No abstract

The objective was to produce alpaca embryos in laboratory due to its potential role for the multiplication of genetically superior animals and for conservation purposes. Ovaries were collected from an alpaca abattoir located in the Central Highlands of Peru and transported in a thermos flask with warm saline and antibiotics to the laboratory located 200 km away on the coast. Alpaca epididymal sperm to be used for fertilization was previously frozen by diluting in a TRIS-Fructose based extender with 10% glycerol and frozen as pellets in liquid nitrogen vapor. From 31 ovaries, 262 cumulus–oocyte complexes (COCs) were collected (mean of 8.5 COCs per ovary) which were matured in TCM-199 supplemented with 10% heat inactivated FCS plus epidermal growth factor (EGF), FSH, LH, estradiol, and cysteamine for 30 h incubation at 38.5°C, 5% CO2 and 90% humidity. The selected oocytes post-maturation were fertilized with the frozen/thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugation and resuspension in TALP-IVF medium supplemented with 20 μm D-penicillamine, 10 μm hypotaurine, 1 μm epinephrine and 1.1 μg mL–1 of heparin. The oocytes were inseminated with a concentration of 10 × 106 spermatozoa per drop of 100 mL of fertilization medium containing 30 oocytes each and incubated for 24 h at 38.5°C, 5% CO2 and 90% humidity. The presumptive zygotes were transferred to 200 μL drops (30 zygotes per drop) of SOFaa media supplemented with 5% heat-inactivated FCS which was replaced by SOFaa plus 1% heat-inactivated FCS on day 5 after fertilization. The incubation period post-fertilization was up to day 7 at 38.5°C, 5% CO2 and 90% humidity, when the embryos were inspected and graded. The cleavage rate was evaluated at 72 h post-fertilization and embryo development was evaluated on day 5 and 7 post-fertilization. The cleavage rate was 27.1% (71/262) and the percentage of oocytes that reached the stage of morula and blastocyst was 8.0% (21/262). The percentage of blastocyst that hatched when incubated after day 7 was 14.28% (3/21). The in vitro embryo production in alpacas was successful and suggests the possibility for application in intensive reproduction for conservation of South American camelids and for genetic improvement. Research was partially funded by contributions of BIONICHE and SAIS TUPAC AMARU, Junin, Peru.

No abstract

The objective of the study was to evaluate 4 superovulatory regimes in terms of the quantity of transferable embryos recovered. A total of 48 female alpacas, 3 to 5 years of age and located at Malkini Alpacas Farm (4100 m elevation), were distributed into 4 treatments. In treatment 1, 13 female alpacas received on Day 0 an intravaginal device containing 0.78 mg of progesterone (Cue Mate®, Bioniche Animal Health, Belleville, Ontario, Canada) followed immediately by an i.m injection of estradiol (1 mg of estradiol benzoate) and an i.m. injection of PGF2α (Veyx®, 0.25 mg of cloprostenol). The intravaginal device was removed on Day 7, performing at removal time an i.m. injection of estradiol. From Days 8 to 16, the alpacas received an i.m injection twice per day and 12 hours apart of pFSH (FolltropinV®, Bioniche Animal Health) in decreasing doses totaling 420 mg of pFSH; on Day 16,300 IU of eCGi.m. (Pregnecol®, Bioniche Animal Health) was injected. In treatment 2, 13 alpacas received on Day 0 an intravaginal device of progesterone followed by an i.m. injection of PGF2; from Days 5 to 9, alpacas received injections twice per day of decreasing doses of pFSH (porcine FHS) totaling 320 mg; on Day 7, the intravaginal device was removed and 500 IU i.m. of eCG was injected. In treatment 3,13 alpacas received on Day 0 an intravaginal device of progesterone followed immediately by an i.m injection of GnRH (Conceptal®, 0.0042 mg of acetate of busereline); pFSH was injected i.m. from Days 5 to 9 in decreasing doses twice per day, totaling 440 mg; the intravaginal device was removed on Day 7. In treatment 4, 9 female alpacas received on Day 0 an i.m. injection of GnRH after verifying the presence of a preovulatory follicle (>8.0 mm diameter). On Day 2, the alpacas received 1000 IU i.m. of eCG followed on Day 7 by an i.m. injection of PGF2. In all cases, the donor alpacas were evaluated by ultrasonography. The matings for treatments 1, 2, and 3 were performed twice per donor alpaca at 12-hour intervals between Days 5 and 8 of the initiation of the pFSH treatments, whereas in treatment 4 the matings were made the following day after the application of the PGF2. In treatment 1, the donor alpacas received at time of first mating an i.m injection of 3.75 mg of LH (Lutropin®, Bioniche Animal Health); in treatments 2, 3, and 4, the donors received an i.m. injection of GnRH. In all treatments, embryo collection was performed by nonsurgical method 6.5 days after first mating. There were significant differences between treatments (P < 0.05) in the mean number of CL, with treatment 4 being the highest (4.7 ± 2.63, 4.1 ± 3.05, 1.8 ± 1.8, and 6.0 ± 3.16 for treatments 1 to 4, respectively). The total number of blastocysts recovered per treatment was 7, 16, 2, and 18 for treatments 1 to 4, respectively. The superovulatory strategy followed for treatment 4 showed to be the one resulting in the highest number of transferable embryos. Further comparative evaluations between FSH and eCG treatments are recommended. Research was partially funded by the contributions of Bioniche Animal Health.

The guinea pig (Cavia porcellus) has been used as a laboratory animal since the late 18th century and still remains essential in many research areas. It also plays an important role in the Andes societies as a source of protein for many low-income highlanders and as part of rituals and traditional medicines. Thus, the conservation of genetic diversity is a long-term issue that must be considered. To establish an embryo cryobank, it is necessary to develop a method of embryo transfer. Up to now no pregnancies after surgical embryo transfer into synchronized females have been reported in guinea pigs. The aim of this work was to design a standard embryo transfer method in this species. Eight normally cycling female guinea pigs from the Maria-Marcela Farm (Puente Piedra, Peru), weighing from 1 to 1.5 kg, were used in this study. Females were housed under farming conditions and fed on commercial pellets and tap water ad libitum. Three donor females were superovulated using 15 IU of human menopausal gonadotrophin (hMG, Massone®, Buenos Aires, Argentina) and mated as soon as the vagina opened. Copulatory plug was observed and vaginal smears were taken to guarantee successful mating. Thirty-eight embryos were collected between Days 3.5 and 4.5 after ovulation at the morula and early blastocyst stages. Five recipient females were synchronized by a daily 0.1-mL dose of altrenogest (Regumate® Equine, Intervet, France) per os by means of a syringe for 15 days. Two embryos were transferred into each uterine horn by laparotomy at Day 3.5 and 4.5 after ovulation. Two types of pipettes were tested for embryo transfer: pulled glass pipettes approximately 0.3 mm in diameter in 2 female recipients and plastic open pulled straws (OPS, Minitüb®, Germany) in 3 recipients. Pregnancy diagnosis was detected by observation of no return to oestrus at Day 16 and confirmed by ultrasonography. None of the 3 OPS-transferred females were pregnant. One of the 2 pulled glass pipette–transferred females was diagnosed as pregnant and delivered 2 stillbirths (one per uterine horn). There were no postsurgical complications and the females undergoing embryo transfer returned to normal reproduction. We demonstrated that a classic surgical embryo transfer method is possible under field conditions to obtain pregnancy in this species. We suggest further studies using glass pipettes, which allow a more precise embryo deposition. Future experiments will incorporate the transfer of frozen-thawed embryos on a larger scale.