Sperm cryopreservation, in vitro fertilization and faecal oestrogen enzyme immunoassay validation in the African lion (Panthera leo)

[摘要] English: Eight captive male African lions aged 1 to 4 years (Panthera leo) from Bothaville district (n=6) and Johannesburg Zoo (n=2), as well as 1 lioness from the Bloemfontein Zoo were used during this study. The 8 male lions were chemically immobilized using Zoletil® (Virbac,SA). External sexual organ characteristics were determined by palpation. The length and the width of the testis were recorded. Results showed that 4 lions had testis with a firm texture, while the other 4 animals had either 1 or both testis flaccid. Well developed cauda epididymis were detected in 6 of the lions. The mean length of the testis was 5.3 ± 0.5 cm and the width 3.1± 0.39 cm.Semen was succesfully collected from 6 chemically immobilized male lions by means of electroejaculation. The semen was evaluated after collection for volume, pH, overall motility, linear forward progression, concentration and structural morphology using standard procedures.A mean of 5.28 ± 0.6 ml of semen with a whitish color was collected from the 6 animals. The mean pH of the samples was 7.1± 0.05. A mean of 90 ±1.6% motile sperm with a linear progression of 4 was recorded for the semen samples collected. The semen samples had a mean concentration of 101 x106 ± 3.1 sperm/ml and 65.7 ± 2.14%structurally normal sperm.Five different treatments were used to cryopreserve the collected lion semen. Biladyl®,a Tris-citrate buffered extender, was used in combination with either DMSO or Glycerolat inclusion levels of 4% and 8%. After adding the cryoprotectant, the extended semenwas loaded into 0.25ml straws (n=12) and equilibrated for 2 hours at 4°C. Afterequilibration, the straws were frozen in liquid nitrogen vapour (±-70°C) and stored in aliquid nitrogen flask at -196°. After 5 months of cryopreservation, 2 straws per animal,per cryopreservation treatment were thawed by one of 3 methods: i) room temperature(22°C) for 2 minutes, ii) 36°C for 2 minutes (waterbath) and iii) 50° for 8 seconds(waterbath). The semen was evaluated post-thawing for overall motility and forwardprogression. No post-thaw motility or forward progression was recorded in the semensamples thawed without a cryoprotectant (control). Treatments containig Glycerol orDMSO at 4% and 8% recorded a significant higher motility and forward progression. Amean post-thaw motility of 18±2.4 % was recorded for semen (cryopreserved in DMSOand Glycerol) thawed at three different temperatures. There were no significantdifferences between the 4 treatments containing cryoprotectants or between the 3thawing methods used.A heterologous in vitro fertilization assay was set up to evaluate the fertilizing capacityof the lion sperm cells post-thaw. Semen from the 4 cryoprotectant treatments (4 and8% DMSO and Glycerol) were thawed at 36°C for 2 minutes and used to fertilize in vitromatured zona pellucida free bovine oocytes. The results showed a fertilization rate of25% and 33% for lion semen cryopreserved in 4% Glycerol and DMSO respectively.The sperm cryopreserved in 8% Glycerol showed fertilizing rates of 0% and spermcryopreserved in 8% DMSO a rate of 13% fertlization. Although the fertilzation rateobtained during this study are relatively low, the cryopreservation protocols using 4%Glycerol and DMSO hold promise for in vitro fertilizatrion programs for the African lion(Panthera leo) using in homologous oocytes.A faecal oestradiol- 17β enzyme immunoassay for the African lioness was developedand tested. Faecal samples were collected from one adult lioness on a weekly basis over a period of 3 months. The assay was validated for the African lion and the oestradiol -17β level present in the faeces was determined. The oestradiol - 17β levels ranged between 0.01ng/ml to 3.8ng/ml. An interval of 43 days between oestradiol -17β peaks was determined. A representative oestrogen profile could not be set up due to limited faecal samplesFrom the results presented in this study it could be concluded that lion semen can be successfully cryopreserved in Biladyl® cryodiluent containing either 4% or 8% Glycerol or DMSO. The results of the post-thaw heterologous IVF assay suggest that the 4% Glycerol or DMSO inclusion in the semen extender for cryopreservation, results in better fertilization rates than at concentrations of 8% glycerol or DMSO.

[发布机构] University of the Free State