The ability to accurately evaluate the quality and estimate the fertilizing potential of dog semen has increased in importance as a result of the more widespread use of artificial insemination but remains challenging. The actual conception rate (CR) of a particular male of this polytocous species may provide useful information particularly in a competitive setting of a multi-sire mating or insemination. A crucial part in establishing the actual CR of a male is played by the fecundity of the female-which may ovulate from multi-ovular follicles-as well as breeding timing, breeding technique, and number of spermatozoa inseminated. This thesis determined, in a multi-sire insemination trial using DNA analysis and paternity testing, that the optimal day for surgical insemination using frozen-thawed semen was six days after the rise of the plasma progesterone concentration to between 6 and 9 nmol/L. Concurrently, the frozen-thawed semen used in the insemination trial was evaluated by means of conventional and modern semen evaluation methods, one of which, namely the Merocyanine 540 staining method, was newly validated on fresh dog sperm. Conventional assessment of sperm quality variables included individual progressive motility, viability and morphology using eosin-nigrosin staining. More modern semen evaluation procedures such as epifluorescence microscopy and flow cytometry were used to assess viability (Ethidium Homodimer, Yo-Pro 1), capacitation status (Anti-phosphotyrosine Clone 4G10), membrane destabilization (Merocyanine M540), acrosomal status (FITC-PNA), presence of progesterone receptors (P-BSA-FITC), motility parameters assessed using CASA, as well as the defragmentation index of sperm chromatin (SCSA). Males were ranked according to their CR which was then correlated to 40 sperm quality variables. Two sperm subpopulations, namely the percentage of live sperm which show signs of membrane destabilization (negatively), and the ability of sperm to maintain their viability (positively), did correlate or tended to correlate to in vivo fertility of the males. Another aspect of this thesis estimated the overall probability of a bitch having more than one conceptus derived from a smaller number of follicles, by retrospectively evaluating data of fertility trials as well as collecting data from private practice and welfare organization, thus establishing that the number of corpora lutea of a bitch may be used as a measurement for her fertility, despite the occurrence of multi-ovular follicles in the bitch. The current thesis assessed different aspects of male and female fertility in the domestic dog which, used in conjunction, may increase the ability to accurately estimate the fertilizing potential of frozen-thawed dog semen.