Deep freezing of concentrated boar semen for intra-uterine insemination

University essay from SLU/Dept. of Clinical Sciences

Abstract: Frozen-thawed (FT) boar semen for artificial insemination (AI) in commercial pig production is still only used in exceptional cases, mostly for incorporation of top genetics. Low sperm survival after a freezing procedure that is still considered suboptimal, which results in low farrowing rates and small litter sizes when conventional cervical AI is performed, are among the reasons why FT boar semen is rarely used in AI. Compensatory attempts include AI with large sperm numbers, which, however, reduces the number of doses produced per ejaculate. New procedures for intra-uterine AI are now available, opening up possibilities for the deposition of smaller volumes (thus diminishing semen reflux) and therefore lower total sperm numbers per dose, provided these spermatozoa (spz) can be packed in a higher density and sustain cryopreservation (i.e. provided they have an acceptable survival rate post-thaw). This thesis includes a summarised review of available literature on the history, as well as constraints on and future prospects of the use of frozen boar semen in AI. Also, a trial is reported whose aim was to concentrate (2 x 109 spz/mL) and freeze boar spermatozoa packed in either 0.5 ml volume medium straws (MS) or a multiple FlatPack (MFP) (four 0.7 ml volume segments of a single FlatPack [SFP]) intended as AI doses for intra-uterine AI. A single, well-proved freezing protocol was followed using a SFP (5 x 109 spz/5 ml) as control package and sperm concentration.Sperm motility was measured by computer-assisted sperm analysis (CASA) while plasma membrane integrity was assessed with the SYBR-14/PI assay combined with flow cytometry, and with a rapid hypo-osmotic swelling test (sHOST), in order to establish sperm viability. Sperm motility did not differ statistically (NS) between the test-packages and the control, neither in terms of overall sperm motility (range of means: 37-46%) nor sperm velocity.Percentages of linear motile spermatozoa (LIN), however, were significantly higher in controls (SFP) than in the test packages. Spermatozoa frozen in the SFP (control) and MFP depicted the highest percentages of PMI (54 and 49% respectively) compared to straws (38 %, p<0.05) when measured with flow cytometry. In absolute numbers, more viable spermatozoa were present in the MFP dose post-thaw than in the MS (p<0.05). Inter-boar variation was present, albeit only significant for MS (sperm motility) and SFP (PMI). In conclusion, the results indicate that boar spermatozoa can be successfully frozen if concentrated in a small volume.

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