Embryo survival in the oviduct not significantly influenced by major histocompatibility complex social signaling in the horse.

The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally inseminated them with semen of another stallion, and flushed the uterus 8 days later to test for the presence of embryos. In total 68 embryos could be collected from 97 experimental trials. This success rate of 70.1% was significantly different from the mean pregnancy rate of 45.7% observed 17 days after fertilization using the same experimental protocol but without embryo flushing. Embryo recovery rate was not significantly dependent on whether the mares had been socially exposed to an MHC-dissimilar or an MHC-similar stallion. These observations suggest that MHC-linked maternal strategies affect embryo survival mainly (or only) during the time of fixation in the uterus.

Stallion semen quality depends on major histocompatibility complex matching to teaser mare.

The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm numbers as compared to exposure to an MHC-similar mare. Sperm velocity seemed not affected by the treatment. Overall genetic similarity between stallions and mares (determined from polymorphic microsatellites on 20 different chromosomes) played no significant role here. The MHC type of the teaser mare also affected characteristics of cold-stored sperm after 24 and 48 hr. As expected from ejaculate economics, sperm viability was elevated after exposure to an MHC-dissimilar mare. However, oxidative stress and the percentage of sperm with a high DNA fragmentation were mostly increased after exposure to an MHC-dissimilar mare, depending also on whether the teaser mare was in oestrous or not. We conclude that males can quickly adjust ejaculate quality relative to a female's MHC, and that this male reaction to the social environment can also affect important characteristics of cold-stored semen.

Seasonal changes of DNA fragmentation and quality of raw and cold-stored stallion spermatozoa.

In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca+2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca2+ level of sperm.

Quality of seminal fluids varies with type of stimulus at ejaculation.

The theory of ejaculate economics was mainly built around different sperm competition scenarios but also predicts that investments into ejaculates depend on female fecundity. Previous tests of this prediction focused on invertebrates and lower vertebrate, and on species with high female reproductive potential. It remains unclear whether the prediction also holds for polygynous mammals with low female reproductive potential (due to low litter size and long inter-birth intervals). We used horses (Equus caballus) to experimentally test whether semen characteristics are adjusted to the oestrous cycle of the mare a stallion is exposed to during few moments before ejaculation. We analysed 122 weekly semen samples collected from 16 stallions during exposure to either an oestrous or a dioestrous mare. Semen volume and the rate of motile sperm were higher when stallions were exposed to an oestrous than to a diestrous mare, while total sperm counts and sperm velocity remained unchanged. Sperm collected after exposure to an oestrous mare also showed reduced oxidative degeneration of cell membranes over a period of 48 hours. We conclude that stallions invest more into their seminal fluids when the chance of fertilization is elevated, and that this adjustment of ejaculate quality can happen very quickly.