoeba infection in alpacas (Vicugna pacos) in Shanxi Province, northern China

Intestinal protozoa Eimeria and Entamoeba can infect many animal species including alpacas. However, data on the prevalence and pathogenicity of species of the two genera Eimeria and Entamoeba in alpacas in China is scarce. The current study was carried out to investigate the prevalence of Eimeria and Entamoeba in alpacas in two cities (Taiyuan and Xinzhou) in Shanxi Province, northern China, using PCR-based approaches. Eimeria spp. were only found in Taiyuan city, and the overall prevalence was 1.64%. All samples collected from male alpacas were PCR-negative for Eimeria. Four Eimeria-positive samples were tested positive as Eimeria lamae. The molecular prevalence of Entamoeba in alpacas was 18.03% (66/366), including 16.39% (50/305) in alpacas from Taiyuan city and 26.23% (16/61) from Xinzhou city, respectively. The Entamoeba prevalence in male alpacas (25.00%) was significantly higher than that in female alpacas (15.69%). Entamoeba bovis was the predominant species, and no Entamoeba histolytica infection was detected. Nine unique SSU rRNA gene sequences of Entamoeba were obtained which formed a new cluster. The results showed that sex and location might be the risk factors associated with prevalence of Eimeria spp., and sex might be the risk factor associated with prevalence of Entamoeba spp.. This is the first report of Entamoeba in alpacas worldwide. These findings expand our understanding of the prevalence and genetic diversity of Eimeria and Entamoeba in alpacas.

28 Evaluation invitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n=66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25M sucrose. After 1min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5M sucrose. After 2min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1M sucrose] for 20s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1M sucrose] for 20s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5s and subsequently thawed at 37°C for 50s. Straws were emptied into 1mL of prewarmed holding medium solution (HMS1) containing 1M sucrose for wash and the thawed blastocysts were transferred into a second 1mL of prewarmed HMS1. After 5min incubation at 37°C, the blastocysts were transferred into 1mL of warmed Holding medium solution 2 (HMS2) containing 0.5M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P&amp;lt;0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P&amp;lt;0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed. This research was funded by FONDECYT project no. 149-2017.

Sedative effects and pharmacokinetics of detomidine when administered intravenously and intravaginally as a gel in alpacas

ObjectivesTo evaluate the sedative effects and pharmacokinetics of detomidine gel administered intravaginally to alpacas in comparison with intravenously (IV) administered detomidine. Study designRandomized, crossover, blinded experiment. AnimalsA group of six healthy adult female Huacaya alpacas (70.3 ± 7.9 kg). MethodsAlpacas were studied on two occasions separated by ≥5 days. Treatments were IV detomidine hydrochloride (70 μg kg−1; treatment DET–IV) or detomidine gel (200 μg kg−1; treatment DET–VAG) administered intravaginally. Sedation and heart rate (HR) were evaluated at intervals for 240 minutes. Venous blood was collected at intervals for 360 minutes after treatment for analysis of detomidine, carboxydetomidine and hydroxydetomidine using liquid chromatography–tandem mass spectrometry. Measured variables were compared between treatments and over time using mixed model analysis. Data are presented as the mean ± standard error of the mean, and a p value of <0.05 was considered significant. ResultsOnset of sedation was faster in treatment DET–IV (1.6 ± 0.2 minutes) than in treatment DET–VAG (13.0 ± 2.5 minutes). Time to maximum sedation was shorter in treatment DET–IV (8.3 ± 1.3 minutes) than in treatment DET–VAG (25 ± 4 minutes). Duration of sedation was not different between treatments. There was a significant linear relationship between sedation score and plasma detomidine concentration. HR was less than baseline for 60 and 125 minutes for treatments DET–IV and DET–VAG, respectively. The maximal decrease in HR occurred at 15 minutes for both treatments. The mean maximum plasma concentration of detomidine, time to maximum concentration and bioavailability for treatment DET–VAG were 39.6 ng mL−1, 19.9 minutes and 20%, respectively. Conclusions and clinical relevanceDetomidine administration at the doses studied resulted in moderate sedation when administered IV or intravaginally to alpacas.

108 Use of seminal plasma as ovulation inductor in alpacas (Vicugna pacos) embryo recipient and its effect on pregnancy rate

Alpacas, like other camelids, are classified as induced ovulators because an external stimulus is required for the occurrence of ovulation. Recent studies have identified the β-nerve growth factor (β-NGF), a protein present in the seminal plasma (SP), as responsible for inducing ovulation in this species and having potent luteotropic function. We speculate that SP can be used in reproductive biotechnologies, such as embryo transfer (ET), to increase the number of genetically superior animals produced in breeding programs. The objective of this study was to evaluate the effect of inducing ovulation with SP or a gonadotrophin-releasing hormone (GnRH) analogue on pregnancy rate in recipients of an alpaca ET program. Semen from 5 adult male alpacas was collected with an artificial vagina and diluted 1:1 (v/v) with phosphate-buffered saline (PBS; Gibco-BRL). The diluted semen was centrifuged for 30min at 1200g, and the supernatant or SP was decanted and examined for absence of sperm. Then, the SP was centrifuged again for 20min at 1200×g. The SP was stored at −80°C until the use. Female alpacas (n=38; 6-8 years) with a body condition score of 2.5-3.5 (scale 1-5) were used for the experiment. Animals were evaluated daily by transrectal ultrasonography to determine the presence of a dominant follicle ≥7mm and randomly assigned to one of two groups: (1) GnRH (n=20), 0.04mg of Buserelin acetate IM, or (2) SP (n=18): 1.0mL of seminal plasma IM. Seven days after GnRH or SP treatment (Day 0) the recipients received a fresh embryo. Alpaca donors (n=18) were given GnRH (0.04mg IM) and treated 36h later with 700IU of equine chorionic gonadotropin (ECG). Donors were mated with fertile males 5 days after ECG (Day 0 of recipients), and embryos were recovered 7 days later. Embryos of similar quality were assigned to both groups and transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum. Ultrasonography examinations were performed on Day 2 to confirm ovulation and Day 25 to determine pregnancy in all of the recipients. Data was analysed by chi-squared test. Ovulation rate was not different between groups (100% each). Pregnancy rate was 45% (9 out of 20) and 44% (8 of 18) in GnRH and SP groups, respectively (P=0.77). In conclusion, SP was effective to induce ovulation in alpacas and was able to produce a pregnancy rate similar to that of GnRH as an ovulation-inducing treatment. Thus, SP can be used as an alternative for ET programs in alpacas. Research was funded by the project Role of Seminal Plasma in Reproductive Physiology and Application of Biotechnologies in Camelids (149-2017-CIENCIACTIVA).

228 Effect of seminal plasma on the interval to application of equine chorionic gonadotrophin for the recovery of cumulus-oocyte complexes in alpacas (Vicugna pacos)

Alpacas as other camelids are induced ovulators and require an external stimulus-mounting by the male-for ovulation. NGF-β, protein present in the seminal plasma (SP) is reported as being responsible for stimulating ovulation. However, limited information exists on the effect of ovulation inducers in the new follicular wave emergence with application on protocols of superstimulation. The aim of the study was to determine the effect of two ovulation induced techniques and two different times on the interval to application of equine chorionic gonadotrophin (eCG) for the ovarian superstimulation for the recovery of COCs by ovum pick- up (OPU). Alpacas were evaluated by transrectal ultrasonography with an Aloka SSD 500 ultrasound and transducer (7.5MHz) to determine the presence of a dominant follicle=7mm. A 2×2 experimental design was used with adult female alpacas, 6 to 8 years old, which were assigned to one of the treatments when a dominant follicle=7mm was present. Alpacas were assigned randomly 1 of 2 groups (Day 0) in which follicular ovulation was induced by seminal plasma 1mL IM (SP, n=13; group 1) with application of 650IU of eCG at 36h (n=6) or 48h (n=7) post-application of SP, or by GnRH with 0.008mg of Buserelin IM (GnRH, n=10; group 2) with application of 650IU eCG at 36h (n=5) or 48h (n=5) post-application of GnRH. On Day 7, COCs were counted and the OPU procedure was performed in every group. Data were analysed using ANOVA. Results are presented in Table 1. In conclusion, both inducers of ovulation and both times of application of eCG were effective for producing follicles of 7mm and COCs acceptable for recovery in Alpacas. Table 1.Number of follicles=7mm (top) and number of COCs recovered (mean±s.e.m.) Outcome and group 36 h 48 h Total Follicles GnRH 6.8±1.93 13.4±4.34 10.10±2.50a SP 5.5±2.23 5.29±1.15 5.38±1.25b Total 6.09±1.44 8.67±2.18 7.43±1.33 COCs GnRH 5±0.97 3±1.26 4.33±0.74a SP 1±0.41 0.33±0.22 0.67±0.25b Total 3.29±0.67x 1.4±0.6y 2.5±0.53 a,bValues within columns with different letters differ significantly (P=0.05). x,yValues within columns with different letters differ significantly (P=0.05). This research funded by CIENCIACTIVA-CONCYTEC as part of the project title “Role of seminal plasma in reproductive physiology and application of biotechnologies in camelids” (149-2017).

7 Pregnancy from a vitrified-warmed alpaca pre-implantation embryo

The alpaca (Vicugna pacos) is a ruminant livestock species in the South American camelid family. There are more than 9 million South American camelids globally that make important contributions to the livelihoods of rural farmers through conversion of low quality roughages to high quality food and fibre. Reproductive biotechnologies for alpacas are not well developed compared with those for other ruminant livestock species. In particular, embryo cryopreservation technologies are lacking. The objective of this study was to evaluate under field conditions a vitrification protocol originally developed for old world camels that we adapted for use in alpacas. Potential donors were evaluated for follicular development using a 7.5-MHz ultrasound probe. Hembras (sexually mature female alpacas) with ovarian follicles 7-10mm in diameter were behaviour tested to determine sexual receptivity, and receptive females were naturally mated to a proven herd sire. At the time of breeding, non-superovulated donors (n=4) received 30μg gonadorelin. Embryos were nonsurgically collected 7 days after breeding and handled at 20°C. Diameter of harvested embryos (n=4 quality grade 1 hatched expanded blastocysts) was measured using an eyepiece reticle. All recovered embryos were placed individually into 0.5-mL drops of vitrification solution (VS1: 1.4M glycerol) for 5min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6M ethylene glycol) for 5min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6M ethylene glycol) for 20s, and 0.05-mL drops of VS3 for 20s while loading into open-pulled straws (OPS). Each OPS was plunged directly into liquid nitrogen for storage for 29 days. At warming, each OPS was submerged into a 1-mL drop of warming solution 1 (WS1: 0.5M galactose) for 1min followed by 1min in WS2 (0.25 M galactose) for 5min before being incubated at 37°C in 5% CO2 in humidified air for 21h in 1mL of Syngro holding medium supplemented with 10% (vol/vol) alpaca serum. Embryos that grew during culture (n=2) were transferred individually into synchronous recipients, and embryos that did not appear to grow (n=2) were transferred together as a pair. Prior to embryo transfer, potential recipients were evaluated ultrasonographically as described previously. Hembras with ovarian follicles 7-10mm in diameter were behaviour tested, and sexually receptive females received 30μg gonadorelin 6 days before embryo transfer. Final selection of recipients (n=3) was based on presence of a corpus luteum and nonreceptive behaviour to a herd sire 24h before transfer. Pregnancy was detected ultrasonographically, and fetal heartbeat was detected 29 days post-transfer in one of the three recipients. Ultrasound at 177 days post-transfer revealed that the pregnancy, generated from a 400μm×375μm vitrified-warmed embryo that had grown in culture, was still ongoing. If this pregnancy results in the birth of a live cria (newborn alpaca), it would represent-to the best of our knowledge-the world's first cria born from a cryopreserved alpaca pre-implantation embryo. It would also demonstrate the potential utility of this protocol under field conditions.

112 Comparative study between slow freezing and vitrification on the survival rate of cryopreserved alpaca embryos post-transfer

The objective of this study was to compare the effectiveness of cryopreserving in vivo-produced alpaca embryos by slow freezing v. vitrification. The embryos were produced from 9 female alpacas at Fundo Mallkini, Puno, Peru, located at 4300m elevation. The donor alpacas were synchronized by induction to ovulate with an injection of gonadotropin-releasing hormone (0.0084mg of buserelin acetate) and natural mating with vasectomized males to male receptive donors (day of ovulation induction was considered Day 0). On Day 2, the donors were injected 700IU of eCG. On Day 7, the donors received an injection of prostaglandin F2α (0.25mg of cloprostenol) and were mated on Day 8 by fertile males (2 matings 12h apart: 0600 and 1800h). The embryos were collected at 5.5 days after fertile mating and were graded as per IETS recommendations; most of the embryos were already expanded and hatched blastocysts. Embryos were washed and maintained in holding medium (1L PBS+1g Glucose+36mg sodium pyruvate+0.4% BSA+50mg kanamycin monosulfate) at 23°C for up to 1h and distributed into 2 groups for either slow freezing for direct transfer (n=14 embryos) or vitrification (n=10 embryos). Slow freezing consisted of transfer into freezing medium (9mL of 1.5M ethylene glycol+1mL of 1.0M sucrose prepared in holding media) at 23°C, placing in 0.25-mL straws and subjected to freezing at a rate of −0.5°C/ minute to −35°C and then plunging into LN. Vitrification followed a procedure described for camel embryos whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed periods and were then loaded into an open pull straw and plunged directly into LN for storage. The cryopreserved embryos were transferred into adult alpacas at the Community of Suitucancha, Junin, Peru (1500km from the farm where the embryos were collected and cryopreserved, 4200m elevation). Embryos in the slow-freezing group were thawed in warm water at 37°C for 30s and loaded directly into the embryo transfer gun for direct transfer into 7 alpaca recipients (2 embryos per recipient). Vitrified embryos were warmed by removing the open pull straw from the LN and transferring the embryos to 2 warming solutions at 37°C with decreasing levels of vitricants and containing 0.5M galactose with a final incubation at room temperature in holding media and then transferred into 5 alpaca recipients (2 embryos per recipient). The embryos were transferred into synchronized recipients by transcervical nonsurgical method. Pregnancy diagnosis was made by transrectal ultrasound examination at 45 days post-transfer. The pregnancy rates in the slow-freezing and vitrification groups, respectively, were 2/7 (29%) and 0/5 (0%); the difference was not significant (P&amp;gt;0.05) based on Fisher’s exact test. Twin pregnancies were not detected. We consider the result with slow freezing very promising, as in previous trials we had less than 18% pregnancies. More trials with larger number of embryos per cryopreservation method are being programmed.

Changes in mucins and matrix metalloproteases in the endometrium of early pregnant alpacas (Vicugna pacos)

South American Camelids (SAC) have unique reproductive features, one of which is that 98% of the pregnancies develop in the left uterine horn. Furthermore, early pregnancy is an uncharacterized process in these species, especially in regard to the ultrastructural, biochemical and genetic changes that the uterine epithelial surface undergoes to allow embryo implantation. The present study describes the uterine horn luminal surface and the characteristics of the mucinous glycocalyx in non-pregnant and early pregnant (15 days) female alpacas. In addition, the relative abundance of Mucin 1 and 16 genes (MUC1 and MUC16) was determined, as well as the relative mRNA abundance of matrix metalloproteinases (MMPs) that could be involved in MUC shedding during early pregnancy. Noticeable changes were detected in the uterine luminal epithelium and glycocalyx of pregnant alpacas in comparison to non-pregnant ones, as well as presence of MUCs and MMPs in the endometrial environment. The decrease in glycocalyx staining and in the relative abundance of MUC 1 and MUC 16 transcripts in pregnant females would allow embryo attachment to the luminal epithelium and its subsequent implantation, as has been described in other mammals. These results suggest a crucial role of MUC1 and MUC16 and a possible role of MMPs in successful embryo implantation and survival in alpacas.

96 Follicular Fluid and Serum Biochemical Composition in Alpacas (Vicugna pacos) with Different Nutritional Planes

The importance of nutrition on composition of follicular fluid and oocyte quality is recognised in several species but limited information is available on alpacas and would explain the high rate of early embryo mortality. The aim of this study was to analyse the biochemical composition of serum and follicular fluid (FF) from alpacas (Vicugna pacos) with different nutritional planes. Twelve adult female alpacas between 6 and 8 years old were assigned randomly to either a high (n = 6) or low (n = 6) plane of nutrition. Nutritional planes were defined by the grazing condition of the natural pasture with (high) or without (low) supplementation with 200 g of concentrate/day (total digestible nutrients: 52%, protein: 16%, and metabolizable energy: 2.8 Mcal kg−1). The nutritional conditions were imposed 1 month before the start of the experiment.The concentrations of total glucose, cholesterol, triglycerides, and proteins were determined with a semi-automatic biochemical analyzer (SINOWA, China). When an ovulatory follicle ≥7 mm was detected by ultrasonography using a 5-MHz linear-array transducer (SSD-500, Aloka, Tokyo, Japan), a transvaginal transducer with an attached needle guide (UST-945BP-5, Aloka) was used to collect follicular fluid. Follicular puncture was performed using a disposable 19-gauge # 12 mm hypodermic needle connected to a 50-mL conical tube via a silicon tube. A caudal epidural anaesthesia was induced with 4 mL of 2% lidocaine. Blood samples were obtained by jugular venipuncture into serum vacutainers and kept at room temperature for 30 min. Follicular fluid and serum were centrifuged at 1500 × g for 20 min, decanted, and stored at –20°C until analysed. Results were analysed by Student’s t-test. The results are presented in Table 1. The results suggest differences in biochemical composition of glucose and cholesterol in follicular fluid, which could explain the oocyte quality and embryo survival in alpacas. Table 1.Biochemical composition of follicular fluid (FF) and serum from alpacas (Vicugna pacos) under 2 nutritional planes Research was supported by INNOVATE PERU Grant 405-PNICP-PIAP-2014.