Abstract

Assisted reproductive techniques are becoming more important as an alternative strategy for the breeding management of zoo animals. In this case, semen collected from a currently unrepresented male western lowland gorilla was used to fertilize oocytes collected non-surgically from a parous female gorilla. Pregnancy was not established. Nevertheless, this investigation demonstrates that sophisticated assisted reproductive techniques can help to better manage the captive gorilla population.

Introduction

As management of current captive populations becomes more intense, it stands to reason that reproductive management would become increasingly sophisticated. As a consequence, assisted reproductive techniques are becoming more important as an alternative strategy for the breeding management of zoo animals. Although the AZA Gorilla SSP has one of the largest founder populations of any management program, there are still 21 (12 males, 9 females) potential founder animals that have no living offspring. One such male founder, SB #268, was housed alone at the Gladys Porter Zoo and was treated chronically with steroids for an apparent immune-mediated disease. Due to ill health and social problems, this gorilla male had never reproduced in 40 years and was, therefore, considered a good candidate for assisted reproductive techniques. Semen was collected from this male on three occasions using rectal probe electrostimulation and the samples were cryopreserved using standard techniques. Probably due to the long-term lack of sexual activity, the sperm count and motility were extremely poor making in vitro fertilization by intracytoplasmic sperm injection (ICSI) the only option for embryo production since this requires only a single viable sperm per oocyte.

Methods

A 17-year-old, parous female gorilla (SB #376) housed at the Gladys Porter Zoo, was given an oral monophasic estrogen/progesterone pill (OvCon 35, Bristol-Myers Squibb Co.) daily for four months to control her menstrual cycle. Daily urine was collected to monitor urinary estrogen and progesterone, and to synchronize her ovarian cycle. Immediately following pill withdrawal, her urine was tested daily for occult blood (Hemastix, Bayer Corporation, Elkhart, IN) to detect the onset of menses (day 1). The ovarian stimulation protocol was as follows: on days 3 and 4, 225 IU FSH (Follistim, Organon Inc., West Orange, NJ) and 75 IU hFSH + 75 IU hLH (Pergonal, Serono, Inc., Rockland, MA) were administered; on days 5–10, she received 300 IU FSH (Follistim); on days 8–11, 25 mg GnRH (Antagon, Organon Inc., West Orange, NJ) was included, and on day 11 she received 150 IU FSH (Follistim) and 150 hFSH + 150 IU hLH (Pergonal). Each of these treatments was administered IM by hand injection. Final oocyte maturation was stimulated on day 11, 12 hours following the last stimulation injection, by administering 10,000 IU hCG (Pregnyl, Organon) IM.

Thirty-six hours post-hCG administration, the female was immobilized, and the oocytes were retrieved using transvaginal, ultrasound-guided aspiration. Approximately 10–12 maturing follicles (10–15 mm diameter) were aspirated and six oocytes were recovered. The oocytes were transported in HEPES-buffered transport media in a portable incubator at 37°C by airline immediately following retrieval from Brownsville to Dallas, Texas. Less than six hours post-retrieval, the oocytes arrived at the Center for Reproductive Medicine in Dallas, Texas, and were fertilized by intracytoplasmic sperm injection (ICSI) using the cryopreserved sperm from the male SB #268. Fertilized oocytes were cultured in Gardner’s Sequential Media at 37°C in 6% CO₂. Three cleaved and by 115 hours post-ICSI, two of these developed into quality blastocysts five days post-retrieval. Both blastocysts were then transported back to the Gladys Porter Zoo and transferred transcervically using ultrasound guidance into the oocyte donor. At the time of embryo transfer, she was given 500 mg progesterone IM and then oral progesterone 400 mg BID, PO (Prometrium, Solvay Pharmaceuticals, Inc., Marietta, GA). Urine was monitored weekly using Ovuquik test strips for pregnancy detection. Unfortunately, pregnancy was not established.

Results and Discussion

In spite of the fact that pregnancy was not established, this investigation demonstrates that ICSI is a viable option for assisted reproduction in the western lowland gorilla, a species that commonly is found to have poor semen quality. Further investigation into this and other techniques for assisted reproduction (including the use of sex-sorted sperm to produce female embryos) are currently in development as an alternative means to manage the captive gorilla population. In the future, such sophisticated techniques may offer population management programs a tool for genetic enhancement of captive populations. Sex-sorting sperm prior to fertilization of oocytes offers yet another opportunity for SSPs to help create a more manageable population. For instance, those species where there is currently a surplus of males that create a housing problem, such as gorillas, elephants, and chimpanzees. Zoo veterinary staff are the key and must have an open mind towards such alternative means of animal management.