Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 µg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Effect of expression of estrus and treatment with GnRH on pregnancies per AI in beef cattle synchronized with an estradiol/progesterone-based protocol.

Three experiments evaluated the effects of expression of estrus and gonadotropin releasing hormone (GnRH) treatment on pregnancies per AI (P/AI) in beef cattle that were treated with an estradiol/progesterone (P4)-based protocol for fixed-time artificial insemination (FTAI). In Experiment 1, 20 non-lactating beef cows were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of P4. Seven days later, P4 devices were removed and all cows received prostaglandin F2 alpha (PGF2α) and 0.5 mg estradiol cypionate (ECP). Estrus was detected using tail paint and cows that did not show estrus by 48 h after P4 device removal were randomized to receive GnRH or no treatment. Ovulation, as determined by ultrasonography, occurred earlier in cows that showed estrus (68.0 ± 2.5 h) than in cows that did not (82.0 ± 2.1 h, P < 0.05). Furthermore, cows that received GnRH ovulated earlier (78.0 ± 2.6 h) than those that did not (86.0 ± 2.0, P < 0.05). Experiment 2 determined whether expression of estrus and the administration of GnRH to animals that did not show estrus increased P/AI. Non-lactating beef cows and heifers (n = 1356) were treated as in Experiment 1 (P4 device removal, PGF2α and ECP administration on Day 7) or extended until Day 8. All animals in estrus by 48 h after P4 device removal were inseminated and those not showing estrus received GnRH or no treatment and were FTAI 8 h later (i.e., at 56 h). P/AI were greater (P < 0.01) in animals that were observed in estrus by 48 h (56.4%) than in those that did not show estrus (46.5%). Likewise, animals that did not show estrus but were treated with GnRH had greater P/AI (53.8%, P < 0.04) than those that did not receive GnRH (37.9%). Experiment 3 was designed to determine the effect of delaying GnRH treatment to the time of FTAI (at 56 h) in cows not showing estrus by 48 h after P4 device removal. Suckled beef cows (n = 969) were treated as in Experiment 1, except that all cows also received 400 IU of eCG at the time of P4 device removal on Day 7. Cows that showed estrus by 48 h or 56 h had greater P/AI (62.3%, P < 0.05) than those did not show estrus (51.5%). Furthermore, when cows that did not show estrus by 48 h were analyzed separately, P/AI were greater (P < 0.05) in those that received GnRH at 48 h and were FTAI by 56 h (64.9%) than in those that received GnRH concurrent with FTAI by 56 h after device removal (54.6%). In summary, expression of estrus was associated with earlier ovulations and resulted in greater P/AI in cows and heifers treated with an estradiol/P4-based protocol for FTAI. Furthermore, GnRH treatment in animals that did not show estrus hastened the time of ovulation and increased P/AI.

Effect of estrus expression or treatment with GnRH on pregnancies per embryo transfer and pregnancy losses in beef recipients synchronized with estradiol/progesterone-based protocols.

Two experiments were designed to determine the effect of expression of estrus or GnRH treatment on pregnancies per embryo transfer (P/ET) and pregnancy losses in beef recipients that were synchronized with estradiol/progesterone based protocols for fixed-time embryo transfer (FTET). Experiment 1 evaluated the effect of expression of estrus and GnRH treatment in the absence of estrus on P/ET. Beef cows (n = 729) were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of progesterone. Devices were removed 8 d later and all cows received prostaglandin F2α (PGF2α), 400 IU eCG, and 0.5 mg estradiol cypionate (ECP) at that time. Expression of estrus was determined at 48 and 56 h after device removal using tail-paint and cows that did not show positive signs of estrus by 48 h received GnRH or no treatment at random. The overall estrus rate was 76.0% (554/729); 68.0% had positive signs of estrus by 48 h after progesterone device removal and 28.0% of those not in estrus by 48 h showed estrus by 56 h. The proportion of recipients receiving in vivo-derived (IVD) or in vitro-produced (IVP) embryos and P/ET were greater in recipients that showed estrus by 48 and 56 h (94.0% and 48.4%, respectively) than in those that did not show estrus 41.0% and 29.0%, respectively; P < 0.01). However, GnRH treatment of recipients not showing estrus by 48 h did not improve P/ET. Experiment 2 evaluated the effect of expression of estrus on P/ET and pregnancy loses up to parturition in recipients synchronized with two estradiol-based protocols. Beef cows (n = 403) were divided at random to receive the same synchronization protocol as in Experiment 1 (ECP) or a J-Synch protocol (device removal on day 6 and without using estradiol cypionate to induce ovulation). In this experiment, pregnancy was determined at 30 and 60 d by ultrasonography, and all pregnant recipients were followed until parturition to determine pregnancy losses during gestation. Although the number of recipients receiving IVP embryos was greater in the ECP group (90.5% vs. 83.5%; P = 0.03), P/ET did not differ (ECP: 37.0% and J-Synch: 39.0%; P = 0.43). Overall, 88.0% (357/407) of the recipients synchronized showed estrus and a greater P/ET (P = 0.05) was found in the recipients that showed estrus (39.0%) vs. those that did not show estrus (26.0%), regardless of treatment group. Pregnancy losses were lower (P = 0.004) and the calving rate was higher (P = 0.01) in recipients that showed estrus (25.0% and 29.3%, respectively) than in those that did not (88.8% and 2.9%, respectively). In summary, expression of estrus was associated with a greater P/ET in recipients treated with two different estradiol/P4-based synchronization protocols. The expression of estrus was associated with a greater proportion of recipients receiving embryos, P/ET and calving rate. Treatment with GnRH did not improve P/ET in the recipients that did not show estrus, questioning the its use in recipients synchronized with estradiol/progesterone based FTET protocols.

Effect of FSH treatment on cumulus oocyte complex recovery by ovum pick up and in vitro embryo production in beef donor cows.

This study was conducted to evaluate oocyte recovery and in-vitro blastocyst production of donor cows superstimulated for ovarian follicular development with FSH administered as twice-daily injections in saline or a single injection diluted in 0.5 % hyaluronan before oocyte aspiration. In Experiment 1, cows were treated with 160 mg of Folltropin-Vdiluted in saline, administered in four twice-daily i.m. injections for 2 days (Multiple FSH group); 160 mg of Folltropin-V diluted in hyaluronan and administered in a single i.m. injection (Single FSH group); or no FSH treatment (Control). In Experiment 2, donor cows were treated with either a single FSH i.m. injection or there was no treatment (Control) before ovum pick up (OPU) was performed. In both experiments, COCs collected using OPU were classified, matured, fertilized and cultured at 38.8 °C in a humidified atmosphere for 7 days. In Experiment 1, the number of follicles aspirated and COCs recovered were greater (P < 0.05) in cows treated with multiple and single doses of FSH. Number of blastocysts produced, however, did not differ among groups. In Experiment 2, mean number of follicles aspirated and COCs recovered were also greater (P < 0.05) in FSH-treated cows. Nevertheless, number of blastocysts produced did not differ. In summary, single and multiple FSH administrations induced similar follicular stimulation for OPU. Furthermore, with both FSH treatments there was induction of development of a larger number of follicles to be aspirated and COCs recovered by OPU compared with these values for donor beef cows with no FSH treatment for follicular stimulation.

Superovulation and embryo transfer in wood bison (Bison bison athabascae).

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥ 7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥ 9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.

Superovulation of beef cattle with a split-single intramuscular administration of Folltropin-V in two concentrations of hyaluronan.

Three experiments were designed to evaluate the superovulatory response of beef cows following two intramuscular (IM) administrations 48 h apart of Folltropin-V diluted in reduced concentrations of hyaluronan (Split-single IM administrations; Experiment 1-300 mg Folltropin-V on the first day and 100 mg 48 h later; Experiment 2-200 mg Folltropin-V on the first day and 100 mg 48 h later). In Experiments 1 and 2, superovulatory response and ova embryo/embryo production did not differ between donors receiving twice daily IM of Folltropin-V over 4 days and those given a Split-single IM administration of Folltropin-V diluted in 10 mg/mL hyaluronan solution. Experiment 3 compared Split-single IM administration of Folltropin-V diluted in two hyaluronan concentrations (5 or 10 mg/mL) with Folltropin-V diluted in saline and administered twice-daily over 4 days. Beef cows (17 Angus and 12 Simmental) were randomly assigned to one of three treatment groups to be superstimulated three times in a cross-over design, so that all cows received all treatments. A total dose of 300 mg Folltropin-V was divided into twice-daily IM over 4 days, or in two IM treatment 48 h apart (200 mg on first day and 100 mg 48 h later) in the hyaluronan groups. Mean (± SEM) numbers of transferable embryos did not differ among treatment groups (Control: 4.0 ± 0.8; 10 mg/mL hylauronan: 5.0 ± 0.9; 5 mg/mL hyaluronan: 6.1 ± 1.3). We concluded that the Split-single IM administration of Folltropin-V diluted in either concentration of hyaluronan resulted in a comparable superovulatory response to the traditional twice-daily protocol.

Superstimulation of ovarian follicular development in beef cattle with a single intramuscular injection of Folltropin-V.

The need to inject FSH twice daily for superstimulation of ovarian follicular development in cattle necessitates frequent attention by farm-personnel and increases the possibility of failures due to mishandling and errors in administration of treatments. A series of three experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single intramuscular (IM) injection of Folltropin-V diluted in a hyaluronan-based slow-release formulation (SRF). In Experiment 1, cows were assigned to one of three treatment groups to compare two methods of injection as compared to the twice daily IM injection protocol. Superovulatory response of cows (n=6) treated with twice daily IM injections over 4 days (Control) was greater than of cows treated with a single subcutaneous (SC) injection in SRF (n=6), while superovulatory response of cows treated with a single IM injection in SRF (n=6) was intermediate. Experiment 2 was designed to compare two concentrations of SRF (20mg/mL hyaluronan, 100% compared to 10mg/mL hyaluronan, 50%) in a single IM injection protocol. The mean number of corpora lutea (CL) were not significantly different (P≥0.05), but the numbers of total ova/embryos (P<0.05), fertilized ova (P<0.01) and transferable embryos (P<0.001) were greater in cows treated with FSH in 100% SRF (n=20) than cows treated with FSH in 50% SRF (n=20). Experiment 3 was designed to compare superovulatory response in Red Angus donor cows treated with a single IM injection of Folltropin-V diluted in 100% solution of SRF with those treated with the traditional twice-daily IM injection protocol over 4 days. Mean (±SEM) numbers of CL (13.7±1.2 compared to 13.8±1.2), total ova/embryos (12.3±1.5 compared to 13.7±2.1), fertilized ova (7.2±1.1 compared to 8.4±1.4) and transferable embryos (4.9±0.8 compared to 6.4±1.3) were not significantly different between Control (n=29) and Single injection (n=29) groups, respectively. In summary, superstimulation of beef donor cows with a single IM injection of Folltropin-V diluted in 100% solution of SRF resulted in a comparable superovulatory response to the traditional twice-daily IM administration of Folltropin-V diluted in saline over 4 days.

New approaches to superovulation in the cow.

There is continuing need to simplify bovine superovulation protocols without compromising embryo production. The control of follicular wave emergence and ovulation has facilitated donor management, but the most commonly used treatment, oestradiol, cannot be used in many parts of the world and mechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include gonadotrophin-releasing hormone (GnRH) or LH, but efficacy in groups of randomly cycling animals is variable. Another alternative is to increase the response to GnRH by inducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferable embryos following superovulation during the first follicular wave did not differ from that achieved 4 days after oestradiol benzoate and progesterone. To further simplify superovulation, FSH has been administered as a single intramuscular injection. Superovulation of beef donors with a single intramuscular injection of Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) diluted in a slow-release formulation resulted in embryo production comparable to that obtained using the traditional twice-daily protocol. The single intramuscular injection has the potential to reduce labour and handling and may be useful when handling stress is an impediment to success. These alternatives provide ways of facilitating widespread application of embryo transfer technologies.