Alpaca embryo transfer on a private Canadian farm

Introduction

The alpaca is one of two domestic New World camelids that originated in the Andes of South America (1). In Canada, the alpaca is exploited mainly for the quality of its fiber (2). Alpaca fiber diameter varies from 20 to 34 μm, making it one of the finest in the world (3). Fiber length, strength, high insulation, and hypoallergenic properties, and the wide variety of natural colors make alpaca fiber highly desirable for the textile industry (2–4). Fiber quality, therefore, has been the priority trait for alpaca breed improvement throughout the world (3,5). However, delayed sexual maturation (18 mo) and long gestation (11 mo) compared to other small ruminants make genetic improvement programs using natural mating inefficient in alpacas (6).

Superovulation and embryo transfer (ET) have contributed significantly to genetic progress in cattle (7). However, these techniques have not been widely applied in alpacas because of their reproductive peculiarities (6,8). In recent years, ET has been used with relative success in alpacas in South America (9,10) and Australia (8). Protocols for superovulation and embryo collection in South American camelids have been developed using equine chorionic gonadotrophin (eCG) and follicle stimulating hormone (FSH) (9,11). Embryos remain in the oviduct for 5 d after mating (12) and are usually collected by uterine flush 6 to 8 d after mating (1,10). Embryos collected by uterine flush are usually hatched blastocysts, and are classified according to the appearance of the trophectoderm (8). Attempts at embryo cryopreservation have been hampered by the relatively large size and advanced developmental stage of camelid embryos compared with other species; hence, alpaca embryos are transferred fresh to recipient females (10).

In Canada, livestock registration is recommended as a method to verify pedigree, which certifies the value of high quality alpacas (2). Until 2015, however, alpaca offspring obtained from ET were not permitted to be registered in the Canadian Llama and Alpaca Association Herdbook. The recent lifting of the ban means that alpaca crias produced from ET can now be registered, following confirmation with a DNA paternal test (13). The use of superovulation and ET in alpacas has not been reported in Canada, although an experimental ET trial in llamas at the University of Saskatchewan resulted in the birth of 1 offspring in 2012 (Dr. Gregg Adams, personal communication). The application of technologies such as superovulation and ET may be important in the establishment of genetic improvement programs in Canadian alpacas to enhance reproductive performance and promote fiber quality. Therefore, our objective was to determine the feasibility of a superovulatory and embryo transfer protocol on a private alpaca farm in Canada.

Materials and methods

Facilities and alpacas

The trial was performed at Arriba Linea farm, Uxbridge, Ontario, Canada (44°09.20034′N, 79°07.24830′W). Alpacas were maintained with free access to alfalfa/orchard grass hay and fresh water. Donors and recipients were 3 to 7 y of age and had an average body condition score of 3.5 (scale of 1 to 5) (14).

Donor superovulation and embryo collection

Ovarian synchrony was induced among 5 donor alpacas by 2 intramuscular treatments of GnRH (Gonadorelin acetate, Fertiline; Vétoquinol NA, Lavaltrie, Québec), 50 μg given 12 d apart. Ovarian superstimulation was initiated 2 d after the second GnRH treatment (i.e., expected day of new follicle wave emergence), involving once daily IM administration of 40 mg of FSH (Folltropin-V; Vétoquinol NA) for 5 d (11). On the last day of FSH treatment, cloprostenol (Estroplan; Vétoquinol NA), 250 μg, IM, was given in the morning and afternoon to induce luteolysis. Two days later, donors were mated (≥ 15 min) with mature males and given a single dose of GnRH. On Day 7 (Day 0 = day of breeding), the ovaries were examined by transrectal ultrasonography to determine the superovulatory response, and the uterus was flushed to collect embryos.

Embryos were collected non-surgically by transcervical uterine flush. Briefly, epidural anesthesia was induced with 2 mL of 2% lidocaine hydrochloride and epinephrine (Bimeda-MTC Animal Health, Cambridge, Ontario) given at the sacro-coccygeal junction. After disinfection of the vulva, a 16 French silicone catheter with a stainless-steel stylet (Partnar Animal Health, Ilderton, Ontario) was introduced through the cervix and placed at the base of a uterine horn using transrectal manipulation. The balloon cuff was inflated with 5 to 8 mL of flushing medium to fix the catheter in place and prevent reflux during flushing. The uterine horn was flushed using a 35-mL catheter-tip syringe containing 15 to 25 mL of commercial flushing medium (Complete Flush; Vétoquinol NA). Each uterine horn was flushed with 6 or 7 syringe volumes of medium, and the aspirate was passed through a 75-μm filter (Emcon filter; Agtech, Manhattan, Kansas, USA). Embryos were located by stereomicroscopy and classified according to the appearance of the trophecthoderm (based on 8) as Grade 1 (Excellent; rounded and uniform surface), Grade 2 (Good; rounded but surface not uniform), Grade 3 (Poor; not rounded and/or shrunken appearance), and Grade 4 (Embryo with holes). Embryos were selected for transfer and placed in a 35-mm Petri dish with holding medium (MOFA; Verona, Wisconsin, USA) kept on a plate-warmer at 35°C. Donors were given a luteolytic dose of cloprostenol immediately after collection.

Recipient synchronization and embryo transfer

Ovarian synchrony was induced among 8 alpaca recipients using 2 doses of GnRH given 12 d apart, as described for the donors. The second administration of GnRH was given the day before the donors were mated (Day –1) to induce ovulation and corpus luteum (CL) formation. Embryos were transferred into recipients 8 d after the second GnRH treatment. Recipients were examined by transrectal ultrasonography on the day of embryo transfer to determine presence and size of the CL. A single embryo was transferred to each recipient no more than 1 h after being collected. The embryo was loaded into a 0.25-mL straw in succeeding columns of medium and air in the following pattern: medium, air bubble, medium with the embryo, air bubble, and medium. The ET gun (YT gun; Yamanetech, Matsumoto-City, Nagano, Japan) containing the loaded embryo was introduced transcervically into the uterine horn ipsilateral to the ovary bearing the CL, and the embryo was deposited in the middle part of the horn.

Pregnancy diagnosis

Sexual receptivity was tested in recipients 15 d after embryo transfer. Individual recipient females were transferred to a pen with a single male alpaca, and if the female spit, kicked, and/or ran away from the male, she was considered a suspect for pregnancy; a recipient that remained quiet and lay down in the presence of the male was considered non-pregnant. All recipients were examined by transrectal ultrasonography 2 mo after ET to determine pregnancy status.

Results

Ovarian responses and embryo collection results in alpaca donors are displayed in Table 1. One alpaca donor did not respond to superovulatory treatment and was not flushed. Ten transferable embryos were collected from 4 donors (Figure 1). A CL was not detected in 2 recipient alpacas, thus they were not used for embryo transfer. In the remaining 6 recipients, the CL was 10.7 ± 0.7 mm (mean ± SEM) in diameter.

Figure 1

Embryos collected from a single alpaca donor on Day 7 (Day 0 = day of mating). G1 — Grade 1 embryos; G2 — Grade 2 embryo; and G3 — Grade 3 embryo.

Pregnancy outcomes are displayed in Table 2. Although 4 of 6 recipients were positive to the behavioral test (suspected pregnant), only 1 alpaca was confirmed to be pregnant by ultrasound examination 2 mo after transfer. The recipient gave birth unassisted to a healthy male 349 d after ET (Figure 2).

Figure 2

First alpaca cria obtained through embryo transfer in Canada; unassisted birth of a live male on August 25, 2017 at Arriba Linea Farm, Ontario.

Discussion

In the present trial, 4 of 5 alpaca donors were superovulated using a customized hormone protocol. From the superovulated alpacas that were flushed, 12 embryos were collected, of which 10 were classified as transferable. Six of 8 recipient alpacas were successfully synchronized with the donors and received a single embryo, and 1 pregnant dam delivered a live offspring 349 d after ET. To our knowledge, this is the first alpaca cria obtained through embryo transfer in Canada.

The superovulation protocol used in the present trial resulted in an average of 5.2 CL per donor, confirming the effectiveness of FSH for inducing superovulation in alpacas, previously reported in Australia where approximately 6.5 CL per donor were observed (8). In the present study, an average of 3 embryos were collected from each of 4 donors, which is intermediate between the 2.5 embryos reported in alpacas in Australia (8) and the 4.8 in llamas in Peru (15). Technical differences, such as a larger rectum in the llama, may have allowed better manipulation of the uterus during flushing of the embryos. Therefore, the size of the alpaca and diameter of the practitioner’s hand must be taken into account when working with embryo transfer technologies in this species (10).

Due to a limited number of recipients, not all the embryos were used in this trial. The average size of the CL in recipients was 10.7 mm at the time of embryo transfer (Day 7), similar to previous reports in llamas and alpacas after natural breeding or hormonal treatment (16,17). Despite this, however, only 1 alpaca recipient became pregnant after transfer of a single fresh embryo, which is lower than the 41% pregnancy rate reported in the Australian study involving ~4500 recipients (8). The reason for the low pregnancy rate in the present study is not clear, but may be a result of imprecise donor-recipient synchronization. Maternal recognition of pregnancy, as indicated by a rescue and resurgence of the CL, occurs between Days 8 to 10 (16). Hence, slight asynchrony between the age of the embryo and the developmental stage of the CL may be expected to have a marked impact on pregnancy rate.

In conclusion, superovulation and embryo transfer are feasible techniques that can be applied on private alpaca farms in Canada. This is the first report of alpaca embryo transfer in Canada that resulted in the birth of a live offspring. Improvement of these techniques may be achieved with application on a larger scale, and may be of benefit to the genetic improvement program of Canadian alpaca producers. CVJ