Effects of heat stress on sperm quality of French Bulldogs.

This study aimed to evaluate the effects of heat stress (HS) and its duration on semen quality, serum testosterone, pulsatility and resistibility index of the testicular artery of French Bulldogs. Eight male French Bulldogs, 3-7 years old, 12.63 ± 1.8 Kg were adapted and trained for two months. Room temperature was 21 °C. Semen was collected by digital stimulation. The median of four andrological evaluations was T0. Heat was applied to the scrotum using an electrical heat pad at 40 °C for 11 min. Rectal temperature (RT) and scrotum temperature were evaluated using a mercury thermometer and an infrared thermography camera before and after HS. Semen was evaluated immediately (T1) and after seven (T7), 14 (T14), 21 (T21), 28 (T28) and 60 (T60) days after HS. Semen parameters included macroscopic (volume, color and viscosity) and microscopic (sperm motility and vigor, percentage of morphologically normal or defected spermatozoa, sperm concentration and total number of sperm cells) aspects. A pulsed colored doppler ultrasound was performed on the testicular artery at the spermatic cord and epididymis region before and immediately after HS. Serum testosterone was analyzed before, 48 and 96 h after HS. Data was analyzed by ANOVA using SAS. There was a 1.23 °C increase on RT and a 4.98 °C increase on thermograph after HS. Sperm motility decreased at T1 (P < 0.05) and tended to stay lower at T7 (P = 0.056). It improved at T14, but reduced again at T21 (P < 0.05). At T28 and T60 motility was normal. Vigor was lower at T1 (P < 0.05), normal at T7 and T14, but decreased at T21 (P = 0.054), at T28 and T60 it was not different than T0. Sperm concentration was lower at T1 (P < 0.05) and not different from T0 at other timepoints. Volume color and viscosity were not different. Total sperm per ejaculate was reduced at T1 and T7 (P < 0.05) and tended to be lower at T14 (P = 0.057). T21, T28 and T60 were not different than T0. There was a decrease in normal sperm cells and an increase in defected sperm at T7. There was no difference within T14, T21, T28 and T60. The raise in pathologies at T7 was from an increase in minor defects (P < 0.05). There was no difference in serum concentration of testosterone nor pulsatility and resistivity index before and after HS. In conclusion, induction of HS directly to the testis reduces sperm quality in French Bulldog. This impairment is immediately and transitory.

Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers.

Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression.

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10  ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at day 15 of pregnancy.

Colony-stimulating factor 2 inhibits induction of apoptosis in the bovine preimplantation embryo.

PROBLEM: Addition of colony-stimulating factor 2 (CSF2) to culture medium increases post-transfer survival of bovine embryos. Here we provide evidence that one mechanism by which CSF2 affects the embryo is through inhibition of apoptosis. METHOD OF STUDY: In the first experiment, genes and pathways whose expression were regulated by CSF2 were identified by microarray analysis. Embryos were treated with 10 ng/ml CSF2 or vehicle at Day 5 after insemination; morulae were selected for microarray analysis at Day 6. In a second experiment, antiapoptotic effects of CSF2 were determined. Embryos were treated with CSF2 or vehicle at Day 5. On Day 6 (24 h after treatment), morulae were cultured for 15 h at either 42°C (a temperature that induces apoptosis) or 38.5°C (cow body temperature). RESULTS: In the first experiment, a total of 214 genes were differentially regulated and 160 of these could be annotated (67 upregulated genes and 93 downregulated genes). Differentially expressed genes could be placed in 13 biological process ontologies in four functional groups (development and differentiation process, cell communication, apoptosis and cell adhesion). Antiapoptotic effects of CSF2 were confirmed in the second experiment because the magnitude of the increase in TUNEL positive cells caused by heat shock was reduced by CSF2. CONCLUSION: CSF2 blocks apoptosis in bovine embryos through actions associated with regulation of genes controlling apoptosis.