Identification of lactoferrin and glutamate receptor‐interacting protein 1 in bovine cervical mucus: A putative marker for oestrous detection

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous‐specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non‐oestrous and controlled internal drug release (CIDR)‐induced oestrous‐stage mucus proteins were purified and subjected to surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI‐TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor‐interacting protein 1 (GRIP1) showed a twofold increase during the CIDR‐induced oestrous stage compared to the levels in non‐oestrous stage in bovine cervical mucus. The RT‐PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non‐oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.     

An Inter‐Subspecies Cloned Buffalo (Bubalus bubalis) Obtained by Transferring of Cryopreserved Embryos via Somatic Cell Nuclear Transfer

The aim of this study was to explore the feasibility of cryopreservation of inter‐subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum‐starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross‐bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13‐month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter‐subspecies cloned embryos is feasible to produce buffalo offspring.     

Prevalence of BVDV infection in Greek dairy herds

Thirty-nine Greek dairy herds, totalling 6333 cattle, enrolled in a voluntary bovine viral diarrhoea virus (BVDV) eradication programme based on the identification and removal of persistently infected (PI) animals. The aim of this study was to estimate the prevalences of BVD antigen-positive and PI animals, and investigate the significance of the associations between the prevalence estimates and herd size. Initially, all animals were bled and examined for BVDV, using an antigen ELISA. A second sample was collected from the positive animals, after a period of at least three weeks. Animals retested positive were classified as PI. Antigen positive and PI animals were detected in all herds. The respective mean prevalences, adjusted for the test's accuracy and the herd-clustering effect, were 14% (95%CI: 11-18%) and 1.3% (0.8-1.8%), respectively. Herd size was not associated with the prevalence of antigen-positive or PI animals.     

A matched case-control study of factors associated with retention of fetal membranes in dairy ewes in Southern Greece

We defined retention of fetal membranes (RFM) in dairy ewes as failure to expel the placenta within 6h after lambing the last lamb and designed a matched case-control study to identify factors that affect the risk of retention. For each ewe with RFM, the next ewe in the flock that lambed and expelled the placenta in <6h after lambing the last lamb was selected as control. Data analyzed included 92 pairs of ewes from 25 flocks comprising a total of 7275 ewes (median flocksize 270 ewes). Factors investigated for associations with RFM were induction of lambing, obstetrical assistance because of dystocia, parity, the number of liveborn lambs, the occurrence of stillbirth(s), the mean weight of the litter on the third day post-lambing and the occurrence of neonatal death in the litter. Conditional logistic regression indicated (1) that the risk of RFM increased linearly with increasing number of liveborn lambs and (2) that the risk of retention was 4-fold higher in ewes that received assistance at lambing than those that lambed normally.     

Incidence risk and clinical features of retention of foetal membranes in ewes in 28 flocks in southern Greece

In a field investigation of 28 flocks in southern Greece, 7660 lambings were monitored. Retention of foetal membranes (defined as failure to expel the foetal membranes within 6 h of lambing the last lamb) was recorded in 92 ewes. The median within-flock incidence risk was 1.25% (range: 0-1.9%). The heterogeneity of the risk among flocks was not significant (p = 0.99); no correlation was found between the incidence risk and the flock size (r(sp) = 0.33, p = 0.09). Overall, the median duration of retention was 72 h (range: 9-288 h); it did not differ among flocks (p = 0.89) and was not correlated with flock size (r(sp) = 0.24, p = 0.27). During the initial stage of retention the membranes were fleshy and humid; the cotyledons were thick and congested. Progressively, the membranes became thin, dry and stringy; the cotyledons shrunk and were pale. Finally the membranes dropped as a mass. In ewes with retention for >12 h, a variety of accompanying signs was recorded: straining (in 22% of the ewes with retention), vulval oedema and reddening (in 16%), anorexia (in 13%), recumbency (in 13%) and increased temperature (in 12%) were the most frequent ones. Overall, the median clinical score of the disorder was '2' (range: '1'-'5'); it did not differ among flocks (p = 0.98) and was not correlated with flock size (r(sp) = 0.26, p = 0.25). In 4% of the ewes with retention for <4 days accompanying signs were recorded (median clinical score: '2'), whilst in 80% of the ewes with retention for > or =4 days accompanying signs were recorded (median clinical score: '3'). This difference in the prevalence of clinical signs was significant (p < 0.0001).     

Follicle Ablation Improves the Ovarian Response and the Number of Collected Embryos in Superovulated Cows During the Early Stages of Lactation

A field study was designed to compare ovarian response and embryo yield in cows during early lactation when gonadotropin administration followed one of four treatments. In group 1A (n = 19) and 1B (n = 9), the estrouses were synchronized by two prostaglandin F2alpha (PG) injections given 11 days apart, and starting from day 9 of the synchronized cycle superovulation was conducted with eight decreasing dose of FSH. In group 1B, ablation of all follicles >3 mm was carried out on day 8. In group 2A and 2B (each n = 9), a progesterone plus oestradiol intravaginal device (PRID) was inserted for 11 days and gonadotropin administration started on day 9, while cows from group 2B had a follicle ablation on day 8. In all groups, two PG injections were given along with the sixth and the seventh dose of FSH, and the cows were twice inseminated 12 and 24 h after estrus detection. Embryos were collected on day 7. In cumulative results from aspirated and non-aspirated cows, follicular ablation significantly improved: the ovarian response (10 +/- 1.23 vs 6.69 +/- 0.60 corpora lutea per donor), the mean collected embryos (6.57 +/- 0.94 vs 2.46 +/- 0.53) and the mean transferable embryos (4.43 +/- 0.89 vs 2.18 +/- 0.47). Group 1B and 2B cows had better ovarian response than 1A (6.44 +/- 0.81, 12.25 +/- 4.11 and 9.44 +/- 0.93, for groups 1A, 1B and 2B, respectively, p < 0.05). Similarly, from groups 1B and 2B more (p < 0.05) embryos were collected in comparison with their respective group, while the mean transferable embryos from group 2B (5.22 +/- 1.13) was greater (p < 0.05) than that of group 1A (1.67 +/- 0.35), and tented to be greater than those of groups 2A (3.44 +/- 1.19, p = 0.062) and 1B (3.00 +/- 1.78, p = 0.066). The highest (p < 0.05) transferable embryo collection rate was recorded in group 2B (55.29%), followed by that of group 1B (41.33%). In summary, early in lactation, an acceptable number of transferable embryos can be collected from high producing dairy cows, when follicle ablation prior to superovulation is combined with progesterone and oestradiol administration.     

Genetic diversity and thermotolerance in Holstein cows: Pathway analysis and marker development using whole-genome sequencing

Heat stress causes extensive losses in the dairy sector, due to negative effects on milk production and reproduction. Cows have evolved a series of protective mechanisms, (physiological, biochemical, behavioural) to cope with the thermostressing environments, which have allowed the preservation of productive and reproductive potential of specific animals during summer; these animals are considered thermotolerant and could be used to design programs of selective breeding. These programs, targeting the generations of a population of heat-resistant animals, would increase the frequency of the desired phenotypes, tackling the financial losses on one hand and reducing the carbon footprints of the dairy sector on the other. The development of genomics techniques has enabled genome wide variant calling, to detect SNPs associated with the desired phenotypes. In this study, we used a comparative genomics approach to detect genetic variation associated with thermotolerance and to design molecular markers for characterizing the animals as tolerant/sensitive. A total of 40 cows from each group were split in four sequencing pools and a whole-genome sequencing approach was used. Results and conclusion: Genome-wide genetic variation between groups was characterized and enrichment analysis revealed specific pathways which participate in the adaptive mechanisms of thermotolerance, implicated into systemic and cellular responses, including the immune system functionality, Heat Stress and Unfolded Protein Response. The markers made a promising set of results, as specific SNPs in five genes encoding for Heat Shock Proteins were significantly associated with thermotolerance.     

Genetic Diversity Based on AIIozyme Alleles of Chinese Cultivated Rice

Genetic diversity was analyzed with 6 632 core rice cultivars selected from 60 282 Chinese rice accessions on the basis of 12 allozyme loci, Pgil, Pgi2, Amp1, Amp2, Amp3, Amp4, Sdhl, Adhl, Estl, Est2, Est5 and Est9, by starch gel electrophoresis. Among the materials examined, 52 alleles at 12 polymorphic loci were identified, which occupied 96.3% of 54 alleles found in cultivated germplasm of O. sativa L. The number of alleles per locus ranged from 2 to 7 with an average of 4.33. The gene diversity （He） each locus varied considerably from 0.017 for Amp4 to 0.583 for Est2 with an average gene diversity （Ht） 0.271, mid Shannon-Wiener index from 0.055 to 0.946 with an average of 0.468. The degree of polymorphism （DP） was in a range from 0.9 to 46.9% with an average of 21.4%. It was found that the genetic diversity in japonica （Keng） subspecies was lower in terms of allele＇s number, Ht and S-W index, being 91.8, 66.2 and 75.7% of indica （Hsien） one, respectively. Significant genetic differentiation between indica and japonica rice has been appeared in the loci Pgil, Amp2, Pgi2, and Est2, with higher average coefficient of genetic differentiation （Gst） 0.635, 0.626, 0.322 and 0.282, respectively. Except less allele number per locus （3.33） for modern cultivars, being 76.9% of landraces, the Ht and S-W index showed in similar between the modem cultivars and the landraces detected. In terms of allozyme, the rice cultivars in the Southwest Plateau and Central China have richer genetic diversity. The present study reveals again that Chinese cultivated rice germplasm has rich genetic diversity, showed by the allozyme allele variation.