Synchronization of estrus and superovulation in goats

The synchronization of donor and recipient goats is important to perform the technique of laparoscopic oocyte aspiration. The protocols differ in the exogenous hormones used, but are similar in duration and application (^{Souza-Fabjan et al., 2014}).

Superovulation is achieved with exogenous hormones that promote follicular development and growth. To improve the collection of oocytes during the AOL in the ET, prior synchronization in the recipient and donor female is required; besides superovulating the donor female. Synchronization manipulates the estrous cycle and superovulation generates more oocytes for collection and evaluation. The synchronization of estrus (recipient-donor) is performed by intravaginal devices impregnated with P₄ (^{Abecia et al., 2012}).

The synchronization and superovulation protocols differ according to the suction means and the anesthetics to be used (^{Souza-Fabjan et al., 2014}, Table 1).

^{Baldessarre (2007)} evaluated different protocols to synchronize donor females with intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate (AMP, Veramix®, Upjohn Laboratories, Canada) for 10 days, and 48 hours before the AOL, applied 125 μg of cloprostenol (Estrumate®, Malinkrodt Laboratories, Canada). For super-ovulation, he compared two protocols; in the first, he injected follicle stimulating hormone (FSH, Folltropin®, Bioniche, Canada) and in the second, he combined FSH with equine chorionic gonadotropin (eCG, Pregnecol®, Bioniche, Canada), which he administered in a single dose 36 h before perform the AOL (protocol "OneShot", ^{Baldessarre, 2007}). The results were similar (P> 0.001) between the treatments of multiple injections and the "OneShot" treatments for the AOL (goats from 60 to 90 d and from 90 to 150 d of age: 84 and 80% recovery, respectively). However, for the young females, the protocols were identical to those used in the adults, due to the fact that they were not cycling and, therefore, estrous was not synchronized (intravaginal + luteolytic sponge, ^{Baldessarre, 2007}).

^{Avelar et al. (2012)} synchronized donor goats using an intravaginal sponge with 60 mg of AMP (Syntex, Buenos Aires, Argentina) for 11 d, and on day eight, they injected 50 μg of d-cloprostenol (Ciosin, Coopers, São Paulo, Brazil). For ovarian stimulation, the goats were divided into three treatments: 1) 120 mg of NIH-FSH-P1 (standard swine FSH, National Institute of Health for standard FSH, Folltropin-V, Vetrepharm, Belleville, Canada), given in five injections (5D) intramuscularly (30, 30, 20, 20, and 20 mg, respectively) with intervals of 12 h; 2) 120 mg of NIH-FSH-P1 divided into three injections (3D) with intervals of 24 h (60, 40 and 20 mg, respectively); and 3) a single injection (1D) of 70 mg of NIH-FSH-P1 plus 200 IU of eCG (Novormon, Syntex, Buenos Aires, Argentina), 36 h before removing the sponge.

The results were similar (P> 0.05) between treatments for the number of follicles observed in each ovary; however, the highest percentage (P <0.05) of oocyte recovery was obtained with the 5D treatment (84.1%, 211/251 females), with respect to that of the 3D treatments (68.2%, 182/267 females) and 1D (72.4%, 184/254 females), respectively (^{Avelar et al., 2012}).

The percentage of recovery of oocytes differs among authors, according to the number of females used per protocol and the response by animals or population (Table 2).

^{Souza-Fabjan et al. (2013)} synchronized nulliparous and multiparous goats, using intravaginal sponges impregnated with 60 mg of AMP (Progespon, Syntex, Buenos Aires, Argentina) for 11 d. On day eight, they delivered 75 μg of d-cloprostenol (Prolise, Pfizer Animal Health, São Paulo, Brazil), and 36 h before removing the sponge, they applied a single dose of 70 mg of porcine FSH (FSHp, Folltropin-V Vetrepharm, Ontario, Canada) plus 200 IU equine chorionic gonadotropin (eCG, Novormon, Syntex). Oocyte collection was performed six days after the end of treatment. No differences were found (P> 0.05) for the response of estrus (40.0% vs. 84.7%), the onset of estrus (62.0 ± 15.5 vs. 50.7 ± 19.2 h, mean0020± EE), the duration of estrus (25.0 ± 16.1 vs. 30.0 ± 15.1 h) and the percentage of ovulation (60.0% vs. 85.7%) in the study animals (^{Souza-Fabjan et al., 2013}).

^{Souza-Fabjan et al. (2014)} synchronized goats with intravaginal sponges impregnated with 45 mg of fluoromethane acetate (AFG, Chronogest CR, Intervet Schering-Plow Animal Health, Angers, France) for 11 d; and on day eight, they injected a dose of prostaglandins (Cloprostenol 50 μg, Intervet Schering-Plow Animal Health); at the same time, they stimulated with 16 mg of FSHp (Stimufol, highly purified porcine FSH, kindly provided by Prof. JF Beckers, Liège, Belgium) at 12 h intervals with decreasing doses (4, 4, 3, 3, and 2 mg , respectively). With this protocol, the authors found similarities between the oocytes obtained by AOL and those originating from trace for intrinsic quality and development competence through parthenogenetic activation; however, they suggested that the oocytes obtained by AOL are less competent for fertilization under conditions of maturation and in vitro fertilization (^{Souza-Fabjan et al., 2014}).

^{Anakkul et al. (2013)} synchronized goat recipients and donors, using intravaginal sponges with 65 mg of mexodroxyprogesterone acetate (AMP, Ovejero®, Spain) for 13 d, and an eCG injection (Folligon®, Intervet Schering-Plow Animal Health, Holanada). In estrus recipients, they administered a single dose of 200 IU of hCG (Chorulon®, Intervet Schering-Plow Animal Health, Holland) to induce ovulation. In addition to the P₄ analog (Sincro-gest sponges, 65 mg of Medroxyprogesterone, Ovejero®, Spain) in the donors, stimulated follicular development with seven doses of FSH (50, 25, 25, 25, 25, 25 and 25 mg, respectively Folltropin V®, Bioniche Animal Health, Vetrepharm, Canada) at 12-h intervals.

The first dose was applied on day 10 of the treatment. For the sixth and seventh doses, they injected 125 μg mL^-1 of cloprostenol (Estrumate®, Schering-Plow Animal Health, NJ, USA). They detected estrus every 6 h and to induce ovulation, they applied 200 IU of hCG (Chorulon®, Intervet Schering-Plow Animal Health, Holland). Thirty embryos from 11 donors (Black Bengal: BB=7, 50%; Australian Melaan: AA=4.0%, recovery percentages in three donors) were transferred in 30 recipient females. 30% of gestation was obtained (9/30) from where nine offspring were born and one of them presented black color.

The results suggest that artificial insemination and TE by laparoscopy can be combined to improve the genetic potential that codes for the black color of the skin (^{Anakkul et al., 2013}).

^{Perera et al. (2008)} synchronized donor goats with vaginal devices (45 mg, Cronolone, Intervet) for 17 d; applied 2.5 ml of FSHp (Folltropin-V, 20 mg/ml NIH-FSH-P1, BIONICHE, Canada); and ovine FSH (FSHo; Ovagen™, 0.88 mg/mL NIADDK-oFSH-17-Standard, ICPbio Limited, New Zealanda) on eighth day. In addition, they injected 300 IU of eCG (Folligon, Intervet International BV, Boxmeer-Holland), during the night of the eight. They stimulated folliculogenesis and follicular maturation with injections of 1.25 ml of FSHp or FSHo in the morning and on the evening of day 9 and 10. On the morning of ninth day, they injected 197 mg of prostaglandin F_2α (PGF_2α, cloprostenol sodium; PGF Veyx fort, Veyx Pharma, Schwarzenborn) and removed the vaginal devices on the night of day 10. On the morning of day 11, they applied 1.25 ml of FSHp and FSHo, and at night they applied 1 ml of luteinizing hormone-releasing hormone (LHRH; μL/ml, Depherelin Veyx Pharma, Schwarzenborn). After this injection, they kept the goats in contact with a goat for natural hunts for 48 h (Figure 1).

(A) ^{Baldessarre, 2007}; (B) ^{Avelar et al., 2012}; (C) ^{Souza-Fabjan et al., 2013}; (D) ^{Souza-Fabjan et al., 2014}; (E) ^{Anakkul et al., 2013}; (F) and (G) ^{Perera et al., 2008}. AMP: medroxyprogesterone acetate, AFG: fluoromethane acetate, MP: mexodroxyprogesterone, FSH: Follicle stimulating hormone, NHI-FSH-P1: Standard porcine follicle stimulating hormone, FSHp: Porcine follicle stimulating hormone, FSHo: Ovine follicle stimulating hormone, eCG: Equine chorionic gonadotropin hormone, hCG: Human chorionic gonadotropin hormone, PGF_2α: Prostaglandin F2 alpha.

Figure 1 Synchronization and superovulation protocols in goats. 

No significant differences were found in the number of embryos per animal in both protocols (FSHp: 4.3 ± 2.0 and FSHo: 4.25 ± 2.0, respectively). Possibly the protocols are adequate; nevertheless, it is necessary to perfect the AOL technique (^{Perera et al., 2008}).

Laparoscopic oocyte aspiration (AOL)

Laparoscopy allows vision and manipulation of the abdomino-pelvic cavity by means of the laparoscope, which is introduced through a small incision and consists of a light source transmitted by an optical fiber; by means of which a monitor is projected (^{Baldassarre, 2007}). To perform the AOL technique, donor females are required to be fasted for at least 24 hours beforehand to reduce the risk of accidental punctures in the intestines, rumen or urinary bladder during the procedure. The female is depilated and disinfected with iodized alcohol in the abdominal area, placed on a dorsal decubitus stretcher to tilt it to an angle of 30 to 45 °, so that the viscera move in the cranial direction and allow to visualize the reproductive tract of the female (^{Alexander et al., 2010}). The AOL requires highly qualified personnel, a laparoscopic team consisting of a laparoscope of 5 mm in diameter, an atraumatic clamp and an aspiration pipette of 3.5 mm in diameter, with their respective trocars (^{Baldassare, 2007}).

The endoscope is inserted 2 to 3 cm cranial to the mammary gland, and 2 to 3 cm to the left of the midline in the abdominal cavity through a first trocar; a second trocar is inserted in the right side of the abdomen (opposite to the first) to introduce the applicator-tracer, with which the uterus is located. Next, a third trocar is inserted in the midline, in order to pass the oocyte retrieval needle (^{Souza-Fabjan et al., 2014}). Once the three trocars were inserted and the laparoscope was inserted, the atraumatic clamp and the aspiration pipette, follicular puncture was performed (^{Cortez-Romero et al., 2011}, Figure 2).

Figure 2 Aspiration of oocytes by laparoscopy (Adapted from ^{Cortez-Romero et al., 2011}). 

To perform it, the ovary is discovered by lifting the fimbria with the atraumatic clamp and pulling in different directions, to expose the different areas on its surface. The procedure is repeated in both ovaries and before removing the equipment, both ovaries are washed with heparinized physiological solution at 37 °C (^{Baldassare, 2007}).

The embryo transfer method requires synchronization between the donor and the recipient, ensuring that the embryo is collected in the appropriate stage of development (morula or early blastocyst), and then transferred to the recipient in the proper state of the estrous cycle. Laparoscopic methods of embryo collection and transfer have replaced surgical laparotomy methods, reducing the risk of postoperative complications of the reproductive tract (^{Garza-Arredondo et al., 2015}).

The technique of laparoscopy is convenient for ovaries with a small number of follicles, and has the advantage of repeating aspirations at short intervals. For ovaries that present a large number of follicles after stimulation by gonadotropins, abdominal laparotomy under general anesthesia makes aspiration more efficient, and the recovery rate of oocytes is higher (^{Córdova-Izquierdo et al., 2008}).