Study On The Practical Use Of Cryopreserved Boar Semen With High Density In 0.25mL Staws

Artificial insemination(AI) with frozen-thawed semen is an efficient and convenient way in animal reproduction and breeding.AI technique furnishes an important tool for the preservation of male genetic resources.Firstly,it's convenient for exportation of potencial male genetypes around' the world without the limitation of time and space, which enables animal exchange provincially and internationally and improves the efficiency of good males.More importantly,cryopreservation of semen extends the utilization duration of male genentic resources and is also important for preservation and supply of semen,blood lineage renewal,breed introduction,low productive cost and so forth.In addition,before the wide-bound clinical generalization of semen,lots of message about genetic background and sickness can be obtained,which insure the stability of genetic progression and biological security.Althoug AI with frozen-thawed bull semen is an integral biotechnological component of the cattle industry worldwidely, the use of frozen semen for AI in pigs is very limited due to poor post-thaw quality and deficiency of efficient insemination technology for frozen semen.Based on our previous study,the frozen-thawed procedures of boar semen were further optimized and the post-thaw quality of frozen semen was improved in this study,focusing on the post-thaw survival duration of frozen semen and artificial insemination so as to establish an efficient technique system for practical use of frozen boar semen and to accelerate the wide-bound clinical generalization of frozen semen.Experiment 1.Cryopreservation of boar semen with 0.25 mL strawsThe purpose of this experiment is to establish an optimal protocol to cryopreserve boar semen.The results showed that the optimal procedure should be operated as follows. Firstly,boar semen were pre-diluted with ZO solution and pre-equilibrated at room temperature for 1 h.After adding the extenderⅠ,spermatozoa were equilibrated at 5℃for 1.5 h and then equal volume of the extenderⅡwas added,holding 2 h for equilibration. Resulting spermatozoa were loaded into 0.25 mL straws and equilibrated for 10 min at 3 cm above the surface of liquid nitrogen(LN) and then submerged into LN promptly. When thawing,straws were put into water bath at 37℃for 30 s.The above procedures yielded the highest post-thaw motility of 0.584±0.03 and the plasma integrity of 63.24±1.2%,together with the normal acrosome was 51.4 4±2.6%.The abnormality of spermatozoa after freezing was the lowest at 14.0±3.0%.Experiment 2.Comparison of motility of post-thaw boar spermatozoa at different timeThis experience mainly focused on the effects of temperature,egg yolk supplement, seminal plasma supplement,anti-oxidant supplement,energy substance ATP supplement and egg yolk-seminal plasma complex on motility of post-thaw spermatozoa preserved for 4 h at room temperature.The thawed semen were divided into groups according to examination time,marked as 0,1,1.5,2,3 and 4 h respectively and the motilily of spermatozoa were compared.The results showed that post-thaw preservation temperature, yolk and seminal plasma supplement were the key factors impacting spermatozoa survival duration.The initial motility of semen was 0.6,after preservation for 1h,the motility of semen placed at room temperature(22℃—25℃) were significantly higher than the group placed at 37℃(respectively 0.45 and 0.10).Preserving for 4 h at room temperature,the motility of the group supplemented with 40%seminal plasma + 20% egg yolk was significantly higher than other groups supplemented with 40%seminal plasma or 20%egg yolk only(0.52 vs 0.45 and 0.10).The results indicated that supplement simultaneously with seminal plasma and egg yolk,placing at room temperature could improve survival duration of boar frozen-thawed semen significantly, which will provide reliable technological support for the wide-bound clinical generalization of boar frozen semen.Experiment 3.Study on deep intra-uterine insemination(DIUI) with smaller volume and higher density of boar frozen semenIn this experience,oestrus female sows were inseminated using deep intra-uterine insemination method with boar frozen semen,which were thawed in the ZO supplemented with 40%seminal plasma or not.DIUI was conducted for once or twice, the number of spermatozoa was about 5×10~8 spermatozoa and the dosage of insemination was 5 ml,resulting in pregnancy rate of 33.3%(6/18) from the group supplemented with 40%seminal plasma but no pregnant from the non-supplement group.In addition, insemination twicely yielded higher pregnancy rate(50%) and were superior to insemination once(20%).It showes that it's feasible to do DIUI with smaller volume and higher density of boar frozen semen to some extent but need to be further improved.