Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

卵巢玻璃化冷冻保存研究进展

卵巢玻璃化冷冻是一种操作简单、冷冻效果好的卵巢组织冷冻降温技术,对动物繁殖能力保存、物种保护、生物多样性保存,以及动物胚胎工程和人类临床医学研究与应用等具有重要意义。卵巢组织冷冻保存较卵母细胞冷冻保存,具有明显的优点。在冷冻保护剂的选择、冷冻材料、冷冻平衡及冷冻方法上都有其特殊之处。卵巢组织冷冻保存技术已在人类医学临床上发挥其应用价值。     

Technology and prospects of cryopreservation of germplasm

Summary Entire plants have recently been regenerated from frozen cultures of protoplasts, cells, tissues and organs and stored at super-low temperatures for various lengths of time. This suggests the possibility of utilizing cryogenic methods for the conservation of important and rare germplasm, especially of the vegetatively propagated plants, which at present do not have any satisfactory method for their preservation. In addition, the freeze preservation offers a number of significant uses and prospects; (i) tissue cultures often show chromosomal variations, nuclear and ploidy changes, and mutations. Such genetic variables and cell lines which do not occur in nature can be picked out, banked and used in response to research needs; (ii) to maintain a cell line, it has to be transferred to a fresh medium periodically; the freeze storage would suppress cell division and thus the need for subculturing is avoided; (iii) at super-low temperatures the cells are in a non-metabolic state, which delays or virtually stops the ageing process, and thus the morphogenetic potential of cultures is retained; (iv) pathogen-free stocks could be frozen, revived and propagated when desired, this would be ideal for the international exchange of such materials; and (v) the freezing of pollen would solve some of the problems encountered with the incompatibility and pollen longevity.Ever increasing population pressures demand improving and evolving new cultivars. However, with this enthusiastic search for obtaining new plants, and rapid increase in their number, it is not only becoming difficult but at times impossible to maintain or preserve some of the stocks which at present, are not needed for breeding purposes. Thus, some of the germplasm which may not seem to be of importance today, but might be needed in future, is ignored or completely lost. Therefore it is for the purpose of preservation of materials which are threatened with extinction that Germplasm Banks need to be established.It is envisaged that freeze storage and tissue cultures would be a meaningful tool in experimental biology and agricultural research for the preservation and international exchange of important and pathogen-free germplasm.     

Reproductive performance of immobilized cryopreserved bovine semen used for timed artificial insemination

The SpermVital^® technology comprises embedding of spermatozoa within an alginate gel to facilitate release of sperm cells over a prolonged period in utero after AI. The aim of this study was to examine whether the survival time of spermatozoa is extended when applying this immobilization technology in combination with cryopreservation. Sperm cell survival (acrosome and plasma membrane integrity) was studied in vitro for 48 hr at physiological temperature. One dose of SpermVital^® (SV) semen was compared with single doses of Biladyl^® (B) processed semen as well as double doses of B (B double). B double was obtained by adding a second B dose the following day, thereby mimicking double AI. Furthermore, reproductive performance applying single early timed AI (TAI) with SV following oestrus synchronization was studied in a field trial. Double insemination (TAI on two consecutive days) with B semen served as control. Number of acrosome‐intact live sperm cells decreased over time in vitro for all treatments (p < .05). There was no difference between SV sperm cell survival and B double after 24 hr (p > .05). However, after 48 hr, SV sperm cell survival was higher than B double (p < .05). Moreover, multivariate analysis showed that the outcome of single early TAI with SV was not significantly different from B double (p > .05). Likelihood of pregnancy and calving in the heifer group was higher than in the cow group (p < .05). These results imply that spermatozoa immobilized in alginate gel have prolonged survival.