Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

Is the Production of Embryos in Small‐Scale Farming an Economically Feasible Enterprise?

The present assay attempts to evaluate the feasibility of using embryo transfer in small community farmers by in vivo study and by modelling the results obtained. From the total of 59 donor cows, 62.7% responded to treatment, with a significant difference (p = 0.002) in the percentage of the response between breeds, being 90.5% (19/21) in Holstein and 47.4% (18/38) in Brahman. A total of 283 embryos were graded as transferable, while 141 as non‐transferable, without difference in the percentage of transferable embryo by breed (p = 0.18). The mean of transferable embryos graded as class I and II was not different between Holstein and Brahman (p = 0.96 and p = 0.92, respectively); besides, no differences were observed in the other grades (non‐transferable). The highest difference in costs, regardless of its quality by breed, was seen in the lower levels of probable fertility of the embryo transferred, even reaching several hundred dollars. When modelling the expected costs for embryo produced and transferred, values can reach nearly $2000.00 when the probable fertility is only 10%. However, when the probable fertility was 60%, embryo cost was close to $300.00. This technology seems to be viable on average or high‐scale systems, having a superovulatory response between 60 and 80% with 4–6 transferrable embryos. Yet, in small‐scale farming, due to the reduced number of donors and/or recipients, the costs surpass the economical feasibility of the technique.     

Unilateral scrotal pyocele in ram caused by Staphylococcus capitis

A massive unilateral scrotal pyocele caused by Staphylococcus capitis in a 6‐year‐old ram is reported. Ultrasound examination of the right hemiscrotum showed an irregular hyperechoic mass in an anechoic fluid. A dense exudate was collected from the scrotum for microbiological analysis. Grossly, there was an extensive greenish purulent exudate and a completely atrophied right testis. Coagulase‐negative S. capitis was isolated in pure culture. To the authors' knowledge, this is the first report of genital infection by S. capitis in rams. This microorganism should be included in the differential diagnosis of ovine genital infections.     

The intensity of resistance by mature Merino ewes against Haemonchus contortus and Tichostrongylus colubriformis in single-species and combined-species infection

Three-year-old, non-lactating and non-pregnant Merino ewes, raised on pasture under a program of strategic treatment with anthelmintic and found to be extremely resistant to "trickle" infection with Haemonchus contortus, were given single-dose infections with either H. contortus or Trichostrongylus colubriformis or both species together. The purpose was to ascertain the intensity of protective immunity against the 2 parasites in sheep with immunity acquired from a presumably slight exposure to infection. To provide a criterion, some infected ewes were immunosuppressed with corticosteroid, dexamethasone. Untreated ewes were extremely resistant to challenge infection with either 15,000 or 150,000 H. contortus or 15,000 T. colubriformis. Surprisingly, when mixed infection was given, egg counts for H. contortus were significantly elevated compared with infection by that species alone. Antibody to antigens from infective larval and adult H. contortus was measured in serum by enzyme-linked-immunosorbent assay (ELISA) during the course of infection. Serum titres against larval antigens were significantly depressed when infections with either H. contortus or T. colubriformis were permitted by immunosuppression with dexamethasone, whereas those against adult antigen were depressed when infection with T. colubriformis was permitted.     

Percentage of Ubiquitinated Spermatozoa does not Correlate with Fertilizing Capacity of Thawed Bovine Semen

In the spermatozoa of some species, the ubiquitin–proteasome system detects altered proteins and tags them for elimination by the proteasome. In some species' ejaculates, a high proportion of ubiquitinated spermatozoa (i.e. those having ubiquitin bound to the altered or damaged membrane proteins) has been related to infertility. The aim of this study was to assess whether the percentage of ubiquitinated spermatozoa relates to fertility of dairy bulls and whether ubiquitination increases during protein remodelling that occurs during in vitro spermatic capacitation. Thirty‐two frozen semen straws from four high‐fertility (ReproMax^®) and four normal‐fertility (Normal) Holstein‐Friesian sires were evaluated. Ubiquitinated and capacitated spermatozoa were quantified by sperm ubiquitin tag immunoassay and chlortetracycline stain, respectively. Fertilizing capacity of sires was assessed by in vitro fertilization. No differences were found between Normal and ReproMax^® sires with regard to the observed percentage of ubiquitinated spermatozoa (42.97 ± 3.69% and 49.68 ± 9.27%, respectively; p > 0.05). Additionally, no differences were found in the percentage of ubiquitinated spermatozoa as a consequence of spermatic capacitation in either Normal (42.97 ± 3.69% before capacitation vs 44.67 ± 7.5% after; p > 0.05) or ReproMax^® sires (49.68 ± 9.27% before vs 45.05 ± 7.51% after; p > 0.05). The percentage of ubiquitinated spermatozoa in a thawed sperm samples did not correlate with its in vitro fertilizing capacity; thus, this assay does not prove useful to detect in vivo fertility differences between sires. Additionally, protein degradation occurring during remodelling of the spermatozoon plasma membrane during the capacitation process does not seem to involve the ubiquitin–proteasome system.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

疑似碘酊致牛死亡一例

笔者在临床上曾遇到一疑似“碘酊”致牛死亡的病例,由于未曾见过碘酊致牛死亡的报道,查阅资料也终未得到答案.笔者将其整理如下,供同行探讨。     

Assessment of Suitable Porcine Semen for Freezing,According to the Ejaculate Characteristics in the Iberico × Landrace Breed

A total of 35 ejaculates were studied in order to assess the suitability of porcine semen for freezing according to the ejaculate characteristics. The effects of the freezing procedure were identified; a decrease in motility and acrosome quality was found after thawing. The best results on motility were linked to the ejaculates with a volume of less than 100 ml of the sperm‐rich fraction, a concentration lower than 450 × 10⁶ spermatozoa/ml and an agglutination score below 2. However, the best normal apical ridge (NAR) was found when the volume of the sperm‐rich fraction was greater than 150 ml. For this reason, an intermediate volume of the sperm‐rich fraction of the ejaculate for the best motility and the best NAR, a concentration lower than 450 × 10⁶ spermatozoa/ml and a rate of agglutination below 2 should provide the best quality after freezing. This study also attempted to determine whether a positive effect of ejaculate selection on the overall freezing performance might be expected.