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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 7-10 mm 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 30 μg 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: 1.4 M glycerol) for 5 min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6 M ethylene glycol) for 5 min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6 M ethylene glycol) for 20 s, and 0.05-mL drops of VS3 for 20 s 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: 0.5 M galactose) for 1 min followed by 1 min in WS2 (0.25 M galactose) for 5 min before being incubated at 37°C in 5% CO2 in humidified air for 21 h in 1 mL 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 7-10 mm in diameter were behaviour tested, and sexually receptive females received 30 μg 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 24 h 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 400 μm × 375 μm 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.

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 4300 m elevation. The donor alpacas were synchronized by induction to ovulate with an injection of gonadotropin-releasing hormone (0.0084 mg 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 700 IU of eCG. On Day 7, the donors received an injection of prostaglandin F2α (0.25 mg of cloprostenol) and were mated on Day 8 by fertile males (2 matings 12 h apart: 0600 and 1800 h). 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 (1 L PBS + 1 g Glucose + 36 mg sodium pyruvate + 0.4% BSA + 50 mg kanamycin monosulfate) at 23°C for up to 1 h 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 (9 mL of 1.5 M ethylene glycol + 1 mL of 1.0 M 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 (1500 km from the farm where the embryos were collected and cryopreserved, 4200 m elevation). Embryos in the slow-freezing group were thawed in warm water at 37°C for 30 s 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 0.5 M 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 > 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.

The aim of the study was to improve the pregnancy rate in recipient alpacas using nonsteroidal anti-inflammatpry druds (NSAIDs) at time of embryo transfer. Because most NSAIDs are non-selective inhibitors of cyclooxygenases, which are the rate-limiting enzymes in the formation of prostaglandins, such treatment could temporarily block the production of prositaglandin F2α (PGF2α) and hence maintain corpus luetum (CL) activity long enough to support embryo development and pregnancy. The experiment was carried out in the Peruvian southern highlands (4,100 m elevation). Thirty-one adult alpaca donors were subjected to superovulation and embryo flushing as described previously (Vivanco-Mackie 2013 Proc. 29th Annu. Mtg. AETE, Istanbul, pp. 43-74; http://www.aete.eu/index.php/publications-aete/proceedings/2013/file). From the collected embryos, 20 grade A embryos were selected and transferred fresh into the recipients of the 2 experimental groups. All embryos were collected and transferred at 6.5 days post-mating of the donors with one embryo transferred per recipient. Recipient alpacas (n = 20) were synchronized and induced to ovulate after a selection made by ultrasonography, selecting as recipients the alpacas with follicles >8 mm and then exposing them to vasectomized males followed by IM injection of gonadotropin-releasing hormone (GnRH, 0.0084 mg of acetate of buserelin). Embryo transfers were made by laparoscopically aided laparotomy 6.5 days after ovulation induction as this method has been demonstrated to be more effective in previous trials compared with transcervical non-surgical transfers. At the time of embryo transfer, the recipients were randomly assigned to 1 of the 2 treatments according to the NSAID injected immediately after embryo transfer: Treatment 1 (10 alpacas) was an IM injection of meloxicam at 0.5 mg/kg of body weight; treatment 2 (10 alpacas) was an IM injection of tolfenamic acid at 3 mg/kg of body weight. At the pregnancy test by ultrasound scanning on Day 58 post-transfer, 30% (3/10) of the recipients had a live fetus in treatment 1, whereas treatment 2 had only 10% (1/10).The difference was not significant (P > 0.05) based on Chi-squared analysis. Th historical pregnancy rate obtained with fresh embryos transferred using the same technique and on the same farm where the comparison between NSAIDs was performed was 28.6% at 58 days post-transfer (Vivanco-Mackie et al. 2015 Reprod. Fertil. Dev. 27, 173 abst). Results suggest that there is no difference between tolfenamic acid and meloxicam in their effect on pregnancy rates in alpacas receiving fresh embryo transfers. Compared with historical data of nontreated recipients, results of the present experiment may indicate that the use of NSAIDs at the time of embryo transfer does not improve pregnancy rates in alpaca fresh embryo recipients. However, additional research studies with greater numbers of recipients and an untreated control group are necessary to confirm the preliminary results of the present study. The study was funded by the ‘INNOVATE PERU’ program of the Peruvian Government.

The aim was to improve the pregnancy rate in recipient alpacas following embryo transfer. Two experiments were carried out, Experiment 1 in the southern highlands (4,100 m elevation) and Experiment 2 (E2) in the central highlands (3,200 m elevation) of the Peruvian Andes. In both experiments, a group of alpaca donors was subjected to superovulation and embryo flushing as per procedures already described (Vivanco 2013 29th Annu. Mtg. European Embryo Transfer Association, 43–74). The synchronization of the recipient alpacas and ovulation induction in both experiments was made by ultrasonography selection of the alpacas with follicles greater than 8 mm and their exposure to vasectomized males followed by an injection of GnRH (0.0042 mg of acetate of buserelin, Buserelina Zoovet®, Laboratorio Zoovet, Santa Fe, Argentina). The embryo transfers in both locations were made by laparoscopically aided laparotomy 6.5 days after ovulation induction. In Experiment 1, 29 recipient alpacas were distributed in 2 treatments: treatment 1 (n = 14) was the control (no hormonal supplementation); alpacas in treatment 2 (n = 15) received an injection of 300 IU of eCG (Novormon®, Partnar Animal Health, Port Huron, MI, USA) and the insertion of an intravaginal sponge containing 60 mg of medroxyprogesterone acetate (Progespon®, Syntex SA, Buenos Aires, Argentina) 72 h after the injection of the GnRH applied at ovulation induction, the sponges remained in the vagina of the recipients up until Day 21 post-transfer. In Experiment 2, ten recipient alpacas were distributed into 2 treatments, treatment 1 as the control (n = 5, no hormonal supplementation) and treatment 2 (n = 5) in which the recipient alpacas received a dose of a nonsteroidal anti-inflammatory (2 mg/kg of tolfenamic acid, Tolfén L.A. 8%®, Agrovet Market Animal Health, Lima, Peru) 12 h before the embryo transfer with the aim of blocking PGF2α secretion and preventing corpus luteum (CL) regression. At time of transfer, control recipients in Experiment 1 showed in average 0.71 ± 0.71 follicles and 1.14 ± 0.53 CL and the treatment 2 recipients showed 2.73 ± 2.52 follicles and 1.20 ± 0.56 CL, respectively, and there was no significant differences (P > 0.05) between treatment groups in the pregnancy rates 60 days post-transfer, being 35.7% (5/14) and 26.7% (4/15) for treatment 1 (control) and 2 (hormonally supplemented), respectively. In Experiment 2, the number of follicles and CL were not recorded; the differences between treatments for pregnancy rates were also nonsignificant (P > 0.05), the pregnancy rates were 20% (1/5) and 40% (2/5) for treatment 1 (control) and treatment 2 (injected with anti-inflammatory) respectively. In the first experiment, the hormonal treatment increased the number of non-ovulatory follicles; this could have negatively influenced its effectivity on increasing pregnancy rates. In the second experiment, a tendency can be observed for better holding rates in alpacas receiving the anti-inflammatory treatment; it will be necessary to increase the number of observations in a new experiment in order to determine if this will have a significant effect on improving holding rates in alpacas. These studies were funded by INNOVATE PERU and by the FONDECYT of the Peruvian government.

The objective of this applied field study was to assess the feasibility of successfully performing bovine embryo transfer in the Republic of Kosova—a feat that had not yet been accomplished in this newly independent (2008) eastern European country. Three Holstein heifers at the Iowa State University dairy farm were superovulated with a conventional descending dose regimen of FSH (Folltropin). Approximately 12 and 24 h after the observed onset of oestrus, heifers were inseminated with semen from a single Red Holstein bull. Embryos were non-surgically collected and washed in accordance with IETS procedures for sanitary handling of embryos. Embryos were cryopreserved for subsequent direct transfer. After obtaining an import permit from the Kosovo Food and Veterinary Agency, embryos were approved for export to the Republic of Kosova by the US Department of Agriculture, Animal Plant Health Inspection Service. Embryos were shipped via an express courier service. A total of 19 embryos were received in the Republic of Kosova. Recipients were monitored for signs of naturally occurring oestrus, and immediately before transfer, embryos were thawed by holding in air for 3 to 5 s followed by placement into a 37°C water bath for 25 to 30 s. The first-ever bovine embryo transfer calf in the Republic of Kosova was born July 6, 2015. A total of 9 calves were born from the 19 embryos transferred (47.4% embryo survival rate). Results of this applied field study show that bovine embryo transfer is feasible in the Republic of Kosova. Embryo transfer will be used to improve the quality of dairy cattle genetics in the Republic of Kosova and to subsequently increase the national supply of milk, decrease dependence on milk imports, and increase food security of the nation.

The objective of the study was to determine the embryo survival up to calving of fresh and cryopreserved (frozen and vitrified) alpaca embryos transferred into alpaca recipients by nonsurgical transcervical embryo transfer and by surgical laparoscopically aided embryo transfer. For this report we have compiled the information from 127 embryo transfers in alpacas done by our group at Mallkini, Puno, Peru, at 4200 m elevation. The embryos have been collected from superovulated donor alpacas flushed at 6.5 days post mating, some were transferred as fresh and some were cryopreserved; the recipients (3 to 7 years old) were selected based on presence of functional corpora lutea at ecosonographic examination and subjected to ovarian cycle synchronization and ovulation induction as per Vivanco (2013). From a total of 133 alpacas selected, 127 were used, from which 82 received fresh, 32 frozen, and 13 vitrified embryos. All embryos were classed as A-class expanded blastocysts at time of transfer. By nonsurgical transcervical embryo transfer, 33 embryos were transferred fresh, 22 were frozen/thawed embryos, and 13 were vitrified/warmed embryos. By surgical laparoscopically aided method, 49 embryos were transferred fresh and 10 embryos were frozen/thawed; no vitrified embryos were transferred by this method. Results are detailed in Table 1. Pregnancy losses occured at up to 9 weeks (63 days) of gestation, the heaviest loss occurs in the first 3 weeks. After 9 weeks of gestation, no losses were registered. In average, 22% of fresh embryos transferred were represented as crias born. None of the cryopreserved embryos survived up to 11 weeks post-transfer. There is no difference in percentage of crias born between nonsurgical transcervical embryo transfers and surgical laparoscopically aided embryo transfers.The heavy embryo losses could be related to nutrition and high-altitude limitations; however, it is difficult to make comparisons with others because reports to date lack information on the actual crias born from embryo transfers in alpacas; most of the reports are based on pregnancy reports up to 30 to 60 days post-transfers. To date, no births from cryopreserved alpaca embryos have been reported. Furher studies on causes of embryo/fetal losses are necessary. Table 1.Results of embryo transfer This study was financed by the Peruvian Fund for Innovation, Science and Technology (FINCYT).