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).

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>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 alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 μg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20–22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

Alpacas are induced ovulators, responding to copulation and (or) exogenous application of ovulation-inducing hormones. The objective of this study was to determine the efficiency of the injection of a gonadotropin-releasing hormone (GnRH) agonist versus LH in the induction of ovulation and the presence and size of non-ovulated follicles at the time of embryo collection and its relationship to the yield of transferable embryos in superovulated alpacas. Twenty-one adult (3 to 7 years old) female alpacas under extensive grazing at 4300 m elevation in the Peruvian Andes that had been synchronized and treated for superovulation were induced to ovulate 6 days after the application of the superovulatory hormone (1000 IU of eCG, Folligon®, Intervet International BV, Boxmeer, the Netherlands) by mating with fertile males and injection immediately after copulation of either an IM dose of 0.0084 mg of buserelin acetate (Buserelina Zoovet®, Laboratorio Zoovet, Santa Fe, Argentina) to 10 alpacas (T1) or an IM dose of 5-mg Armour standard of LH (Lutropin®, Bioniche Animal Health, Belleville, ON, Canada) to 11 alpacas (T2). All alpacas had a second mating 12 h after the first mating and were subjected to ovarian inspection by ultrasonography and previous embryo collection by nonsurgical transcervical embryo flushing 6.5 days after the first mating. On average, the embryo recovery rate for T1 was 34.6% and there were 7.8 ± 3.99 corpora lutea (CL), 2.7 ± 4.08 collected embryos, and 3.6 ± 2.95 total, 0.5 ± 0.85 small (<6 mm), 1.8 ± 1.99 medium (≥6 but ≤14 mm), and 1.3 ± 2.11 large (≥15 mm) non-ovulated follicles. For T2, the embryo recovery rate was 59.4% and there were 6.73 ± 1.49 CL, 4.0 ± 2.57 collected embryos, and 0.64 ± 0.81 total, 0.0 ± 0.0 small, 0.36 ± 0.67 medium, and 0.27 ± 0.47 large non-ovulated follicles. The differences between treatments were nonsignificant (P > 0.05) for all the parameters; however, there was a clear tendency for a better recovery rate, more embryos collected, and lower number of non-ovulated follicles in T2. The Pearson correlation coefficient between the presence of large follicles in the ovaries at the time of embryo collection and the total number of embryos collected was negative (r = –0.26) and highly significant (P ≤ 0.001). The use of LH for ovulation induction of superovulated alpacas seems to be more advisable than the use of GnRH agonist; further comparisons with larger number of observations per treatment are recommended. This study was financed by the Peruvian Fund for Innovation, Science and Technology (FINCYT).

The objective of the present study was to determine the ovarian response of alpacas to different treatments for follicular development and superovulation. Twenty-nine mature, lactating alpacas, between 31 and 56 days postpartum, managed in the Peruvian highlands (altitude = 4100 m) were randomly distributed into 4 experimental groups. Groups 1 (n = 7) and 3 (n = 8) received a homemade intravaginal sponge containing 60 mg of medroxyprogesterone acetate (MAP; Sigma Chemical Co., St Louis, MO, USA) plus 2 mL of PGF2α (IM; Illiren�; Intervet International, Boxmeer, The Netherlands) on Day 0. Groups 2 (n = 7) and 4 (n = 7) received 2 mL of PGF2α (IM) on Day 0, but did not receive a MAP sponge. All groups received 6 injections (IM) of FSH (Folltropin V�; Bioniche Animal Health, Beltsville, Ontario, Canada) in decreasing dosages of 50, 50, 30, 30, 20, and 20 mg, respectively, every 12 h (at 0700 and 1900 h each day), plus 300 IU of eCG (IM; Folligon�; Intervet International) at the time of the last FSH treatment, with the aim of increasing LH levels. The FSH treatments started on Days 7, 5, 9, and 7 (from Day 0) in groups 1–4, respectively. MAP sponges were removed at the time of the last FSH treatment in groups 1 and 3. All alpacas were naturally mated twice at 12 and 24 h after the last FSH treatment. Alpacas in groups 1 and 2 received 3000 IU of hCG (IM; Corulon�; Intervet International) and alpacas of groups 3 and 4 received 2.5 mL of GnRH (IM; Conceptal�; Intervet International) immediately after the first mating. Seven days after the first mating, ovaries of all alpacas were examined by transvaginal ultrasonography. Ovarian response was estimated by determining the number of CL present on each ovary. The numbers of follicles that were at least 8 mm in diameter were also counted. Data were analyzed as a complete randomized design with 4 treatments. The average number of CL per alpaca was 1.3, 1.00, 1.00, and 0.9 for groups 1 to 4, respectively (P &gt; 0.05). The average number of follicles that were at least 8 mm in diameter per alpaca was 9.4, 20.4, 0.9, and 3.9 for groups 1 to 4, respectively (P ≤ 0.05) with females in group 2 showing the highest response. We conclude that progestin treatment did not affect ovulatory response of lactating alpacas to exogenous gonadotropins. An effective ovarian stimulation strategy for achieving superovulation in alpacas remains to be developed.