Influence of Seminal Characteristics in Male Tigers on Strategies for Genome Resource Banking

Shelley E. S. Sandmaier^1,2, Jason R. Herrick^1,3

1. Department of Reproductive Sciences, Omaha's Henry Doorly Zoo & Aquarium, Omaha, NE, USA
2. Current Address: Department of Reproductive Sciences, San Diego Zoo Wildlife Alliance, San Diego, CA, USA
3. Current Address: Colossal Foundation, Dallas, TX, USA

Abstract Text: The Association of Zoos and Aquariums (AZA) maintains three subspecies of tigers within North American zoos, all of which have exhibited reduced fecundity in recent years. To aid in population sustainability efforts, the Tiger Species Survival Plan has established a genome resource bank (GRB) to store cryopreserved spermatozoa from all adult males. For the genetics of each individual male to be fully represented in a GRB, there must be sufficient spermatozoa to produce 10 offspring through assisted reproduction. In the process of collecting samples for the GRB, we analyzed 104 ejaculates collected from 69 individuals (n = 32 Amur tigers, Panthera tigris altaica; n = 14 Malayan tigers, Panthera tigris jacksoni; n = 23 Sumatran tigers, Panthera tigris sumatrae) housed at 48 AZA accredited institutions. The results provided the opportunity to evaluate how the quantity and quality of spermatozoa produced in a single collection will affect strategies to capture a male’s genetics in the GRB. Semen was collected via electroejaculation (90 to 100 stimulations, 3-7 volts), subjected to analysis (volume, pH, concentration, motility, and morphology), and cryopreserved. Season did not have any effect on any of the parameters measured (P >0.05). While total sperm count per ejaculate was not influenced by subspecies (population average = 175.2 x 10⁶ ± 22.5 x 10⁶), the volume of ejaculate was significantly higher in Sumatran tigers (8.29 ml ± 0.98 ml) compared to both Amur (4.80 ml  ± 0.49 ml) and Malayan (5.23 ml ± 0.67 ml) tigers (P < 0.05). Although most spermatozoa were motile (population average = 67.6% ± 2.3%), the number of spermatozoa exhibiting normal morphology was very low (average = 14.5% ± 1.0%). This percentage of morphologically normal sperm is much lower than that of previous studies of the North American population, as well as a recent study of Bengal tigers (P. t. tigris) in Thailand. The reason for the decline in sperm quality in this population of tigers over the last three decades is unclear. However, it is known that reduced genetic diversity, as well as nutrition and stress, can affect semen quality.  A subset of cryopreserved samples (n = 13) were thawed and evaluated for motility (19.9% ± 3.8%). The average ejaculate from a male tiger will result in 23.6 million motile sperm after cryopreservation and thawing (175.2 million sperm per collection * 67.6% pre-freeze motility * 19.9% post-thaw motility). Laparoscopic oviductal inseminations in felids require ~5 million motile spermatozoa per attempt and are successful in ~30% of attempts.  To produce 10 offspring with current technologies, there should be enough spermatozoa for 30 inseminations or 150 million motile sperm after thawing. Based on our data, that would require approximately 750 million motile sperm pre-cryopreservation or approximately 7 to 8 collections per male. Population managers must plan for frequent collections throughout the reproductive lifespan of the male (~12 years). In addition, further studies of in vitro fertilization technologies, which require fewer spermatozoa (thousands per procedure or less), would be advantageous for males not available for frequent collections and increase the number of assisted reproductive procedures that could be done with a single sample.