Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart.

This study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5-8 mm, third group 9-15 mm and fourth group 16-20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5-8 mm, 9-15 mm and 16-20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

Establishment a protocol for total RNA isolation from buffalo fresh and frozen semen for molecular applications.

To date, there is no an established protocol for total RNA isolation in Egyptian buffalo spermatozoa. The present study aimed (I) to establish a defined protocol for total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality and quantity from different extraction methods and studying gene expression. Warm and standard room temperature modified QIAzol Lysis Reagents were used for total RNA extraction. The quality and quantity of extracted RNA were checked, and subsequently qRT-PCR was performed using androgen receptor-like and three reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/µl) and frozen spermatozoa (110.59 ± 4.43 ng/µl), compared to standard room temperature modified QIAzol (421.26 ± 7.18 ng/µl) and (29.07 ± 5.25 ng/µl), for fresh and frozen semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and frozen isolated semen by warm method respectively. The integrity of RNA was good and appeared as a sharp band on 2% agarose gel. The most stable reference gene was 18S. Reliable extraction method of high quality RNA yield could be a step forward for understanding mechanisms of spermatogenesis for improving male fertility.

PCR-RFLP of bone morphogenetic protein 15 (BMP15/FecX) gene as a candidate for prolificacy in sheep.

Bone morphogenetic protein 15 (BMP15/FecX) gene is considered one of the major genes and a candidate marker for the reproduction in farm animals, especially sheep. The present study aimed to detect the genetic polymorphisms of BMP15 gene in sheep using PCR-RFLP technique. In the present study, 115 ewes were assigned into high and low prolificacy categories according to their reproductive history. In high prolific group (n = 20), ewes produced twins more than single births. In the low prolific type (n = 95), the ewes produced single births more than twins. DNA was extracted from blood samples of all ewes, subjected to PCR-RFLP analysis and confirmed by sequence analysis. The PCR products of 356 bp size were cut with HinƒI restriction enzyme. Three digested fragments of 70, 117 and 169 bp were obtained in both types of sheep. All animals were homozygous with CC genotype. In conclusion, the accessible findings did not detect any mutation in FecX gene in sheep, regardless their prolificacy. Therefore, further attempts are necessary to detect other SNP for BMP-15 gene in Egyptian sheep breeds.