Impact of inclusion of post-spermatic ejaculate fraction in boar seminal doses on sperm metabolism, quality, and interaction with uterine fluid.

Boar ejaculate is composed of sperm cells and seminal plasma (SP) and is emitted in different fractions (pre-sperm fraction; spermatic-rich fraction; intermediate fraction; post-spermatic fraction), with different composition of SP and volume, which could influence the sperm quality during seminal doses preparation, conservation, and interaction with the female reproductive tract. In artificial insemination (AI) centers, seminal doses are usually prepared with the spermatic-rich and intermediate fractions, but the inclusion of other ejaculate fractions, although controversial, is beginning to be applied. The objective was to evaluate the synergic effect of accumulative ejaculated fractions on sperm functionality during seminal doses preparation, throughout storage and after incubation with uterine fluid (UF). For this purpose, a total of 57 ejaculates were collected, and the following experimental groups were prepared (n = 19 per group): (F1) spermatic-rich fraction; (F2) F1 plus intermediate fraction; (F3) F2 plus post-spermatic fraction. Each group was stored for 5 days at ∼16 °C, and the following parameters were evaluated: sperm metabolism of pure and diluted semen (day 1), sperm quality parameters (days 1, 3, 5), thermal-resistance test (TRT) and incubation with uterine fluid (UF) (day 5). Sperm metabolic rates between accumulative ejaculate fractions from pure and diluted semen did not show differences. Also, sperm quality parameters were not affected by the ejaculate fraction during storage. However, sperm subjected to TRT showed similar results except for progressive motility, which was better in F2 and F3 than F1. When sperm were incubated with UF, the quality decreased in each group, but sperm from F2 and F3 were less affected than those from F1. In conclusion, the post-spermatic fraction can be included in seminal doses for their use in AI-centers, with functionality of sperm of different SP origins not being impaired throughout the storage, and responding better to thermal and UF stress. However, further research in AI-centers is necessary to test the sperm behaviour under presented conditions.

Should All Fractions of the Boar Ejaculate Be Prepared for Insemination Rather Than Using the Sperm Rich Only?

Boar ejaculate is released in several well-characterized fractions, differing in terms of sperm concentration, seminal plasma volume, and composition. However, the inclusion of the last part of the ejaculate for artificial insemination (AI) purposes is still under debate due to its controversial effects. Thus, there is a need to study the potential synergistic impact of the different ejaculate fractions. We aimed to evaluate the effect of accumulative ejaculate fractions on sperm conservation, AI performance, and offspring health. Ejaculates (n = 51) were collected and distributed as follows: F1: sperm-rich fraction; F2: sperm-rich + intermediate fractions; F3: sperm-rich + intermediate + poor fractions. Each group was diluted in a commercial extender, packaged in seminal doses (2000 × 106 sperm/60 mL), and stored at ~16 °C. On day 3 of conservation, sperm were analyzed and used for AI (n = 174). High sperm quality was observed after storage without a significant difference between the groups (p > 0.05). Moreover, no differences were obtained for AI performance (pregnancy and farrowing rates, and litter size; p > 0.05) and offspring health (growth and blood analysis; p > 0.05). Conclusively, the presence of all ejaculate fractions within the seminal doses does not impair the reproductive performance, reporting important economic savings according to the economic model included here.

Relevance of the Ejaculate Fraction and Dilution Method on Boar Sperm Quality during Processing and Conservation of Seminal Doses.

During boar semen processing and distribution, maximizing the work protocols in the laboratories becomes essential for the conservation of seminal doses. One of the recent implementations in the boar studs to improve efficiency has been semi-automatic semen collection systems, which do not allow to discard fractions of the ejaculate. The objective of this work was to evaluate the dilution method and vibrations (simulating delivery transport) effect on sperm quality (motility, viability, morphology, thermo-resistance test) according to the fraction of ejaculate collected. Two different fractions of the ejaculate were obtained [rich fraction (RF); total fractions (TF)] from six boars, and two dilution methods applied [pouring the extender over the semen (control; ES); pouring the semen over the extender (reverse; SE)]. The seminal doses (2000 × 106 sperm/50 mL) were preserved for 5 days. The results showed that the fraction collected affects sperm quality (better total and progressive motility, and faster sperm in TF; p < 0.05) regardless of the dilution method applied. However, these differences diminished after submitting the semen to the thermo-resistance test, with only differences in sperm viability being observed (p < 0.05). When seminal doses were subjected to vibrations, the sperm viability was more affected in the TF than in the RF group (p < 0.05). In conclusion, using the TF ejaculate leads to comparable results to the RF in sperm quality during storage regardless of the dilution method applied. However, the vibrations of seminal doses are more affected in doses prepared with TF than with RF, although more factors should be included to approach the real conditions during transport.

In vivo measurement of pH and CO2 levels in the uterus of sows through the estrous cycle and after insemination.

The pH-CO2-HCO3- system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assisted Reproductive Technologies (ARTs). However, in situ measurements of these parameters in the uterus are scarce or null. This study describes a non-invasive method for in situ time-lapse recording of pH and CO2 within the uterus of non-anesthetized sows. Animals were at three different reproductive conditions, estrous with no insemination and two hours after insemination, and diestrous. From pH and CO2 data, HCO3- concentration was estimated. The non-invasive approach to the porcine uterus with novel optical probes allowed the obtaining of in situ physiological values of pH, CO2, and HCO3-. Variable oscillatory patterns of pH, CO2 and HCO3- were found independently of the estrous condition. Insemination did not immediately change the levels of uterine pH, CO2 (%) and HCO3- concentration, but all the values were affected by the estrous cycle decreasing significantly at diestrous condition. This study contributes to a better understanding of the in vivo regulation of the pH-CO2-HCO3- system in the uterus and may help to optimize the protocols of sperm treatment for in vitro fertilization.