Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls.

The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = -7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A(3) (CMA(3)) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis.

A duplex real-time RT-PCR for the detection of bluetongue virus in bovine semen.

The control measures prescribed by the World Organization for Animal Health (OIE) for international trade in extended semen implicate repeated free testing of the donor's blood for bluetongue virus (BTV). The aim of this study was to validate a real-time RT-PCR for the direct testing of semen for artificial insemination (AI). The amplification of the BTV target was combined with an internal control target in duplex format. Optimal RNA recovery and efficient removal of PCR inhibitors was established using Trizol-based RNA extraction. The total assay was highly repeatable, the preliminary analysis of the specificity was 100% (95% CI: 92-100%) and the limit of detection was -0.36 log(10)TCID(50) ml(-1) (95% CI: -0.53 to -0.18 log(10)TCID(50) ml(-1)) in BTV-8 spiked extended semen. The protocol was evaluated using 89 extended semen samples from 19 bulls showing typical clinical signs of a natural BTV-8 infection. Forty-eight samples were positive, 30 were doubtful and 11 were negative. Infectious BTV-8 was isolated. Based on varying real-time RT-PCR results of additional straws from cut-off samples it is highly recommended to analyse at least five straws per semen batch before declaring semen free of BTV. In conclusion, the partially validated assay presented has the potential to be used for the control of semen for international trade through direct testing of the semen.