In the present study particular importance was addressed to precise classification of the post-ovulatory period regarding sperm distribution and oocyte maturation. For this, ovulation was determined by real-time ultrasound examination combined with progesterone analysis. Pre-ovulatory follicle diameters matched with the data described by other authors[28, 34, 35]. The mean progesterone concentration of 26.1 nmol/L measured at the time of sonographically indicated ovulation was within the average range of 12.7-31.8 nmol/L described in the literature (see introduction). Variation of progesterone values (15.9-41.0 nmol/L) between bitches was probably due to inconsistency in pre-ovulatory follicle luteinisation as well as to the variability in the time span between the pre-ovulatory LH-surge and ovulation, which has been shown to range from 24 to more than 96 hours. Furthermore, differences in ovulatory progesterone concentrations between studies may partly result from different assay systems. The reliability of our methods applied for diagnosing ovulation is verified indirectly by the maturation stages of oocytes recovered 2 and 4 days later. On day 2 all oocytes were still in prophase-I stage, which has been shown to exist for up to 48 hours after ovulation[15, 16], whereas on day 4 metaphase-II oocytes, which can be expected the earliest 48 to 54 hours after ovulation[15, 16], were identified. Despite progesterone concentrations clearly above 47.7 nmol/L, as described by Concannon et al. (1975) at the time of fertilization, signs of fertilization were missing probably due to a delay observed by Reynaud et al. (2005) at least up to 83 hours after ovulation resulting in fertilization the earliest 90 hours after ovulation.
In accordance with previous reports[8, 9] our results show that the uterine glands are a significant site of sperm storage in the dog which may, however, act primarily as sperm barriers by retaining a specific population of spermatozoa. By this the uterine glands may play an important role as selection mechanism for a viable sperm population, which is provided in the glandular region of the utero-tubal junction throughout the protracted time of post-ovulatory oocyte maturation and fertilization[8, 9].
In the cat, a species with coitus induced ovulation of fertilizable metaphase-II oocytes, the uterine crypts and the utero-tubal junction have been shown to serve as sperm reservoirs prior to ovulation whereas the oviductal isthmus is suggested to be a site of sperm storage close to the time of ovulation and fertilization.
Sperm binding to the reproductive epithelium is the crucial mechanism for slowing destabilization of sperm membranes as part of the capacitation process[38, 39] and allowing the timely maturation of sperm in relation to physiological stimuli from the bitch. The latter may include a rise in progesterone concentrations and factors deriving from the maturing oocytes[10, 40, 41]. In our study despite the low sperm numbers detected in oviduct flushing, viable sperm were found both 2 days and 4 days after ovulation with a maximum proportion of 60% motile spermatozoa (group 2) similar to the in vitro findings of England and Pacey (1998) and Pacey et al. (2000).
Two days and 4 days after ovulation, progesterone concentrations had reached high mean levels of 67.7 nmol/L and 93.8 nmol/L, respectively. In this period spermatozoa within the uterine horns were gradually eliminated, as indicated by decreasing absolute sperm numbers (Table1) as well as by the shift from glands containing more than 5 sperm to glands containing less than 5 or even no sperm, also described by Rijsselaere et al. (2004).
Elimination of spermatozoa from the genital tract has also been observed in the progesterone-influenced post-ovulatory period of the cat, 48 and 96 hours after mating corresponding to approximately 16 to 23 hours and 64 to 71 hours after ovulation, respectively. It is suggested that in the bitch spermatozoa detaching from the uterine glands due to the increased progesterone concentrations may be eliminated from the genital tract whereas at least some of the sperm released at the same time from the utero-tubal junction may reach the site of fertilization.
In our study the reduction of sperm number in the utero-tubal junction was similar to that observed by Rijsselaere et al. (2004) in four and three bitches, respectively, from day 1 to day 3 to 4 after ovulation, although all seven bitches had been inseminated only 24 hours prior to ovariohysterectomy, indicating that during this period the rate of both sperm binding and detachment may depend primarily on the concomitant progesterone rise and oocyte maturation rather than on the time of insemination.
Sperm detachment in the utero-tubal junction from day 2 to day 4 after ovulation (Table1 and2) was accompanied by a slight decrease of sperm numbers in the caudal oviduct. This finding may be supported by Urhausen et al. (2011), who found a significant increase of apoptotic cells in the epithelium of the utero-tubal junction and caudal oviduct from day 2 to day 4 after ovulation, suggesting apoptosis to be an underlying mechanism of sperm detachment. The concomitant slight increase of the sperm number in the mid oviduct may provide evidence of a shift of spermatozoa to the site of fertilization. Redistribution of spermatozoa from the isthmus to the site of fertilization has also been shown to be influenced by ovulation and/or by mature oocytes in rabbits and pigs[45, 46].