Although the cheetah (Acinonyx jubatus) routinely lives for more than 12 yr in ex situ collections, females older than 8 yr reproduce infrequently. W

The objective was to examine the influence of animal age, season and captivity status on seminal quality in wild-born cheetahs (Acinonyx jubatus) in Namibia, Africa. Animals were divided into three age categories: juvenile (14–24 months; n = 16 males, 23 ejaculates); adult (25–120 months; n = 76 males, 172 ejaculates); and aged (>120 months; n = 5 males, 5 ejaculates). Seasons were categorised into hot–wet (January–April), cold–dry (May–August) and hot–dry (September–December). A comparison between freshly wild-caught (n = 29 males, 41 ejaculates) and captive-held cheetahs (n = 68 males, 159 ejaculates) was also conducted. Raw ejaculates contained 69.0 ± 1.1% motile spermatozoa (mean ± s.e.m.) with 73.6 ± 1.5% of these cells containing an intact acrosome. Overall, 18.4 ± 0.9% of spermatozoa were morphologically normal, with midpiece anomalies being the most prevalent (~39%) defect. Juvenile cheetahs produced ejaculates with poorer sperm motility, forward progressive status, lower seminal volume and fewer total motile spermatozoa than adult and aged animals. Spermatogenesis continued unabated throughout the year and was minimally influenced by season. Proportions of sperm malformations were also not affected by season. Ejaculates from captive cheetahs had increased volume and intact acrosomes, but lower sperm density than wild-caught counterparts. In summary, Namibian cheetahs produce an extraordinarily high proportion of pleiomorphic spermatozoa regardless of age, season or living (captive versus free-ranging) status. Young males less than 2 years of age produce poorer ejaculate quality than adult and aged males. Because (1) all study animals were wild born and (2) there was little difference between freshly caught males and those maintained in captivity for protracted periods, our results affirm that teratospermia in the cheetah is mostly genetically derived. It also appears that an ex situ environment for the Namibian cheetah can ensure sperm quality comparable with that for free-living males.

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.

Approximately 30% of the Association of Zoos and Aquariums cheetah population (~350 total animals) is unlikely to breed naturally due to advanced age, health, or behavioral issues. Aging cheetah females (≥9 y old) are unlikely to become pregnant via natural breeding if they are nulliparous. We previously demonstrated that oocytes recovered from aged females were of similar quality compared with those recovered from younger females (2–8 y old). We hypothesize that transfer of 4–8 cell embryos produced by in vitro fertilization with oocytes from old donors could result in pregnancy after transfer into younger recipients. Female cheetahs (n = 3 aging donors and n = 3 young recipients) received 300 IU equine Chorionic Gonadotropin (eCG) and 3000 IU Luteinizing Hormone (LH) while fecal metabolites of estrogens and progestogens were closely monitored. At 28 h post-LH injection, oocytes were aspirated laparoscopically from donors and inseminated in vitro with cryopreserved sperm. After 48 h of in vitro culture, resulting embryos (4–8 cells) were transferred into the oviducts of recipient females. Pregnancy was confirmed in one recipient via ultrasound 32 days after transfer and by radiograph 62 days after transfer. Two cubs were born naturally after 90 days of gestation, representing the first cheetah births resulting from transfer of embryos produced in vitro.

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim‐up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST‐yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0–5). Sperm motility index (SMI) was calculated as (% M + [FPS × 20]) ÷ 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P < .05) for Accudenz (range, 36%–39%) compared with control (30%–33%) and multistep (29%–33%) treatments. In study 2, a modified Accudenz protocol was compared with traditional washing and was found to improve (P < .05) SMI (range, 52–64) compared with controls (range, 41–52) at each time postthaw after centrifugation. In study 3, swim‐up processed sperm were compared with those treated by centrifugation through Accudenz and traditional sperm washing for improving sperm morphology. The percentage of structurally‐normal sperm recovered postthawing increased (P < .05) for both the Accudenz (38%) and swim‐up (33%) treatments compared with controls (21%). Percent IA and SMI also were improved (P < .05) for Accudenz (range, 39%–47% and 46–59, respectively) compared with controls (range, 26%–33% and 40–53, respectively). Results indicate that using Accudenz for glycerol removal from cryopreserved cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze‐thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.