Assessment of Viral Presence in Semen and Reproductive Function of Frozen-Thawed Spermatozoa From Pallas’ Cats (Otocolobus manul) Infected with Feline Herpesvirus
Feline herpesvirus type 1 (FHV-1) infection can produce acute clinical disease characterized by upper respiratory tract and ocular signs or a latent carrier state with periodic viral reactivation and shedding. It is unknown if this virus is shed in semen of infected cats, as has been reported for herpesviruses in other mammalian species. In the present study, our objectives were to 1) assess in vitro motility, acrosome status, and function of frozen-thawed Pallas’ cat spermatozoa, and 2) investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa of Pallas’ cats, inseminated domestic cat oocytes, and hybrid embryos formed by heterologous in vitro fertilization (IVF).
Over a 3-yr period (2000–2003) semen was collected periodically from four male Pallas’ cats infected with FHV-1. A total of 33 ejaculates were recovered from anesthetized males using a standardized electroejaculation protocol.1 Aspermic ejaculates (n=16) were centrifuged (1700 x g) and cell-free supernatants frozen for FHV-1 DNA detection using PCR. Spermic ejaculates (n=17) were diluted in cryoprotectant (Test egg yolk w/ 4% glycerol), slowly cooled to 4°C, and frozen by pelleting on dry ice before storage in liquid nitrogen. For IVF, frozen sperm samples were thawed, centrifuged, and resuspended in equilibrated Ham’s F10 medium. Sperm motility and acrosome status were assessed in microdrops (2x106 motile sperm/ml) under oil (38°C; 5% CO2 in air) at 0–6 h post-thaw and a sperm motility index [SMI=% progressive motility + (20 x rate of progressive motility)/2] calculated for each time point. Mature domestic cat oocytes, recovered laparoscopically from females treated with exogenous gonadotropins, were inseminated with frozen-thawed spermatozoa (5x105 motile sperm/ml; 10–17 oocytes/sample), cultured (38°C; 5% CO2 in air) for 48 h, and then evaluated for embryo cleavage. All oocytes, embryos, and aliquots of frozen-thawed spermatozoa were frozen for detection of FHV-1 DNA using PCR. For FHV-1 PCR, DNA was extracted from cell-free seminal fluid samples (16 ejaculates), frozen-thawed spermatozoa (17 ejaculates), noncleaving domestic cat oocytes (n=107), hybrid embryos (n=89) formed by heterologous IVF, and bilateral conjunctival biopsies (n=28) and analyzed for presence of a 322 base-pair fragment of the FHV-1 thymidine kinase gene.2 This protocol reliably detects ≥240 copies of FHV-1 DNA.
Sperm pellets from each ejaculate were thawed to assess post-thaw sperm motility and function. For pre-freeze ejaculates, SMI averaged (±SEM) 71.6±1.4 and 94.8±1.0% of acrosomes were intact. Immediately post-thaw, SMI was decreased (p<0.05) to 52.8±2.7 with fewer (p<0.05) acrosomes (39.0±3.4%) classified as intact. Values for both parameters continued to decline (p<0.05) over 6 h of culture (20.7±1.4, SMI; 32.1±3.5%, acrosome intact). All frozen-thawed samples (n=16) used for IVF fertilized domestic cat oocytes, based on embryo cleavage within 48 h of insemination. Cleavage percentage ranged from 13–80%, with a mean value (±SEM) of 46.1±6.0%. PCR analysis of seminal fluid, frozen-thawed spermatozoa and inseminated oocytes/embryos did not identify FHV-1 DNA in any sample. However, FHV-1 DNA was identified in all four males from a total of 13 conjunctival biopsies collected at the time of electroejaculation.
In conclusion, we were unable to detect cell-associated or non-cell-associated FHV-1 DNA in semen collected from male Pallas’ cats chronically infected with FHV-1. We also demonstrated that frozen-thawed Pallas’ cat spermatozoa exhibit adequate function after thawing to fertilize viable cat oocytes. Together, these results suggest that frozen-thawed spermatozoa from FHV-1-infected male Pallas’ cats may be used in females with minimal risk of transmitting FHV-1 to naïve individuals. These findings also may have positive implications for other cat species, such as domestic cats and cheetahs, which are infected with FHV-1.
1. Swanson W.F., J.L Brown, and D.E. Wildt. 1996. Influence of seasonality on reproductive traits in the male Pallas' cat (Felis manul) and implications for captive management. J. Zoo Wildl. Med. 27:234–240.
2. Weigler B.J., C.A. Babineau, B. Sherry, and M.P. Nasisse. 1997. High sensitivity polymerase chain reaction assay for active and latent feline herpesvirus-1 infections in domestic cats. Vet. Rec. 140:335–338.