Effect of chemical activators after intracytoplasmic sperm injection (ICSI) on embryo development in alpacas.

Low motility and low sperm concentration are characteristics of alpaca semen. Thus, the intracytoplasmic sperm injection (ICSI) technique represents an alternative to improve the reproductive capacity of the male. However, the effect of post-ICSI activation in alpaca is not yet known. The aim of the present study was to compare the effect of chemical activators on alpaca embryo development after ICSI. Alpaca ovaries were collected from a local slaughterhouse and transported to the laboratory. Category I, II and III oocytes were matured for 30 h at 38.5 °C. After ICSI, injected oocytes were randomly divided and activated as follows: i) 5 µM ionomycin for 5 min, ii) 7% ethanol for 4 min, iii) 5 µM ionomycin for 5 min, window period 3 h plus 7% ethanol for 4 min, iv) 5 µM ionomycin for 5 min, window period 3 h, a second ionomycin treatment for 5 min, followed by 1.9 mM 6-DMAP for 3 h, v) 10 mM SrCl2 for 3 h. Culture was carried out for 5 days in SOFaa at 38.5 °C. The cleavage rate was the lowest in the SrCl2 group, morula development was the lowest in the SrCl2 and without activation groups, and blastocyst stage was not different between groups (P<0.05). The rates with SrCl2 were lower in total embryos produced, whereas in transferable embryos they were lower with 2Io/6-DMAP and with SrCl2 (P<0.05). In conclusion, alpaca oocyte activation is more efficient with ionomycin and ethanol to produce transferable embryos.

Specific imprinted genes demethylation in association with oocyte donor's age and culture conditions in bovine embryos assessed at day 7 and 12 post insemination.

The production of bovine embryos through in vitro maturation and fertilization is an important tool of the genomic revolution in dairy cattle. Gene expression analysis of these embryos revealed differences according to the culture conditions or oocyte donor's pubertal status compared to in vivo derived embryos. We hypothesized that some of the methylation patterns in oocytes are acquired in the last step of folliculogenesis and could be influenced by the environment created in the follicles containing these oocytes. These altered patterns may not be erased during the first week of embryonic development in culture or may be sensitive to the conditions during that time. To quantify the changes related to culture conditions, an in vivo control group consisting of embryos (Day 12 post fertilization for all groups) obtained from superovulated and artificially inseminated cows was compared to in vitro produced (IVP) embryos cultured with or without Fetal Bovine Serum (FBS). To measure the effect of the oocytes donor's age, we also compared a fourth group consisting of IVP embryos produced with oocytes collected following ovarian stimulation of pre-pubertal animals. Embryonic disk and trophoblast cells were processed separately and the methylation status of ten imprinted genes (H19, MEST, KCNQ1, SNRPN, PEG3, NNAT, GNASXL, IGF2R, PEG10, and PLAGL1) was assessed by pyrosequencing. Next, ten Day 7 blastocysts were produced following the same methodology as for the D12 embryos (four groups) to observe the most interesting genes (KCNQ1, SNRPN, IGF2R and PLAGL1) at an earlier developmental stage. For all samples, we observed overall lower methylation levels and greater variability in the three in vitro groups compared to the in vivo group. The individual embryo analysis indicated that some embryos were deviant from the others and some were not affected. We concluded that IGF2R, SNRPN, and PEG10 were particularly sensitive to culture conditions and the presence of FBS, while KCNQ1 and PLAGL1 were more affected in embryos derived from pre-pubertal donors. This work provides markers at the single imprinted control region (ICR) resolution to assess the culture environment required to minimize epigenetic perturbations in bovine embryos generated by assisted reproduction techniques, thus laying the groundwork for a better comprehension of the complex interplay between in vitro conditions and imprinted genes.

Alpaca embryo transfer on a private Canadian farm.

This study evaluated the feasibility of using an embryo transfer protocol in an alpaca farm in Canada. Alpaca donors and recipients were synchronized with 2 doses of gonadotrophin-releasing hormone (GnRH), 12 days apart. In donors (n = 5), superstimulation was induced with follicle stimulating hormone (FSH) given daily (40 mg) for 5 days beginning 2 days after the second GnRH treatment. Cloprostenol was given on the last day of FSH, the donors were bred 2 days later, embryos were collected 7 days after breeding. In recipients (n = 8), the second dose of GnRH was given the day before donor mating, and embryos were transferred on the day of donor collection. On average (± SEM), 5.2 ± 1.4 corpora lutea were detected and 2.5 ± 1.2 transferable embryos were collected in the donors. A mature corpus luteum was detected in 6/8 synchronized recipients and a single embryo was transferred to each. One recipient alpaca became pregnant and delivered a healthy baby 349 days after embryo transfer. This is the first report of successful embryo transfer in alpacas in Canada.

Transfert d'un embryon d'alpaga dans une ferme privée canadienne. Cette étude a évalué la faisabilité de l'utilisation d'un protocole de transfert d'un embryon dans une ferme d'alpagas au Canada. Les alpagas donneurs et récipiendaires ont été synchronisés avec deux doses d'hormone de gonadolibérine (GnRH), à 12 jours d'intervalle. Chez les donneurs (n = 5), la super-stimulation a été induite avec une hormone follicostimulante (FSH) administrée quotidiennement (40 mg) pendant 5 jours deux jours après le deuxième traitement de GnRH. Le cloprosténol a été administré le dernier jour de FSH, les donneurs ont été accouplés 2 jours plus tard et les embryons ont été prélevés 7 jours après l'accouplement. Chez les récipiendaires (n = 8), la deuxième dose de GnRH a été administrée la journée avant l'accouplement des donneurs et les embryons ont été transférés le jour du prélèvement du donneur. En moyenne (± SEM), 5,2 ± 1,4 corpora lutea ont été détectés et 2,5 ± 1,2 embryons transférables ont été prélevés des donneurs. Un corpus luteum mature a été détecté chez 6/8 récipiendaires synchronisés et un seul embryon a été transféré à chacun. Un alpaga récipiendaire est devenu gravide et a donné naissance à un petit en santé 349 jours après le transfert de l'embryon. Il s'agit du premier rapport d'un transfert d'embryon réussi chez des alpagas au Canada.(Traduit par Isabelle Vallières).

Effect of extending FSH treatment on superovulation and embryo production in wood bison (Bison bison athabascae).

The effect of extending the length of the FSH treatment protocol on superovulatory response and embryo production was investigated in wood bison during the anovulatory and ovulatory seasons. In Experiment 1 (anovulatory season), follicular wave emergence was synchronized by follicular ablation (Day -1) and bison were assigned randomly to two groups (n = 14/group) and given 200 mg FSH on Day 0 and Day 2 (non-extended group), or 133 mg FSH on Days 0, 2, and 4 (extended group). Human chorionic gonadotropin (hCG; 3000 IU) was given on Day 5 and Day 6 in the non-extended and extended groups, respectively, and bison were inseminated 12 and 24 h later. Ova/embryos were collected 8 days after hCG treatment. In Experiment 2 (ovulatory season), bison were synchronized and superstimulated as in Experiment 1 (n = 12/group), but prostaglandin was given to control CL development. Data were compared by t-test and Chi-square test. In Experiment 1, no differences in ovarian response or embryo production between groups were detected. In Experiment 2, there was no difference in the ovarian response between groups, however, a greater number of ova/embryos (4.3 ± 0.8 vs. 2.3 ± 0.4; P ≤ 0.05) and freezable embryos (2.5 ± 0.6 vs. 1.2 ± 0.4; P ≤ 0.05) were obtained in the extended group. The number of freezable embryos was greater during the ovulatory vs anovulatory season (1.8 ± 0.4 vs. 0.3 ± 0.2; P ≤ 0.05). In conclusion, extending the FSH treatment in wood bison did not improve the superovulatory response during the anovulatory season, but resulted in twice as many freezable embryos during the ovulatory season. The number of freezable embryos collected during the anovulatory season was <20% that of the ovulatory season.

Effects of eCG and progesterone on superovulation and embryo production in wood bison (Bison bison athabascae).

Experiments were done to determine if inclusion of eCG and progesterone in the superstimulation protocol will increase the ovarian response and embryo production in wood bison, and to provide preliminary information regarding the effect of season. In Experiment 1 (anovulatory season), bison (n=26) were synchronized by follicular ablation (Day -1) and given FSH on Days 0 and 2, and assigned to 3 groups: Progesterone (Days 0-4), eCG (Day 3), or progesterone+eCG. On Day 5, bison were given hCG and inseminated 12 and 24h later. Ova/embryos were collected 8days after hCG. In Experiment 2 (ovulatory season), bison (n=24) were synchronized and assigned randomly to two groups in which superstimulation was induced with FSH, either with or without eCG, as in Experiment 1. No differences among groups were found in ovarian response or embryo production in either experiment. The follicular count at wave emergence was positively correlated with the number of large follicles at the end of superstimulation in all groups. A significantly greater number of follicles present at wave emergence in the anovulatory vs. ovulatory season was associated with a greater number of CL at the time of embryo collection, but only half the number of freezable embryos. In conclusion, the number of transferable embryos collected (1-2/bison) was higher than in any previous report, but was not attributable to the inclusion of eCG or progesterone in the superovulatory protocol. The apparent effect of season on oocyte competence, and not superovulatory response, is worthy of further investigation.

In vitro-production of embryos using immature oocytes collected transvaginally from superstimulated wood bison (Bison bison athabascae).

Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1-3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro. Although follicles were larger (P < 0.05) in the 4-day FSH starvation group, there was no effect of starvation period on the distribution of COC morphology; overall, 112/194 (57.7%) were compact, 29/194 (26.3%) were expanded, 39/194 (20.1%) were denuded, and 14/194 (7.2%) were degenerated (P < 0.05). Similarly, there was no effect of starvation period on embryo development. Compact good COC had the highest cleavage (88%) and blastocyst rates (54%; P < 0.05), followed by compact regular COC at 73% and 25%, respectively. Expanded and denuded COC had low cleavage (40% vs. 59%, respectively) and blastocyst rates (5% vs. 8%, respectively). We conclude that morphologic characteristics of wood bison COC are reflective of the ability of the oocyte to develop into an embryo in vitro. Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture.

In vitro embryo production in wood bison (Bison bison athabascae) using in vivo matured cumulus-oocyte complexes.

Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P < 0.05). In conclusion, an additional short period of in vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison.

In vivo and in vitro maturation of oocytes collected from superstimulated wood bison (Bison bison athabascae) during the anovulatory and ovulatory seasons.

Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day -1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days -9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60-70% of oocytes were at the MII stage 24h after in vitro maturation while only 25-27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.

Superovulation in wood bison (Bison bison athabascae): Effects of progesterone, treatment protocol and gonadotropin preparations for the induction of ovulation.

Experiments were done to determine the ovarian response and embryo production following superstimulation of wood bison. In Experiment 1 (Anovulatory season), the efficacy of pLH vs. hCG for inducing ovulation was compared in wood bison superstimulated with a single dose of pFSH in 0.5% hyaluronan and the effect of exogenous progesterone (PRID) on superovulatory response and embryo quality was examined. In Experiment 2 (Ovulatory season), the efficacy of pLH vs. hCG for the induction of ovulation was compared in wood bison superstimulated with pFSH in a single intramuscular dose vs. a two-dose regimen 48 h apart (split dose) in 0.5% hyaluronan. In Experiment 1, the number of CL was greater (P < 0.05) in bison treated with hCG than pLH (6.6 ± 1.8 vs. 2.8 ± 0.8) and in those that were not given PRID (6.0 ± 1.5 vs. 2.7 ± 1.0). There was no effect of progesterone treatment on embryo quality. In Experiment 2, the number of CL was greater (P < 0.05) in bison treated with hCG than with pLH (6.3 ± 0.8 vs. 3.8 ± 1.2) and in bison superstimulated with split dose vs. single dose of FSH (7.1 ± 0.9 vs. 3.0 ± 0.8). The number of ova/embryos and freezable embryos did not differ among groups in either experiment. In conclusion, hCG induced a greater ovulatory response than pLH in both seasons. Two doses of FSH induced the greatest superovulatory response during the ovulatory season. Exogenous progesterone did not improve embryo quality during the anovulatory season.

Inducing ovulation in wood bison (Bison bison athabascae) during the anovulatory season.

As part of the development of a germplasm biobank to preserve the genetic diversity of threatened wood bison (Bison bison athabascae), a 2 × 2 factorial study was designed to determine the effects of ovulation induction agent and follicle maturity on the ovulatory response in wood bison during the anovulatory season. Bison (n=32) were assigned randomly to four groups (n=8/group) and treated with either pLH or hCG when a growing dominant follicle was either 8-9 mm or ≥10 mm. The ovaries were examined daily by ultrasonography to determine the timing of ovulation, and 7 days post-treatment to assess CL development. The proportion of bison that ovulated was greater in bison treated with hCG than pLH ([15/16] 94% vs. [8/16] 50%; P<0.05), and when the dominant follicle was ≥10 mm vs. 8-9 mm at the time of treatment (88% vs. 56%; P<0.05). The interval from treatment to ovulation was 37.0 ± 1.3h and was not affected by induction agent or follicle size. However, synchrony of ovulation tended to be greater (P=0.10) in the ≥10 mm group vs. the 8-9 mm group, and the ensuing corpus luteum was larger (15.3 ± 0.43 mm vs. 13.4 ± 0.36; P<0.05). In conclusion, both ovulation inducing agent and follicle size influenced the ovulatory response in bison during the anovulatory season. Treatment with hCG was more effective than pLH for inducing ovulation in wood bison, and the effect was greater when treatment was given when the growing dominant follicle was ≥10 mm.

Ovarian superstimulation and oocyte collection in wood bison (Bison bison athabascae) during the ovulatory season.

The objective of the study was to establish an effective ovarian superstimulatory protocol and subsequently obtain oocytes from bison by transvaginal ultrasound-guided follicular aspiration. Two experiments involving 22 wood bison were done during the breeding season (September to December). In experiment 1, the bison were given a luteolytic dose of prostaglandin (Day 0) and underwent follicular ablation (Day 8) to induce ovarian synchrony. Synchronized bison were then assigned randomly to two groups (n = 11 per group) and given either 200 mg FSH diluted in saline sc, or 200 mg FSH diluted in a proprietary slow-release formulation (SRF) im on Days 9 and 11. Prostaglandin was given to both groups on Day 11 followed by 25 mg LH on Day 13. Oocytes were collected by transvaginal ultrasound-guided aspiration of follicles ≥5 mm on Day 14. In experiment 2, bison were synchronized as in experiment 1 and assigned randomly to one of two groups (n = 11 per group) and given either a single dose of 2500 IU eCG im on Day 9, or 200 mg FSH sc on Days 9 and 11. Prostaglandin was given to both groups on Day 11, and LH (25 mg) was given on Day 13. Oocyte collection was done as described in experiment 1. Cumulus-oocyte-complexes (COC) were classified according to morphologic characteristics. In experiment 1, more follicles ≥5 mm were detected on Day 14 in bison treated with FSH versus eCG (12.2 ± 1.73 vs. 5.8 ± 0.52; P < 0.05), and more COC were collected from FSH-treated animals (7.2 ± 1.41 vs. 3.4 ± 0.62; P < 0.05). In experiment 2, the FSH-saline and FSH-SRF groups had a similar number (mean value ± standard error of the mean) of follicles ≥5 mm on Day 14 (12.4 ± 1.49 vs. 13.8 ± 1.24, respectively) and a similar number of COC were collected (6.5 ± 1.13 vs. 6.3 ± 0.96, respectively). The proportion of COC collected per follicle aspirated and the percentage of compact, expanded, and denuded oocytes did not differ between groups in either experiment 1 or 2. In summary, a two-dose regimen of FSH diluted in saline and given sc or in a SRF and given im induced a similar ovarian response in wood bison, whereas a single dose of eCG resulted in a significantly lower ovarian response. Overall, COC were collected from 55% of follicles after transvaginal, ultrasound-guided needle aspiration in wood bison.

Ovarian synchronisation in wood bison (Bison bison athabascae) during the anovulatory season.

Two experiments were performed in wood bison during the anovulatory season to establish an effective protocol for ovarian synchronisation. In an untreated control phase, bison cows (n=19) were examined daily to establish the interval to new follicular wave emergence (4.9±0.7 days) for the purposes of comparison with the experimental treatments. In Experiment 1, bison were treated by transvaginal ultrasound-guided follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol (n=10). In Experiment 2, bison were treated by follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol +100mg, i.m., progesterone (n=10). In Experiment 1, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol-treated groups was 4.9±0.7, 1.1±0.1 and 3.1±0.4 days, respectively (P<0.05). The degree of synchrony was 2.4±0.4, 0.2±0.1 and 0.8±0.2 days, respectively (P<0.05). In Experiment 2, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol + progesterone-treated groups was 4.9±0.7, 1.2±0.2 and 3.3±0.3 days, respectively (P<0.05), and the degree of synchrony was 2.4±0.4, 0.2±0.1, and 0.8±0.2 days, respectively (P<0.05). The degree of synchrony did not differ between ablation and hormone treatment groups in either experiment, but was greater in treatment groups than in the untreated control phase. Both follicular ablation and hormone treatment shortened and decreased the variability in the interval to follicular wave emergence in bison, but wave emergence occurred earlier after follicular ablation.

Superstimulatory response and oocyte collection in North American bison during the non-breeding season.

A 2×2 design was used to compare the ovarian response and oocyte collection characteristics in bison given a superstimulatory dose of eCG or FSH, with or without a follow-up dose of LH. Follicular wave emergence was synchronized by follicle ablation (Day -1) and bison were assigned randomly to two superstimulatory treatment groups (n=10 per group): (i) a single intramuscular dose of 2500IU of eCG given on Day 0, or (ii) two subcutaneous doses of 200mg of FSH given on Days 0 and 2. On Day 4, 200mg of LH was given intramuscularly in 5 bison in each superstimulatory treatment group. The study was done in two replicates (n=20 per replicate) involving a crossover design so that each animal was given the opposite superstimulatory treatment (eCG or FSH) during successive replicates. Cumulus-oocyte complexes (COC) were collected by transvaginal ultrasound-guided follicle aspiration, and were classified according to morphologic attributes as compact, expanded, or denuded. Superstimulatory treatment with FSH (vs. eCG) resulted in the development of more follicles ≥5mm (14.2±1.41 vs. 8.2±0.67; P<0.05; mean±SEM), and more follicles aspirated (12.4±1.3 vs. 6.3±0.6; P<0.04). Follow-up treatment with LH (vs. no LH) resulted in a greater proportion of expanded COC (37% vs. 15%; P<0.05), and a tendency for a higher COC collection rate (61% vs. 54%; P=0.08). In summary, superstimulation with FSH (vs. eCG) resulted in twice as many follicles available for aspiration and nearly twice as many COC collected in bison during the anovulatory season, and follow-up treatment with LH increased the proportion of expanded COC collected.

Surgical translocation and ultrasound bio-microscopy of the ovaries in rabbits.

Experiments were designed to validate the use of ultrasound bio-microscopy (UBM) as a method for assessing ovarian structures in rabbits. In Experiment 1, female New Zealand White (NZW) rabbits (n=4) were given an ovulation-inducing treatment and the ovaries were examined ex situ by UBM using a 25 MHz oscillating sector transducer before being processed for histology. Pairwise correlations revealed strong relationships between UBM and histology in the number (Mean±SEM) of follicles ≥0.6 mm (17.3±2.3 compared with19.0±1.6, respectively; r=0.96; P=0.040), CL (8.5±2.9 compared with 8.8±3.0; r=0.99; P=0.003), the diameter of follicles (1.1±0.05 compared with 1.1±0.03 mm; r=0.96; P=0.035) and CL (2.1±0.7 compared with 1.8±0.6 mm, r=0.99; P<0.001). In Experiment 2, the ovaries of NZW rabbits (n=12) were surgically translocated to a subcutaneous position in the flank region to permit serial examination of ovarian structures in vivo by UBM. Beginning 2 weeks after surgery, the ovaries were examined by UBM daily for at least 18 days, and again 2 months after surgery. Post-operative complications were minor, and both ovaries of each rabbit were identified consistently. The number and diameter of follicles ≥0.6 mm were readily visualized during each examination. Multiple corpora lutea were detected in two rabbits, and serial follicular and luteal dynamics in these two rabbits were used to document the consistency of UBM and the retention of ovarian function after surgery. It is concluded that UBM is a valid tool for instant assessment of rabbit ovarian structures (follicles, corpora lutea, and cumulus-oocyte complexes) ex situ, and for serial assessment in vivo using a transcutaneous approach. Surgical translocation had no apparent untoward effect on ovarian function.