Reproductive Biotechnology and Gene Mapping: Tools for Conserving Rare Breeds of Livestock

Today's livestock diversity originated from the wild ancestor species and was subsequently shaped through the processes of mutation, genetic drift, and natural and human selection. Only a subset of the diversity present in the ancestral species survives in the domestic counterparts. A 2007 report released by UN Food and Agriculture Organization ' The State of the World's Animal Genetic Resources ', compiled from surveys conducted in 169 countries, found that nearly 70% of the world's remaining livestock breeds live in developing countries. The UN report was presented to more than 300 policy makers, scientists, breeders, and livestock keepers at the First International Technical Conference on Animal Genetic Resources, held in September 2007 in Interlaken, Switzerland. The conference aims were to adopt a global plan of action for conserving animal genetic resources as its main outcome. In this paper, the current and potential contributions of reproductive and molecular biotechnology are considered as tools of conserving rare breeds of livestock.     

Comparative Aspects of the Endotoxin- and Cytokine-Induced Endocrine Cascade Influencing Neuroendocrine Control of Growth and Reproduction in Farm Animals

Disease in animals is a well-known inhibitor of growth and reproduction. Earlier studies were initiated to determine the effects of endotoxin on pituitary hormone secretion. These studies found that in sheep, growth hormone (GH) concentration was elevated, whereas insulin-like growth factor-I (IGF-I) was inhibited, as was luteinizing hormone (LH). Examination of the site of action of endotoxin in sheep determined that somatotropes expressed the endotoxin receptor (CD14) and that both endotoxin and interleukin-Iβ activated GH secretion directly from the pituitary. In the face of elevated GH, there is a reduction of IGF-I in all species examined. As GH cannot activate IGF-I release during disease, there appears to be a downregulation of GH signalling at the liver, perhaps related to altered nitration of Janus kinase (JAK). In contrast to GH downregulation, LH release is inhibited at the level of the hypothalamus. New insights have been gained in determining the mechanisms by which disease perturbs growth and reproduction, particularly with regard to nitration of critical control pathways, with this perhaps serving as a novel mechanism central to lipopolysaccharide suppression of all signalling pathways. This pathway-based analysis is critical to the developing novel strategies to reverse the detrimental effect of disease on animal production.     

Epidemic of blindness in kangaroos - evidence of a viral aetiology

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.     

Foraging under uniform risk from different types of predators

Background  

Many animals live in environments where different types of predators pose a permanent threat and call for predator specific strategies. When foraging, animals have to balance the competing needs of food and safety in order to survive. While animals sometimes can choose between microhabitats that differ in their risk of predation, many habitats are uniform in their risk distribution. So far, little is known about adaptive antipredator behavior under uniform risk. We simulated two predator types, avian and mammalian, each representing a spatially uniform risk in the artificial resource landscapes. Voles served as experimental foragers.     

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.     

Significance of Non‐conventional Parameters in the Evaluation of Cooling‐induced Damage to Ram Spermatozoa Diluted in Three Different Media

The objectives of this study were two. First, to compare three base media with different sugar composition as an initial step to achieve a good chemically‐defined extender for ram sperm refrigeration. The second one, to determine which sperm quality parameters may be more useful for revealing differences between sperm samples. One medium contained 200 mm sucrose and 2.8 mm glucose (SM), another only disaccharides (D) such as sucrose, trehalose, maltose and lactose (75 mm each); and the third one (D+M) included a mix of monosaccharides (50 mm glucose, 20 mm fructose and 20 mm galactose,) and the same disaccharides as in D (50 mm each). Ram semen samples diluted in the mentioned media were refrigerated at 5°C for 1 h, and rewarmed upto 37°C in order to mimic the temperature in the female reproductive tract. Addition of monosaccharides to the extender did not produce a better preservation of motility or viability after cooling. The supplementation with other disaccharides apart from sucrose did not enhance the viability either. Thus, after cooling and rewarming, there were no significant differences in sperm viability (membrane integrity evaluated by CFDA/PI staining) or the percentage of progressive motile and rapid sperm (evaluated by CASA) between the three media. However, the percentage of viable non‐capacitated sperm evaluated by the chlortetracycline (CTC) assay was higher and sperm oxygen consumption was lower in SM than in D and in D+M. Although the apoptosis‐like markers [phosphatidylserine exposure assessed by Annexin V/CFDA staining and DNA‐damage evaluated by TUNEL assay] showed a continuous increment throughout the process with all diluents, the percentage of sperm with damaged DNA at the end of the process was significantly lower in SM than in the other two media (p < 0.01). On the basis of these results, we would make two recommendations: the use of an extender supplemented only with sucrose and glucose for ram sperm refrigeration; the inclusion of non‐conventional methods such as oxygen consumption measure, evaluation of capacitation state and apoptosis‐like markers for revealing differences between sperm samples.     

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.     

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.     

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.