Chondrodysplasia in Australian Dexter cattle

Objective To describe the occurrence of chondrodysplasia in Australian Dexter cattle.
Design A pathological and genetic case report.
Procedure Congenital lethal chondrodysplasia was studied in two female Dexter foetuses aborted mid to late gestation. Clinicopathological findings including histological changes in limb bones, and analysis of pedigree information were evaluated.
Results Characteristic features of congenital lethal chon-drodysplasia (Dexter bulldog) include abortion, disproportionate dwarfism, a short vertebral column, marked micromelia, a relatively large head with retruded muzzle, cleft palate and protruding tongue and a large abdominal hernia. Histological changes in limb bones are consistent with failure of endochondral ossification. Dexter chondrodysplasia is considered to be inherited in an incompletely dominant manner with the homozygous form producing the congenital lethal condition. A preliminary minimum estimate of heterozy-gote frequency is 19% within the registered Australian Dexter herd, based on analysis of the contribution of three obligate heterozygotes whose semen has been widely used by artificial insemination in Australia.
Conclusion Dexter chondrodysplasia is present in Australian cattle and further cases of the homozygous form, congenital lethal chondrodysplasia, are likely to occur.
Recommendation It is requested that spleen and liver tissue from bulldog foetuses and blood from their parents be collected to assist research into Dexter chondrodysplasia.     

The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.     

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll^®, Puresperm^® and Bovipure^?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure^? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll^® and Puresperm^® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll^® selected less live and more apoptotic spermatozoa than Puresperm^® and Bovipure^? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

Use of Vaginal Electrical Resistance to Diagnose Oestrus,Dioestrus and Early Pregnancy in Synchronized Tropically Adapted Beef Heifers

The primary objective of this study was to determine whether a single measurement of intravaginal electrical resistance (VER), using the commercially available Ovatec^® probe, can discriminate between dioestrus and oestrus in Bos indicus females, which had been treated to synchronize oestrus. Santa Gertrudis heifers (n = 226) received one of three oestrous synchronization treatments: double PGF_2α 10 days apart, 8‐day controlled internal drug release (CIDR) treatment or CIDR pre‐synchronization + PGF_2α 10 days after CIDR removal. The heifers were inseminated within 12 h following observed oestrus, or, if not observed, at a fixed time approximately 80 h, following the last synchronization treatment. They were palpated per rectum for signs of pregnancy 9 weeks after artificial insemination (AI). Vaginal electrical resistance measurements were taken at the completion of synchronization treatments (presumed dioestrus), immediately prior to AI (oestrus), and then at 3 and 9 weeks post‐AI. Mean VER differed between presumed dioestrus and oestrus (113.7 vs 87.4, p < 0.001). The area under the receiver operating characteristics (ROC) curve was 0.925, indicating that VER was highly discriminatory between dioestrus and oestrus. Vaginal electrical resistance at time of AI was negatively associated with odds of conception when all inseminations were included in the analyses [odds ratio (OR) = 0.97; 95% CI 0.95–1.00; p = 0.018], but not when fixed time AIs were excluded (OR = 1.00; 95% CI 0.97–1.03; p = 0.982). Mean VER readings differed between pregnant and non‐pregnant animals at both 3 weeks (120.5 vs 96.7, p < 0.001) and 9 weeks (124.0 vs 100.3, p < 0.001) post‐AI. However, 3‐ and 9‐week VER measurements were not highly discriminatory between pregnancy and non‐pregnancy (area under ROC curve = 0.791 and 0.736, respectively). Mean VER at time of AI for animals diagnosed in oestrus differed between each of the oestrous synchronization treatments (84.7, 73.6 and 78.9, groups 1–3 respectively, p < 0.001). These findings suggest that measurement of VER may improve accuracy of oestrus diagnoses when selecting cattle for AI following oestrous synchronization programmes involving tropically adapted cattle.     

Does the Amount of Progesterone in Intravaginal Implants Used to Synchronise Oestrus Affect the Reproductive Performance of Brahman Heifers Artificially Inseminated at a Fixed Time

The study tested the hypothesis that reduced intravaginal implant progesterone (P₄) concentration to synchronise oestrus would increase pregnancy rates to fixed‐time artificial insemination (FTAI) in Bos indicus heifers. Brahman heifers (n = 294; 2 year) were body condition scored (BCS), weighed and scanned for presence of a corpus luteum (CL). Only cyclic heifers were selected and allocated randomly within BCS and 25 kg bodyweight category to one of three P₄ treatment groups. On day 10, heifers received a P₄ implant (CueMate‐1‐pod, 0.78g P₄; CueMate‐2‐pod, 1.56g P₄; or CIDR‐B, 1.9g P₄), 2 mg oestradiol benzoate (ODB) intramuscularly (IM) and 250 ug cloprostenol IM. At day 2, the implant was removed, 250 ug cloprostenol was injected IM and tail paint applied. The heifers received 1 mg ODB 24 h later and were FTAI 48–54 h after implant removal (day 0). Ten randomly selected heifers per group were blood sampled and scanned at days 10, 2, 0 and 6 to define the P₄ profiles pre‐ and post‐FTAI. Heifers were heat‐detected 18–20 days post‐FTAI and oestrous heifers AI’d by the AM/PM rule. Bulls joined the heifers on day 27 post‐FTAI. Transrectal ultrasonography estimated conception date on day 72. Statistical analysis examined the effects of treatment, technician, semen, ovarian status, BCS and liveweight, on pregnancy rate (PR) to FTAI. There was no significant difference (p = 0.362) in PR between treatment groups (CueMate 1‐pod, 36.4%; CueMate 2‐pod, 39.6%: CIDR‐B, 28.3%), but PR was higher in those heifers with increased BCS between FTAI and pregnancy diagnosis (p = 0.005). Thirty‐three per cent of monitor heifers had plasma P₄ concentrations of <1 ng/ml on day 6 after FTAI; only 20% of these conceived vs 60% of heifers with P₄ ≥ 1 ng/ml. In summary, no significant difference in PR was identified between treatments but good BCS and a rising plane of nutrition were critical to PR of these pure grade Brahman heifers in northern Australia.     

Overexpression of a fungal laccase gene induces nondehiscent anthers and morphological changes in flowers of transgenic tobacco

Laccases play important roles in the development of fruiting bodies and in lignin degradation by basidiomycetes. In this study, we present novel phenotypes of transgenic tobacco plants with a chimeric gene for fungal laccase under the control of the cauliflower mosaic virus 35S promoter. At the flowering stage, the transgenic plants that produced recombinant laccase had brownish anthers instead of the greenish anthers of wild-type plants. The brownish anthers exhibited male sterility with a nondehiscent phenotype at varying frequencies. The frequency of nondehiscence depended on the temperature at which plants were cultivated and it was higher at 24°C than at 29°C. The cell wall structures of transgenic anther tissues were almost the same as in the wild type, but the stomium was severely deformed, and abnormal components were apparent in cells of the endothecium and epidermis. Furthermore, the pattern of deposition of flavonoids in the transgenic anther epidermis differed from the wild-type pattern. The expression of laccase also induced other phenotypic changes in the flowers of transgenic plants, namely, increased petal number, fused and petaloid stamens, and doubling of floral organs. These results indicate that the ectopic expression of laccase influences various aspects of flower development.     

Antisperm antibodies in repeat‐breeding cows: Frequency,detection and validation of threshold levels employing sperm immobilization,sperm agglutination and immunoperoxidase assay

Antisperm antibodies have been found in repeat‐breeding(RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies (ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization (SIT), sperm agglutination (SAT) and immunoperoxidase (IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat‐breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher (p < .05) in RB cows compared to other groups. The SIT assay was able to identify 61% of RB caused by ASA, more than those employing SAT and IPT. Furthermore, a dilution rate of 1:5 and 1:80 (confirms 59.0 and 57.0% RB+ve)were sufficient to diagnose ASA by SAT and IPT, respectively. Results indicate the presence of __12.6% clumped spermatozoa and __ 2.6%(cut‐off value) peroxidase‐positive spermatozoa at 1:5 and 1:80 dilutions diagnosed with SAT and IPT, respectively, may be considered as repeaters arising out of ASA. Furthermore, study also showed the presence of lower incidence of ASA positivity in other groups of animals (heifer<normal breeder<pregnant animals) compared to repeaters. Study results show that although IPT is more specific and accurate but SAT and SIT are comparatively simple and cost‐effective assays suitable for detecting ASA under field conditions and thus can be recommended for screening of repeaters.     

C, N, and noble gas isotopes in grain size separates of presolar diamonds from efremovka

Nanometer-size presolar diamonds from the Efremovka CV3 chondrite were physically separated into several grain size fractions by ultracentrifugation. The coarsest size fraction is the most enriched in carbon-12; the others have broadly similar carbon isotopic compositions. Measurement of noble gases shows that their concentration decreases with decreasing grain size. This effect is attributed to ion implantation. Such an episode could occur in the envelope of a supernova that produced the diamonds, or in interstellar space; in either case, ions with energies above a certain threshold pass completely through the smaller diamond grains without being captured. Concentrations of nitrogen show only minor variations with grain size, indicating a different mechanism of incorporation into the diamonds.