220 LIVE BIRTH OF A MOHOR GAZELLE (GAZELLA DAMA MHORR) CALF FOLLOWING INTRAUTERINE INSEMINATION OF MOTHER WITH FROZEN–THAWED SEMEN

Abstract

Gazella dama mhorr is an endangered species, and no animals have been observed in the wild since 1968. Assisted reproductive techniques have been used to propagate endangered species. However, no live offspring has been produced after cryopreservation of semen from gazelle species. Therefore, the objective of this study was to evaluate whether cryopreserved Mohor gazelle spermatozoa can fertilize in vivo after artificial insemination. Semen was collected by electroejaculation from four males and centrifuged at 700g for 5 min at room temperature. The supernatant was discarded, and the semen pellet was resuspended with a TEST-1% egg yolk diluent containing 6% glycerol to provide 400 × 10⁶ spermatozoa/mL. The extended semen was loaded into 0.25-mL plastic straws, cooled slowly to 5°C, and equilibrated at 5°C for 2 h. Straws were frozen in nitrogen vapors for 10 min and then plunged into liquid nitrogen. After thawing (37°C, 30 s), the effects of cryopreservation on sperm motility and acrosomal integrity were examined. Percentage of motile sperm in fresh samples was 88.7 ± 3.8% (mean ± SEM), decreased (P < 0.0001) to 58.7 ± 3.8% after freezing and thawing, and then to 40.0 ± 3.8% after 120 min incubation at 37°C. Spermatozoa with normal acrosomes decreased (P < 0.0001) after freezing and thawing (from 94.5 ± 4.2% to 56.0 ± 4.2%), but did not significantly decrease after sperm incubation. Frozen spermatozoa from two males were used in an intrauterine insemination trial. Estrus of females (n = 15) was synchronized with controlled internal drug release (CIDR, InterAg, Hamilton, New Zealand). Single CIDRs (type G, 330 mg progesterone/device) were inserted intravaginally for a total of 13 days. On Day 10, devices were replaced in each animal and all females received an injection of prostaglandin F_2± (PGF_2±; 125 mg/female). At CIDR withdrawal, females received 250-300 IU equine chorionic gonadotropin (eCG: Folligon; Intervet, Salamanca, Spain). After anesthesia with an intravenous injection of xylazine hydrochloride (Rompun; Bayer, Madrid, Spain; 0.8 mg/kg live weight) and ketamine hydrochloride (Imalgene; Leti & Merieux, Madrid, Spain; 2.0 mg/kg live weight), eight females were inseminated with 100 × 10⁶ frozen-thawed spermatozoa 56-57 h after removal of the CIDRs, and seven were inseminated after 64-65 h. Females were inseminated directly into the uterus using laparoscopy. Anesthesia was reversed with yohimbine hydrochloride (0.3 mg/kg live weight). One female in the second group became pregnant. After a 202-day gestation, the female gave birth to one healthy Mohor gazelle male calf. These results demonstrate for the first time the successful use of frozen-thawed semen by means of artificial insemination for the rescue of endangered gazelle species. However, our results reveal that a number of unresolved technical problems remain to be addressed.

This work was supported by the Spanish Ministry of Education and Science (REN2003-1587).