Reproductive efficiency of sows inseminated at single dose fixed time with refrigerated, cryopreserved and encapsulated spermatozoa

Abstract The aim was to assess the reproductive efficiency of different techniques used to preserve spermatozoa in artificial insemination semen doses (AI‐doses) by evaluating refrigeration at 15°C, cryopreservation and encapsulation. Forty‐two hyperprolific sows were treated with buserelin and inseminated once at a single fixed time. The fertility rate, embryonic vesicles viability and the early embryonic mortality (arrested conceptuses) evaluated post‐mortem at 24th day of pregnancy, were analysed in order to assess the effectiveness of each proposed technique. Results show an overall reduction on fertility using the three proposal sperm preservation techniques (69.27%, 60.00% and 78.75% for refrigerated, frozen–thawed and encapsulated AI‐doses, respectively). Total number of embryonic vesicles was very similar among the three treatments; yet, the number of viable vesicles was numerically different among groups, and thus, embryonic viability was 79.25%, 80.0% and 87.15% for refrigerated, frozen–thawed and encapsulated AI‐doses, respectively.


| Seminal doses (AI-doses) preparation
Semen was obtained from two boars from the 'Cinco Villas' AI Center, and pool of ejaculates from these boars were used for preparing AIdoses for post-cervical AI. The AI-doses of the three experimental groups (frozen, refrigerated and encapsulated) were prepared in a volume of 50 ml with a total sperm concentration of 1.5 × 10 9 sperm/ dose. The methodology used in each group was as follows: a. Frozen-thawed AI-doses Ejaculates were collected, evaluated for quality and frozen 30 days before AIs. After collection, samples for further cryopreservation using the extenders and freezing protocol described by Gil et al. (1996). Frozen samples were transported to the farm in a cryogenic storage tank and thawed in a water bath before AIs.
b. Refrigerated semen AI-doses were prepared at the AI Center (Cinco Villas) the day of AIs as usually are prepared for routine distribution to commercial farms. After collection, ejaculates from the two boars were pooled, and the resulting sample was split a half for preparing refrigerated and encapsulated AI-doses.
a. Liquid refrigerated AI-doses After semen quality evaluation, samples were diluted in Duragen® extender (Magapor) and placed in a portable cooler at 15°C until use in AI.
b. Encapsulated AI-doses After semen quality evaluation, pooled sperm samples were encapsulated following the methodology previously described by Vigo et al. (2009) with some modifications. Briefly, after gelification of semen samples the solution was extruded through a peristaltic pump into a Ca ++ -enriched solution that allowed the rapid formation of the capsules. Final size of the formed capsules was 1.5 mm in diameter. As above, semen samples were stored at 15°C until use.
Semen motility in the three experimental groups was subjectively evaluated at the AI Center prior its transport to the farm. The minimum motility required for AI was 80% and 60% for refrigerated (liquid and encapsulated) and frozen-thawed AI-doses, respectively.

| Oestrous synchronization and artificial insemination
At 85 h after weaning, sows received 10 μg of intramuscular buserelin (Porceptal®, MSD), and treated sows were inseminated once 30 h after buserelin injection using a single fixed time AI with a postcervical catheter.

| Embryonic development at slaughter
Sows were slaughtered on day 24 after AI. Genital tracts were transported to the Veterinary Faculty of the 'Universidad de Zaragoza' for further processing. After washing, uterine horns were detached from the mesometrium and longitudinally extended in order to make TA B L E 1 Fertility and embryo viability and embryo mortality (non-viable vesicles with arrested conceptuses) at 24th day of pregnancy with refrigerated, encapsulated and frozen-thawed AI-doses and a single fixed time AI (mean ± SEM)
The effect of semen treatment on fertility, total number of embry-

| DISCUSS ION
The use of refrigerated AI-doses at 15°C is the most widely used system for semen preservation in AI swine programmes due to its high efficacy, ease of application in farms, low cost and wide scope to use (from 1 to 7 days). Yet, semen cryopreservation is not a common The fertility rate achieved in this study is low compared with the results obtained in production farms, which can exceed 90% fertility rate using routine AI techniques, with the application of at least two AI-doses per oestrus. Yet, when a single fixed time AI is used after the application of an ovulation synchronization treatment (using with a GnRH agonist), results are controversial. Thus, similar results have been previously reported with respect routine AI techniques (Driancourt et al., 2013), but also lower (Knox et al., 2014), as in our case. The best fertility results were obtained with encapsulated spermatozoa (78.75%) compared with refrigerated (69.23%) and frozen-thawed semen in which a marked decrease in fertility (60.00%) was observed, but without reaching statistical significance.
Finally, fertility results obtained with the application of frozenthawed AI-doses were similar to those obtained in previous studies (Yeste et al., 2017).
Total number of embryonic vesicles in uterine horns was similar among treatments (from 21.5 to 21.8). Yet, viable embryonic vesicles were numerically higher with the use of encapsulated AI-doses (18.88) compared with the found when using refrigerated AI-doses (17.11) or frozen-thawed (17.00), but differences did not reach statistical significance.
The low fertility results obtained with the three treatments could be explained by an inadequate response to ovulation synchronization treatment with the use of a single AI, since the pooled F I G U R E 1 Viable embryonic vesicle at D24 post-AI F I G U R E 2 Non-viable embryonic vesicles (arrested conceptuses) at D24 post-AI