Effect of ejaculation frequency, prostaglandin F 2 α and cold storage on canine semen yield and postthaw quality

Number of sperm obtained from a single semen collection can vary widely among dogs, often requiring multiple ejaculates to produce a single breeding dose of cryopreserved sperm. Alternative collection techniques and schedules have been reported to increase sperm yield, however, a direct comparison of these methods is warranted. Objective of this study was to determine most effective collection regimen to increase total number of sperm available for cryopreservation without compromising postthaw motility parameters. We hypothesized that multiple collections and administration of prostaglandin F 2  (PGF 2  ) could be used to increase sperm yield without detrimental effects to postthaw semen quality. Sexually mature male dogs (n = 5) were all subjected to following 5 collection protocols in a random order: 1) 1 collection (control); 2) 2 collections 1 hour apart;

3) 2 collections 24 hours apart; 4) 2 collections 48 hours apart; and 5) 1 collection 20 minutes after PGF2  (Dinoprost, 0.05 mg/kg, IM). Total sperm count, total motility (TM) and progressive motility (PM) were assessed for each ejaculate using computer assisted sperm analysis (CASA). Semen was either frozen immediately after collection (Protocols 1 and 5) or stored at 4C, pooled with a second ejaculate (Protocols 2, 3, and 4), and then frozen using a Tris-egg yolk media. Dogs were allowed 7 days of sexual rest between protocols. Frozen samples were evaluated using CASA and flow cytometry to determine TM, PM, viability (VIA) and acrosome integrity (ACR). Data were analyzed using a general linear mixed model and Tukey’s posthoc for pairwise mean comparisons. Significance was set at p < 0.05. All collection protocols resulted in a higher sperm yield compared to control. Postthaw semen parameters (presented in percent) were similar between control and Protocols 2 and 5 (TM: 50.4  5.1, 49.4  5.1,53.8  5.1, PM: 36.8  4.7, 35.4  4.7, 40.2  4.7, VIA: 61.9  3.4, 65.3  3.4, 61.7  3.4, ACR:23.2  2.7, 18.1 2.7, 20.4  2.7), respectively. Protocol 4 resulted in poor postthaw semen parameters (TM: 30  5.1, PM: 20.4  4.7, VIA: 49.8  3.4, ACR: 29.8  2.7). Protocol 3 had intermediate results for all semen parameters (TM: 37.2  5.1, PM: 24.4  4.7, VIA: 55.2  3.4, ACR: 26.6  2.7).

In conclusion, 2 collections performed 1 hour apart with 1 dose of PGF 2  dramatically increased sperm yield and also maintained postthaw quality. Cooled storage before cryopreservation was increasingly detrimental with increasing time between collections. We provided 2 alternative semen collection protocols to practitioners to maximize number of breeding doses cryopreserved in 1 visit. Further, this would enhance client satisfaction while also minimizing costs and veterinary visits.

Keywords: Canine, spermatozoa, yield, cryopreservation, prostaglandin

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.