Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium.

In the present study, the effects of replacing glucose with pyruvate-lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0mgmL(-1) HA, and with either 5.55mM glucose (IVC-Glu) or pyruvate (0.17mM)-lactate (2.73mM) from 0 to 48h post insemination (h.p.i.) and then with glucose from 48 to 168h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7±1.5%) than those cultured with IVC-Glu (14.27±2.75%). At 1.0mgmL(-1), HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0mgmL(-1) HA and IVC-Glu (4.28±0.28% vs 11.01±1.42% and 10.14±2.77%, respectively) and IVC-PL (14.37±1.35% vs 20.96±2.85% and 22.99±1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0mgmL(-1) HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL.

Energy substrate influences the effect of the timing of the first embryonic cleavage on the development of in vitro-produced porcine embryos in a sex-related manner.

In vitro culture conditions and certain events during the earliest stages of development are linked to embryonic survival, possibly in a sex-related manner. In vitro-produced (IVP) porcine embryos cultured with glucose (IVC-Glu) or pyruvate-lactate (IVC-PL) were tested for any relationship between the timing of the first embryonic cleavage and development and sex ratio. The embryos were assigned to IVC-Glu or IVC-PL groups and classified depending on the timing of their first cleavage: 24, 26, 30, and 48 hr post-insemination (hpi). They were cultured separately in vitro and evaluated for cleavage rate and pattern, blastocyst rate and stage, cell number, apoptosis, and sex ratio. Regardless of energy source, the percentage of two-cell stage and fragmented embryos at the time of their first cleavage was, respectively, higher and lower in early-cleaving embryos. Those embryos cleaved by 24 hpi developed to blastocysts at a higher rate (IVC-Glu: 37.90 ± 3.06%; IVC-PL: 38.73 ± 4.08%) than those cleaved between 30 and 48 hpi (IVC-Glu: 5.87 ± 3.02%; IVC-PL: 8.41 ± 3.50%). Furthermore, a shift toward males was seen among embryos first cleaved before 30 hpi, versus towards females among those cleaved later. The early-cleaving embryos, only from the IVC-PL group, had a higher proportion of expanded blastocysts (81.05 ± 6.54% vs. 13.33 ± 13.33%) with higher cell numbers than their late-cleaving counterparts. Moreover, a shift toward males only appeared at the blastocyst stage in IVC-PL embryos. These findings confirm that the timing of the first cleavage influences development of IVP porcine embryos in a sex-related manner, and it depends on the main energy source of the in vitro culture medium.

How do different concentrations of Clostridium perfringens affect the quality of extended boar spermatozoa?

Bacteriospermia in boar fresh and extended semen is a frequent finding that produces alterations on sperm quality and, consequently, causes economic losses in artificial insemination (AI) centres. The present study sought to evaluate the effect of different infective concentrations of Clostridium perfringens on boar sperm quality, assessed as sperm motility (CASA), morphology and viability, through 11 days of storage at 15°C (experiment 1), and after 96h of incubation at 37°C (experiment 2). With this purpose, different seminal doses were artificially inoculated with different infective concentrations of C. perfringens, ranging from 10(2) to 10(8)cfumL(-1). The negative controls were non-inoculated doses. Sperm quality was checked after 0, 1, 2, 3, 4, 7, 8, 9, 10 and 11 days of storage at 15°C in experiment 1, and after 0, 24, 48, 72 and 96h at 37°C in the second experiment. Moreover, the presence/absence of bacteria was detected by PCR analyses during both experiments at different time points. In both experiments, sperm morphology of inoculated samples did not differ from the negative control. Conversely, detrimental effects on sperm viability and motility were observed after 24h of incubation/storage at the highest infective concentrations in both experiments. The deleterious effects observed because of the presence of C. perfringens in semen emphasise the relevance of detecting bacteria in extended doses destined to AI. So, this study suggests that the evaluation of bacterial contamination in semen is a procedure that should be routinely applied while assessing sperm quality in AI centres to avoid the use of doses with low sperm quality and the possible spread of bacterial contaminants.

Sex determination of porcine embryos using a new developed duplex polymerase chain reaction procedure based on the amplification of repetitive sequences.

Polymerase chain reaction (PCR)-based assays have become increasingly prevalent for sexing embryos. The aim of the present study was to develop a suitable duplex PCR procedure based on the amplification of porcine repetitive sequences for sexing porcine tissues, embryos and single cells. Primers were designed targeting the X12696 Y chromosome-specific repeat sequence (SUSYa and SUSYb; sex-related primer sets), the multicopy porcine-specific mitochondrial 12S rRNA gene (SUS12S; control primer set) and the X51555 1 chromosome repeat sequence (SUS1; control primer set). The specificity of the primer sets was established and the technique was optimised by testing combinations of two specific primer sets (SUSYa/SUS12S; SUSYb/SUS12S), different primer concentrations, two sources of DNA polymerase, different melting temperatures and different numbers of amplification cycles using genomic DNA from porcine ovarian and testicular tissue. The optimised SUSYa/SUS12S- and SUSYb/SUS12S-based duplex PCR procedures were applied to porcine in vitro-produced (IVP) blastocysts, cell-stage embryos and oocytes. The SUSYb/SUS12S primer-based procedure successfully sexed porcine single cells and IVP cell-stage embryos (100% efficiency), as well as blastocysts (96.6% accuracy; 96.7% efficiency). This is the first report to demonstrate the applicability of these repetitive sequences for this purpose. In conclusion, the SUSYb/SUS12S primer-based duplex PCR procedure is highly reliable and sensitive for sexing porcine IVP embryos.