Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo ( Bubalus bubalis )

Ashish, Shende; Bhure, S K; Pillai, Harikrishna; Ramteke, Suman; Kutty, V.; Shruthi, N; Kumar, G.V.P.P.S. Ravi; Mahawar, Manish; Ghosh, SK; Sarkar, Mihir

doi:10.1016/j.theriogenology.2017.01.006

Cited by 10 publications

(6 citation statements)

References 34 publications

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“…UXT and MRPL39 are also two of three best internal control genes in the mammary tissue of yak during the lactation cycle [13], indicating that these two genes were the suitable RGs in yak species. HMBS was considered as the stably expressed gene in immature stages, which was similar to previous findings on the spermatozoa of buffalo during freezing and thawing [43]. Therefore, these genes could be selected as the most appropriate pair of RGs in yak testis development.…”

Section: Discussionmentioning

confidence: 59%

Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

Zhou

Chen

et al. 2020

Animals

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Testis has an important function in male reproduction. Its development is regulated by a large number of genes. The real-time reserve transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a useful tool to evaluate the gene expression levels. However, unsuitable reference genes (RGs) can cause the misinterpretation of gene expression levels. Unfortunately, the ideal RGs for yak testis development are yet to be studied. In this study, 13 commonly used RGs were selected to identify the most stable RGs in yak testis at four different developmental stages, including two immature stages (6 months and 18 months) and two mature stages (30 months and 6 years). This study used GeNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder programs to evaluate the stability of 13 candidate genes. The results of RefFinder showed that the stabilities of TATA box-binding protein (TBP) and ubiquitously expressed transcript protein (UXT) were ranked the top two across all developmental stages. TBP and hydroxymethylbilane synthase (HMBS) were stably expressed in immature stages, while mitochondrial ribosomal protein L39 (MRPL39) and TBP had higher stability than other candidate genes in mature stages. This study provided valuable information for gene expression studies to assist further investigation on the molecular mechanisms in underlying yak testis development.

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Section: Discussionmentioning

confidence: 59%

Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

Zhou

Chen

et al. 2020

Animals

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“…Considering the selection of an appropriate housekeeping gene as a reference gene for normalization, several studies demonstrated that the Glut5 gene is highly reliable for analysis of relative gene expression in bovine fresh and frozen‐thawed sperm samples (Ashish et al, 2017). Melt curve analysis was conducted to confirm the specificity of each product.…”

Section: Methodsmentioning

confidence: 99%

“…Half ml DNase I treated cDNA (containing 0.2 µg) was added to 6 µl of SYBR Premix Ex Taq II Mix and 0.75 µM of each specific primer in a total volume of 12 µl. The PCR programme was comprised of 45 cycles of 94°C for 40 s, 60°C-63°C for 30 s (annealing temperature;Table 1) and 72°C for 30 s.Considering the selection of an appropriate housekeeping gene as a reference gene for normalization, several studies demonstrated that the Glut5 gene is highly reliable for analysis of relative gene expression in bovine fresh and frozen-thawed sperm samples(Ashish et al, 2017). Melt curve analysis was conducted to confirm the Sequence and annealing temperature of used primers for RT-PCR…”

mentioning

confidence: 99%

Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm

Nazari

Ahmadi

Hamid

et al. 2020

Veterinary Medicine & Sci

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Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time‐PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non‐frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)‐related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen‐thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.

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“…Two primer pairs were designed using the PerlPrimer program and web tool IDT oligo analyzer, with the specificity of the sequences ensured by using the National Center for Biotechnology Information's (NCBI's) BLAST. Five primer pair sequences were taken from previous studies . ACTB and HMBS were chosen as internal control genes, which were validated for bovine skeletal muscle in a previous study .…”

Section: Methodsmentioning

confidence: 99%

“…Five primer pair sequences were taken from previous studies. 14,24,25 ACTB and HMBS were chosen as internal control genes, which were validated for bovine skeletal muscle in a previous study. 26 All primer pairs tested for the specific length of the amplicons with ethidium-bromide-stained 2% agarose gel electrophoresis.…”

Section: Qpcr Analysismentioning

confidence: 99%

Differential expression analysis of meat tenderness governing genes in different skeletal muscles of bovines

2019

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BACKGROUND The aim of this study was to compare 12 different skeletal muscles of bovine (n = 15) with each other in terms of tenderness and meat‐quality‐related gene expressions. Tenderness values were evaluated by shear force, and ANK1, CAPN1, CAST, HSPB1, HSPA1A gene expressions were analyzed by using quantitative real‐time polymerase chain reaction. RESULTS ANK1 gene showed significant differences between tender and tough muscles (P < 0.001) and was found to be more closely related to meat quality than CAPN1. No difference was found for CAST, HSPB1, and HSPA1A gene expressions between different parts of skeletal muscles (P > 0.05). The results also showed that the most convenient skeletal muscle for the meat quality studies is musculus psoas major. Furthermore, comparative use of musculus longissimus thoracis and musculus extensor digitorum muscles may give the most accurate results, rather than using other muscle groups in comparative studies between tender and tough muscles. CONCLUSION ANK1 gene is a preferable biomarker for the determination of meat quality, and CAPN1 needs further studies. However, CAST, HSPB1, and HSPA1A genes may not be suitable biomarkers for the determination of meat quality based on this study. © 2018 Society of Chemical Industry

show abstract

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo ( Bubalus bubalis )

Cited by 10 publications

References 34 publications

Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm

Differential expression analysis of meat tenderness governing genes in different skeletal muscles of bovines

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