Paul E. Marinari

Age-Dependent Changes in Sperm Production, Semen Quality, and Testicular Volume in the Black-Footed Ferret (Mustela nigripes)

Abstract The black-footed ferret (Mustela nigripes), which was extirpated from its native North American prairie habitat during the 1980s, is being reintroduced to the wild because of a successful captive-breeding program. To enhance propagation, the reproductive biology of this endangered species is being studied intensively. The typical life span of the black-footed ferret is approximately 7 yr. Female fecundity declines after 3 yr of age, but the influence of age on male reproduction is unknown. In this study, testis volume, seminal traits, sperm morphology, and serum testosterone were compared in 116 males from 1 to 7 yr of age living in captivity. Results demonstrated that testes volume during the peak breeding season was similar (P > 0.05) among males 1 to 5 yr of age, reduced (P < 0.05) among males 6 yr of age, and further reduced (P < 0.05) among males 7 yr of age. Motile sperm/ejaculate was similar in males 1 to 6 yr of age but diminished (P < 0.05) in those 7 yr of age. Males at 6 and 7 yr of age produced fewer (P < 0.05) structurally normal sperm than younger counterparts; however, serum testosterone concentrations were not reduced (P > 0.05) in older males. Histological comparison of testicular/epididymal tissue from 5- and 7-yr-old black-footed ferrets confirmed that the interval between these two ages may represent a transitional period to reproductive senescence. In summary, functional reproductive capacity of male black-footed ferrets exceeds that of females by at least 2 yr. Testes and seminal quality are indistinguishable among males 1 to 5 yr of age, with progressive reproductive aging occurring thereafter.

Reproductive inefficiency in male black-footed ferrets (Mustela nigripes)

The black-footed ferret (Mustela nigripes), once considered extinct, has benefited from captive breeding and subsequent reintroduction into native habitat. A high proportion of females (>90%) exhibit estrus in captivity during the spring breeding season. However, many males considered to be prime-breeding age (1-3 years old) fail to sire offspring. Breeding records in 1995 revealed that 40 of 73 males (55%) managed under the Black-Footed Ferret Species Survival Plan did not reproduce, despite being provided opportunity. The present study was conducted to determine the incidence and etiology of male reproductive failure in 1996 and 1997. In 1996, 38 of 69 (55%) 1- to 3-year-old males failed to sire offspring. Likewise, 35 of 60 (58%) males did not reproduce in 1997. Overall, 21% of adult males failed to sire young in three consecutive breeding seasons (1995-1997). Electroejaculate traits (ejaculate volume, sperm concentration, motility, morphology, and acrosomal integrity) from 29 proven breeder males were not different (P > 0.05) from those of 23 males that did not sire young (nonproven breeders). However, six categories of reproductive failure were identified for the 73 prime-breeding age, nonproven males: 1) underdeveloped testes (22%); 2) improper breeding position with the female (25%); 3) excessive aggression toward estrous females (9%); 4) copulation with no sperm detected at postcoital lavage (19%); 5) copulation with sperm in the vaginal lavage but no resulting pregnancy (18%); and 6) copulation with no vaginal lavage performed and no resulting pregnancy (7%). These data indicate that combined behavioral and physiologic factors, but not overall sperm quality, influence reproductive performance in male black-footed ferrets managed in captivity. Zoo Biol 19:517-528, 2000. Copyright 2000 Wiley-Liss, Inc.

VACCINATION WITH F1-V FUSION PROTEIN PROTECTS BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AGAINST PLAGUE UPON ORAL CHALLENGE WITH YERSINIA PESTIS

Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody postvaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862, respectively) were found in all vaccinates up to 2 yr postvaccination, whereas seven control animals remained antibody negative throughout the same time period.

Environment influences morphology and development for in situ and ex situ populations of the black-footed ferret (Mustela nigripes)

For selected species, conservation breeding has become integrated into recovery plans, most often through the production of offspring for reintroduction into nature. As these programs increase in size and scope, it is imperative that conservation managers retain the biological integrity of the species. This study investigated the causes of morphological changes that are known to occur in black-footed ferrets (Mustela nigripes) maintained ex situ. In a previous study, ferrets maintained in captivity were 5–10% smaller in body size than pre-captive, in situ animals. In the present study, the authors compared nine morphological characters among ex situ animals and their in situ descendants. Within the ex situ population, cage types were compared to determine whether housing influenced morphometry. Black-footed ferrets born to reintroduced individuals quickly returned to their pre-captive size suggesting that a diminutive morphology ex situ did not have a genetic basis. Furthermore, cage type affected overall body size and shape; ulnas and tibias were as much as 9% shorter for ex situ animals. The authors hypothesise that small cage size and environmental homogeneity inhibit the mechanical stimuli necessary for long bone development. These findings have ramifications for ex situ managers who need to create artificial captive settings that promote natural physical development. In the absence of such an environment, ‘unnatural’ morphologies can result that may contribute to poor fitness or perhaps even domestication.

Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25 degrees C v. 37 degrees C); (3) type of medium (Hams F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25 degrees C than at 37 degrees C in Hams or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Hams medium, but Hams medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2 degrees C min(-1)) was optimal (P < 0.05) compared to rapid cooling (1 degrees C min(-1)), and ultra-rapid cooling (9 degrees C min(-1)) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

The Black-Footed Ferret: On the Brink of Recovery?

In an attempt to save the species from extinction, the last remaining 18 black-footed ferrets (Mustela nigripes) were trapped up from the wild to initiate a captive breeding program. Nearly 30 years later more than 8,000 black-footed ferrets have been produced in captivity and approximately 4,100 animals have been reintroduced into 20 sites in eight US states (Arizona, New Mexico, Utah, Colorado, Kansas, Wyoming, South Dakota and Montana), Mexico and Canada. However, full recovery of the species has yet to be achieved, mainly due to limited viable habitat, disease and reduced fecundity. This chapter will highlight the advances in the black-footed ferret recovery program over the last 10 years including: (1) adaptive management techniques employed for the captive population; (2) development of new reintroduction sites and associated challenges facing wild black-footed ferrets; and (3) optimization of assisted reproductive techniques to secure the future of this rare species.

Effect of dietary vitamin E and prey supplementation on semen quality in male black-footed ferrets (Mustela nigripes)

Over the recent years, the captive population of the endangered black-footed ferret (Mustela nigripes; ferret) has experienced a decline in normal sperm (NS) morphology (from 50% to 16%), which may be linked to inbreeding depression or it may have been a dietary change. We examined the role of dietary vitamin E, selenium (SE), and vitamin A on serum levels of vitamin E, SE, and vitamin A and semen quality. Ferrets (n = 55 males) were randomly assigned to one of five diet treatments (n = 11 per treatment): (1) horsemeat diet (control); (2) horsemeat diet + vitamin E (400 IU/kg Dry Matter) daily; (3) horsemeat diet + whole prey; (4) horsemeat diet + vitamin E daily + whole prey; and (5) beef diet. Both blood (prediet and postdiet change) and diets were analyzed for vitamin E, vitamin A, and SE concentrations. Electroejaculates were collected monthly and evaluated for sperm concentration, sperm motility index (includes percent motile and forward progression), and percent NS. Results reveal that the beef and horsemeat diets had comparable (P = 0.05) vitamin E and SE concentrations and all diets met most nutrient requirements for small carnivores; however, the horsemeat diet was excessive in vitamin A and the beef diet was deficient in vitamin A. Vitamin E supplementation increased (χ1(2)=25.83; P < 0.001) serum vitamin E. Ferrets fed the beef diet or prey had improved (H4 = 15.596; P = 0.004) sperm motility index than the horsemeat control group, and ferrets fed the horsemeat diet supplemented with vitamin E had the lowest (H4 = 18.408; P = 0.001) NS. In conclusion, the high levels of vitamin A in the horsemeat diet could compete with vitamin E as evidence by serum levels, which may reduce reproductive success in this endangered species.