Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p ≤ 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p ≤ 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.     

Electrocardiographic values in Spanish-bred horses of different ages

SUMMARY The duration of electrocardiograph wave forms and intervals were determined in 179 Spanish-bred (Andalusian) horses aged from 1 month to 17 years. The values were compared with those of other breeds, and the relationship between electrocardiographic data and age was examined. High correlation coefficients were found between PR, ST and QT intervals and the age of the horses, and an inverted relation between heart rate and age was found. A multiple range analysis was made and the results suggest that significant changes in duration values and heart rate ocurred at the age of 6 months and in the second year of life.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

不同区域奶牛子宫内膜炎病原菌的分离鉴定及分布特征

【目的】对中国6个区域奶牛子宫内膜炎病原菌的种类和区域分布特征进行研究,为筛选出最佳治疗药物以减少子宫内膜炎对畜牧业造成经济损失提供数据支持。【方法】分别从赤峰区域、张家口区域、潍坊区域、重庆区域、宝鸡区域和北京区域选择不同规模的奶牛养殖场中患子宫内膜炎的奶牛,用采样器每个区域采集60份(6个区域共计360份)患病牛的子宫内容物,通过革兰氏染色和生化鉴定的方法鉴定分离纯化的奶牛子宫内膜炎病原菌,并分析病原菌的分布特征。【结果】潍坊、赤峰、北京区域引起奶牛子宫内膜炎以单一感染为主。宝鸡、张家口、重庆区域引起奶牛子宫内膜炎以混合感染为主。赤峰、重庆、张家口、潍坊、宝鸡、北京区域引起奶牛子宫内膜炎的病原菌以革兰氏阳性菌为主。赤峰奶牛子宫内膜炎的主要致病菌是葡萄球菌。重庆奶牛子宫内膜炎的主要致病菌是链球菌。张家口奶牛子宫内膜炎的主要致病菌是大肠杆菌。潍坊奶牛子宫内膜炎的主要致病菌是链球菌。宝鸡奶牛子宫内膜炎的主要致病菌是葡萄球菌。北京奶牛子宫内膜炎的主要致病菌是蜡样芽孢杆菌。     

Extended day length in late winter/early spring,with a return to natural day length of shorter duration,increased plasma testosterone and sexual performance in rams with or without melatonin implants

Sixteen rams were used to quantify the effects of long days, imposed during late winter/early spring, with or without exogenous melatonin, on plasma testosterone concentrations and ram serving capacity. Rams were assigned to two groups: photoperiod‐treated rams (Artificial Photoperiod, AP; n = 8), exposed to 2 months of long days (16 hr of light/day) between 22 December and 22 February, and control rams (Natural Photoperiod, NP; n = 8). At the end of the long‐day period, AP rams were returned to the natural photoperiod, and each ram in the two groups either did (+M) or did not (‐M) receive three subcutaneous melatonin implants. Four groups were created as follows: AP+M (n = 4), AP‐M (n = 4), NP+M (n = 4) and NP‐M (n = 4). Thirty days after of the onset of photoperiodic treatment, AP rams (13.5 ± 2.8 ng/ml) had significantly (p < .05) lower testosterone levels than NP rams (36.7 ± 1.0), and similar differences were not apparent at the end of the photoperiod treatment. A month later, AP rams (24.3 ± 7.9) had higher (p < .10) testosterone levels than NP rams (13.1 ± 5.0), with no effect of melatonin treatment. Fifty days after melatonin implantations, rams were exposed for 20 min to three oestrous ewes. AP rams (2.50 ± 0.42) exhibited significantly (p < .05) more serves than did NP rams (1.11 ± 0.39), and melatonin treatment had no significant effect; however, the interaction between treatments was significant. Time to first serve was significantly (p < .05) shorter in AP (2.30 ± 1.20 min) than it was in NP rams (5.58 ± 0.68 min). In conclusion, exposure to 2 months of long days in late winter/early spring, with a return to natural day length of shorter duration, increased plasma testosterone concentrations and sexual performance in rams with or without exogenous melatonin. This particular management is an option if a non‐hormonal reproductive strategy is scheduled; yet, if the use of exogenous hormones is feasible, melatonin implants increase the mating efficiency of rams.     

Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p < 0.05) of this fatty acid in his sperm plasma membranes.     

Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without signs of reproductive disease

Abstract

AIMS: To obtain information and compare the prevalence of Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without clinical signs of reproductive disease.

METHODS: Vaginal swabs and blood samples were collected from animals attending Governmental Veterinary Clinics without (buffalo n=39, cows n=20) and with (buffalo n=63, cows n=53) signs of reproductive disease. Serum samples were tested for antibodies to Chlamydiaceae using complement fixation testing (CFT). Vaginal swabs were tested for Chlamydiaceae following inoculation into Vero cells and 6-day-old embryonated chicken eggs, using modified Giménez and immunoperoxidase staining, PCR analyses targeting the omp2 gene, and Restriction Fragment Length Polymorphism PCR (RFLP-PCR) for species identification.

RESULTS: Antibodies to Chlamydiaceae were detected in 30/39 (77%) and 50/63 (79%) buffalo without and with signs of reproductive disease, respectively, and 10/20 (50%) and 39/53 (74%) of cows with and without signs of reproductive disease, respectively. Positive samples from PCR analysis were identified in 31/39 (79%) and 37/63 (59%) buffalo without and with signs of reproductive disease, respectively, and 12/20 (60%) and 46/53 (89%) of cows without and with signs of reproductive disease, respectively. Using RFLP-PCR, 57/68 (84%) of samples from buffalo, and 47/58 (81%) from cows, were identified as Chlamydophila psittaci and the reminder as Cp. abortus. From the CFT and PCR results there was no significant difference in the prevalence of positive samples between species, or between animals without or with signs of reproductive disease.

CONCLUSION: The presence of anti-Chlamydiaceae antibodies in 77% of the animals with signs of reproductive disease and the detection of Chlamydiaceae in 72% of vaginal swabs of the animals suggest a pathogenic role by Chlamydiaceae in riverine buffalo and cows. The main Chlamydiaceae found in the genital tract of cattle in Egypt were Cp. psittaci and Cp. abortus.

CLINICAL RELEVANCE: Chlamydophila spp. should be included in diagnostic algorithms for reproductive disorders, in order to assess the real burden of Chlamydophila associated disease in buffalo and cattle and to evaluate the potential value of vaccines.     

Prevalence of Candidatus Mycoplasma haemolamae,bovine viral diarrhoea virus,and gastrointestinal parasitism in a sample of adult New Zealand alpaca (Vicugna pacos)

AIM: To determine the prevalence of infection with Candidatus Mycoplasma haemolamae (Mhl), antibodies to bovine viral diarrhoea virus (BVDV), and BVDV antigen, and the prevalence of animals with elevated faecal nematode egg counts (FEC) in a sample of adult New Zealand alpaca (Vicugna pacos).

METHODS: Blood samples were obtained from 175 alpaca, collected from 15 farms around New Zealand, and from 31 samples sent to a diagnostic laboratory for routine haematology. Blood smears (n=170) were examined microscopically for the presence of haemoplasma, and DNA was extracted from whole blood (n=206) for real-time PCR testing for Mhl. Packed cell volume (PCV) was determined for 193 samples. Serum samples (n=195) were tested for BVDV antibody using ELISA, and for BVDV antigen using a real-time PCR assay. Faecal samples were collected from 143 animals; FEC were measured, and samples pooled for larval culture.

RESULTS: No haemoplasma organisms were present on blood smear examination. Of the 206 blood samples, two (from the same farm) were positive for Mhl by real-time PCR testing, giving a prevalence of infection with Mhl of 0.97%. Of the 195 serum samples tested, four (2.1%) were positive for antibodies to BVDV; animals with BVDV antibodies were from 3/15 (20%) farms, none of which farmed cattle. None of the serum samples were positive by PCR for BVDV antigen. The median FEC was 50?epg (min 0, max 4,700), with 55/143 (38.5%) samples having 0?epg, and 33/143 (23.1%) having ≥250?epg. Haemonchus spp. were the most common nematodes present in faecal larval cultures from the North Island. Log₁₀ FEC was negatively associated with PCV (p=0.02), and was higher in males than females (p<0.001), and in animals that were positive compared with negative for Mhl (p=0.022).

CONCLUSIONS AND CLINICAL RELEVANCE: The number of alpaca infected with Mhl was low, as was the seroprevalence of BVDV. Gastrointestinal parasitism was, however, a common finding in this sample of New Zealand alpaca.