Artificial insemination (AI) has been used for avian reproduction due to the discovery of cryoprotectants extending its usefulness both in production of domestic fowl and conservation of wild species. The goal of this study was to assess the effect on domestic and wild fowl pooled semen and individual ejaculate cryopreservation with dimethyl sulfoxide (DMSO) and polyvinylpyrrolidone (PVP). Twenty ejaculates and twenty samples of pooled semen of roosters, pheasants and hawks were frozen in media containing DMSO or PVP. DMSO and PVP cryopreservation are equally effective both for ejaculates and pooled semen. Even PVP is a good alternative since no significant difference was found when compared to DMSO. The fertilizing capacity of fresh and cryopreserved pooled semen was analyzed through AI of hens and female pheasants. Similar fertility rates using DMSO, PVP or frozen-thawed samples demonstrated that reproduction is possible through the use of cryopreserved semen. In the case of female pheasants, the same values were obtained with both cryopreserved and fresh semen.

When Chu-Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 51C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triplestain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology.

Problem statement: Boar sperm cells are sensitive to the freezing process, which compromises viability of frozen-thawed sperm. In a constant search for minimizing or suppressing sperm cell damage caused by the temperature and osmolarity changes during the freezing process, crioprotective and antioxidant substances have been added to the freezing media, such as coconut water, in order to increase the viability of frozen-thawed swine semen. The addition of any substance to the freezing diluent, directly affects osmolarity of the media, which can have positive or negative effects over the sperm cell. Approach: There are no published studies currently that indicate the effect of adding coconut water over the osmolarity of freezing media and their effect over viability of sperm cells, therefore, the objective of the present study was to evaluate the effect of coconut water based diluents osmolarity over the Motility (Mot), Acrosome Integrity (AI), Membrane Integrity (MI) and Mitochondrial Activity (MA) of thawed boar sperm cells. The treatments used were control T1 (LEY with bidistilled water + LEYGO) with an osmolarity range of 296-368 mOsmol Kg −1 , T2 (LEY and deionized coconut water + LEYGO) between 381 and 480 mOsmol Kg −1 and T3 (LEY and in natura coconut water + LEYGO) between 519 and 1041 mOsmol Kg −1 . The Westendorf modified method was the freezing method used. The obtained data were statistically analyzed by GLM, using the SAS software (SAS, 2000). Results: A significant difference was observed on T2 compared to T1 regarding Mot 41.9 Vs 36.9% and MI 58.0 Vs 50.2. T3 had a detrimental effect on all studied variables. Conclusion: Under our study conditions, the osmolarity range of T2, due to the non ionic solutes content, contributed to improve the viability of frozen-thawed sperm cells.

The reproductive tract of Chu-Lin, a giant panda that had died at Madrid Zoo, was removed. Four hours postmortem the epididymides was cut with a blade, and the spermatozoa were collected and immersed in a freezing medium. The sample was kept under refrigeration at 51C. After 4 hr the sperm suspension was aspirated into 0.25-mL straws, which were held for 7 min in nitrogen vapors before they were plunged into liquid nitrogen. To characterize sperm population and distribution, a morphometric study of the triple-stained smears of the thawed semen sample was undertaken with the aid of computerized image analysis. Spermatozoa with intact acrosomes were selected and assessed using an immersion lens (1,000 Â ) and bright-field illumination. The software made it possible to take six basic linear measurements of each spermatozoon: maximum head width (HW), base of the head (BH), maximum head length (HL), midpiece length (ML), midpiece width (MW), and terminal piece length (TL). The system was connected to a data base from which derived parameters could be calculated: head form factor (HL/HW), total flagellum length (TFL), total sperm length (TSL), and the proportion of each of the sections in relation to the whole. The results of the morphometric study did not demonstrate the existence of sperm subpopulations, as the measurements of the 100 spermatozoa analyzed were relatively homogeneous with regard to their distribution for each of the parameters considered. The sperm head of the giant panda displayed a relatively rounded morphology, and the HL/HW was 1.2470.03. Mean head length was