Summary A total of 57 Indian Sahiwal (Bos indicus) cattle and 53 Murrah and 19 Nili‐Ravi (Bubalus bubalis) buffalo were screened using PCR/RFLP to detect the following alleles at growth hormone (GH) and prolactin (PRL) loci: leucine/valine (L/V) for GH/AluI (amino acid 127), (+/+) for GH/MspI (intron III), and A/B for PRL/RsaI (amino acid 103). Among Sahiwal cattle, gene frequencies were 0.96/0.04 (GH/AluI), 0.86/0.14 (GH/MspI), and 0.49/0.51 (PRL/RsaI). In buffalo, animals of both breeds were homozygous for GH loci (LL, ++). In Egyptian buffalo, 11 samples were also monomorphic, indicating that monomorphism at these two loci may be a species characteristic of buffalo. PRL/RsaI frequencies were 0.93/0.07 and 0.84/0.16 in Murrah and Nili‐Ravi buffalo, respectively. Zusammenfassung Bei insgesamt 57 indischen Sahiwal (Bos indicus) und 53 Murrah und Nili‐Ravi Büffeln (Bubalus bubalis) wurde mit PCR/RFLP auf die folgenden Polymorphismen des Wachstumshormon‐ (GH) und Prolaktingens (PRL) untersucht: L/V für GH/AluI (Austausch Leucin/Valin der Aminosäure 127), (‐/+) für GH/MspI (Intron III) und A/B für PRL/RsaI (Aminosäure 103). Bei Sahiwal waren die Genfrequenzen 0,96/0,04 (GH/AluI), 0,86/0,14 (GH/MspI) und 0,49/0,51 (PRL/RsaI). Bei den Büffeln waren die Tiere beider Rassen — wie auch 11 ägyptische Büffel — homozygot für die GH Loci (LL, ++), was darauf hinweist, daß Büffel monomorph an beiden Schnittstellen sind. Die Frequenzen für PRL/RsaI betrugen 0,93/0,07 und 0,84/0,16 bei den Murrah and Nili‐Ravi Büffeln.

We screened 57 Sahiwal cattle (Bos indicus) and 53 Murrah, 19 Nili-Ravi and 11 Egyptian buffaloes (Bubalus bubalis) to detect the polymorphisms at kappa-casein (CSN3) gene using the polymerase chain reaction (PCR). CSN3 A and B alleles were identified by PCR-RFLP using the restriction enzymes that detect the underlying nucleotide changes at codon 136 (Taql) and at codon 148 (HindIII or HinfI) in cattle. The frequency of CSN3 B allele in the Sahiwal cattle was estimated as 0.16 with no homozygous BB animal. Using the same set of primers as used in the Sahiwal cattle, a part of exon IV of buffalo CSN3 gene was amplified, but restriction enzyme analysis using HindIII/HinfI and TaqI did not reveal any polymorphism. However, DNA sequencing of the amplified fragment (GenBank Acc. No. U96662) revealed one polymorphism at codon 135 (ThrACC -->I1eATC) in buffalo; the frequencies of 135 Thr/Ile alleles were estimated as 0.88 and 0.12, respectively.

No abstract

No abstract

Purpose To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat. Methods The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR). Results The viability of isolated testicular cells was>90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers. Conclusion Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats.Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.

The variation in the kidding size of Black Bengal and Sirohi breed of goats makes them an interesting genetic material to study the underlying genetic mechanism of prolificacy. Accordingly, we studied the comparative ovarian morphometry including disparity in numbers of antral follicles of different sizes between these two breeds. Further, we evaluated the differential expression of the important candidate genes (viz., BMP15, GDF9 and BMPR1B) known to influence the ovulation rates and the prolificacy. The ovaries of Black Bengal (n = 20) goat were lighter (p < 0.01) in weight and smaller (p < 0.01) in diameter than those of Sirohi (n = 19) goats but possessed more numbers (p < 0.05) of corpus luteum (CL), large and small antral follicles. Quantitative real-time PCR (RT-qPCR) analysis revealed differential expression of mRNAs encoding for the BMP15 and GDF9. Small antral follicles of Black Bengal goats expressed 2.78-fold more (p < 0.05) of BMP 15 than those of Sirohi goat. Expression of BMP15 (p < 0.01) and GDF9 (p < 0.05) mRNAs was more abundant in the small than the large antral follicles of Black Bengal goat. The more numbers of antral follicles per unit of ovarian mass and differential expression of BMP15 and GDF9 may serve as an important clue for higher prolificacy.

Mycobacterium fortuitum causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

No abstract