Improvement of quality of canine frozen-thawed semen: in vitro and in vivo assays.

During the last decades, the interest in cryopreservation of dog semen has been continuously increasing as the performing of artificial insemination (AI) has become a common practice in canine breeding. However, field data indicate that canine semen freezing and thawing protocols are not yet well defined in order to obtain acceptable results of pregnancy rate. The cause of the lower pregnancy rates obtained with frozen thawed semen with respect to natural mating is the short life span of thawed canine spermatozoa, due to subtle membrane damages affecting sperm cells during freezing and thawing processes. A general overview is given about what happens when a sperm cell is frozen and thawed, as well as a detailed description of factors affecting sperm cell cryopreservation. The addition of substances in the composition of freezing and thawing extenders and the variation of freezing and thawing rates described in international literature are reviewed. Also, the methods used in clinical practice for heat detection and monitoring are reported, as well as ovulation detection and insemination techniques are reviewed. The effects of the various freezing-thawing protocols have to be analysed and compared using different tests in order to assess semen post-thaw characteristics and thus to understand whether adopted procedures caused damages to spermatozoa functionality, and to understand the fertility potential of frozen-thawed spermatozoa. For this reason, a description of the methods of canine semen evaluations and of their predictive value on in vivo fertility is done. The aim of this thesis is to enhance the quality of frozen thawed semen through variations in the cryoprotectant (experiment 1 and 2), through addition of different substances at the time of thawing and through variation of the thawing rate (Experiment 3; Assay I, II, III). Experiments 1 and 2 focus on the substitution of glycerol with ethylene glycol as cryoprotectant. In experiment 1, an in vitro comparison between glycerol and ethylene glycol was done by evaluating: a) motility parameters with a CASA System during incubation at 38°C; b) membrane integrity with the HOS test. In experiment 2, fertility of frozen semen with glycerol and ethylene glycol was evaluated through an in vivo comparison inseminating 9 bitches. Ethylene glycol gave an increase of post- thaw total and progressive motility and velocity parameters at the time of thawing. This effect lasts 1 hour, after which no differences in total and progressive motility were detected between the two cryoprotectants, while other motility parameters, such as VAP, VSL, VCL are consistently higher for ethylene glycol up to 3 hours post-thawing. In experiment 3, the effect of different concentrations of caffeine, pentoxifylline, 2'-deoxyadenosine added at the thawing medium, and the interaction between the thawing rate and the post-thaw addition of caffeine, pentoxifylline and prostatic fluid was evaluated through analysis of motility parameters with a CASA System during in vitro incubation at 37°C for 120 minutes. The motility enhancers used produced an improvement of post-thaw total and progressive motility and generally of the quality of the frozen-thawed semen movement, without reducing sperm longevity. The homologous prostatic fluid did not show a positive effect on the motility parameters measured by the CASA system, while fast and slow thawing rates employed did not affect the motility parameters.