Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

Phosphorus uptake and use efficiency in different varieties of bread wheat (Triticum Aestivum L)

Thirty spring wheat varieties were evaluated and classified into eight different groups on the basis of their grain yield performance and phosphorus (P) uptake using Metroglyph analysis. Significant variability was observed for grain and biomass yield, plant height, P content in grain and straw, total P uptake and phosphorus harvest index and P use efficiency traits. Varieties WH 711 and PBW 343 exhibited high grain yield as well as high P uptake (HGY-HP). WH 283 and UP 2425 with high index score of 19 and 16 respectively, constituted the high grain yield-medium P uptake (HGYMP) group. Both these varieties, though had similar grain yield of 5348 kg/ha, but WH 283 (12.64 kg/ha) utilized much lower P as compared to UP 2425 (16.94 kg/ha). Moreover, WH 283 (81.64) also showed higher values for phosphorus harvest index (PHI) than UP 2425 (67.88%). P uptake of WH 283 was comparable with that of Raj 3765 (10.78 kg/ha) and grouped into high grain yield and low P uptake (HGY-LP) group. The grain yield performance of these two varieties with a relatively low P uptake is reflected in their high index score for P use efficiency thus, earmarking them for low P regimes. Variety HW 2006, despite low grain yields of 4665 kg/ha had high index score of 16 due to its higher value for Phosphorus Biological (PBER) and Economic Yield (PEER) Efficiency Ratio as it has effected least (7.18 kg/ha) P mobilization. In addition high P translocation in the grain was also observed for this variety. Inter-mating of genotypes like HW 2006, UP 2338 and HW 2016 with those belonging to HGY-HP (PBW 343 and WH 711) and HGY-LP (Raj 3765 and WH 283) would be an ideal strategy to develop the cultivars for efficient phosphorus use.     

Positive Effects of Melatonin Treatment on the Reproductive Performance of Young Border Leicester Rams Mated to Merino Ewes in Spring: Preliminary Observations

Poor reproductive performance of Merino ewe flocks when mated to Border Leicester rams during spring may be due to seasonality of the Border Leicester breed. Two approaches were taken to test this assumption. Six young (12 months old) or six mixed‐age (12, 24 and ≥36 months old) Border Leicester rams were either treated or not treated with melatonin implants (2 × 2 design) 6 weeks before the four groups of rams were each put with approximately 300 Merino ewes for an 8‐week mating period. Implants were inserted in early September (experiment 1). The second approach was to yard or not yard ewes and mixed‐age rams on several occasions during the first 3 weeks of the mating period (experiment 2). Pregnancy rate and twinning percentage were assessed by ultrasonography. In experiment 1, melatonin treatment in young rams increased (p < 0.001) pregnancy rate from 5.0% to 92.6%, but mixed‐age rams did not respond (90.7% vs 89.5% for melatonin and non‐melatonin treatments, respectively). Twinning rate was similar (p > 0.05) for ewes mated to either melatonin or non‐melatonin‐treated young rams (36.8% vs 40.0%, respectively), whereas melatonin significantly improved (p < 0.05) twinning rate in those ewes mated to mixed‐age rams (49.1% vs 36.1%). After 6 weeks of melatonin treatment, scrotal circumference was greater (p < 0.05) in both young and mixed‐aged rams than in untreated counterparts. In experiment 2, yarding of ewes and rams overnight on several occasions early in the mating period reduced (p < 0.001) pregnancy rate compared with non‐yarded counterparts (89.5% vs 65.5%). Twinning rate was not affected (37.7% vs 36.1%, respectively). In summary, melatonin treatment of Border Leicester rams significantly improved flock reproductive performance in spring due to improved pregnancy rates with young rams and improved litter size with mixed‐age rams.     

Caudal anaesthesia of the infraorbital nerve for diagnosis of idiopathic headshaking and caudal compression of the infraorbital nerve for its treatment,in 58 horses

Reasons for performing study: Idiopathic headshaking is often a facial pain syndrome, but a diagnostic protocol has not been described. In a previous study, caudal compression of the infraorbital nerve for treatment offered a fair success rate, but low case numbers and short follow‐up time were limitations. Objectives: To describe a diagnostic protocol for headshaking, examining the role of bilateral local analgesia of the posterior ethmoidal nerve (PET block). To report longer‐term follow‐up after surgery of the original cases and further cases and to determine whether changes to the technique influence success rates and complications. Methods: Records of horses that had undergone PET block and caudal compression surgery at 3 hospitals were reviewed. Modifications to the surgical technique included placing additional coils into the infraorbital canal and/or performing concurrent laser cautery of the nerve. Follow‐up information was obtained by telephone contact with owners. Results: The PET block was performed in 27 horses, with a positive result in 23 of 27 (85%). Surgery was performed in 58 horses. A successful outcome was initially achieved in 35 of 57 (63%) horses, but recurrence occurred between 9 and 30 months later in 9 (26%). Surgery was repeated in 10 of 31 (32%) horses. Final success rate, considering only response to the last performed surgery, was 28 of 57 (49%) horses with median follow‐up time of 18 months (range 2–66 months). Nose‐rubbing was reported post operatively in 30 of 48 (63%) horses. This resolved in all but 4 horses, which were subjected to euthanasia. Response to PET block or change in surgical technique did not appear to influence outcome or complications. Conclusions and potential relevance: The diagnostic protocol described is recommended for the investigation of headshakers. Caudal compression offers the best prognosis for a successful outcome compared with other treatments, for horses in which the only alternative is euthanasia. Surgical treatment of the disorder requires refinement, and the pathogenesis of the disorder requires investigation.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

Relationship between endometritis and oxidative stress in the follicular fluid and luteal function in the buffalo

In this study, alteration in the follicular fluid composition and luteal function was investigated in the buffalo with endometritis. Genitalia were classified into cytological and purulent endometritis on the basis of polymorphonuclear cell cut off while non‐endometritis served as control (n = 10/group). In the follicular phase, the number of surface follicles was counted, diameter of the largest follicle was measured and the follicular fluid was assayed for total protein, cholesterol, malondialdehyde (MDA), total antioxidant capacity (TAC), oestradiol (E₂) and progesterone (P₄). The P₄ content of corpus luteum during mid‐luteal phase was estimated by radioimmunoassay. Ovaries from the follicular phase of oestrous cycle showed no significant difference in the total number of surface follicles, size of the largest follicle and volume of follicular fluid in the buffaloes with and without endometritis (p > .05). However, the antral fluid of the largest follicle from the genitalia of buffalo with cytological and purulent endometritis showed a significant decrease in the concentration of total protein, cholesterol, TAC and E₂ and a significant increase in the concentration of MDA and P₄ (p < .05). The results indicated that there is an association between endometritis and decreased ovarian function.     

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.     

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

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.