RESUMO
Lutalyse HighCon (dinoprost tromethamine; Zoetis) has been approved for use both intramuscularly and subcutaneously in lactating dairy cows, although the effect of route of administration on circulating 13,14-dihydro-15-keto-prostaglandin F2α (PGFM), the metabolite of PGF2α, has not been evaluated. Multiparous, lactating Holstein cows were submitted to an Ovsynch protocol in which the last GnRH treatment (G2) was designated as d 0. Cows were fitted with indwelling jugular catheters on d 6 and administered 25 mg of dinoprost tromethamine (2 mL of Lutalyse HighCon) on d 7 either subcutaneously in the neck (SC; n = 6) or intramuscularly in the semitendinosus muscle (IM; n = 6). Blood samples were collected every 15 min after treatment for 1.75 h, then every 2 h for 48 h, and at 60 and 72 h, with the last time point corresponding to when cows would have received timed AI at 72 h within an Ovsynch protocol. Circulating PGFM concentrations were greater for SC than for IM cows from 15 to 90 min after treatment, which resulted in a greater area under the PGFM curve during the first 90 min after treatment (means ± SEM; 1,664 ± 129 pg·h/mL vs. 1,146 ± 177 pg·h/mL for SC vs. IM cows, respectively). This resulted in complete luteolysis in all but one cow in the SC treatment at 56 h, when GnRH would have been administered if dinoprost tromethamine had been administered as part of an Ovsynch protocol for timed AI. For cows that underwent complete luteal regression, circulating P4 did not differ between treatments at any time point. Thus, although SC cows had increased circulating PGFM 15 to 90 min after treatment, there was no difference in circulating P4 during induced luteolysis based on route of dinoprost tromethamine administration.
RESUMO
In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reverse-transcription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A, YWHAE, EIF3E, and PPIB. Independent RT-qPCR analyses were conducted confirming expression stability in another set of CL tissues from pregnancy and regression, with analyses performed for 3 groups of samples: (1) all samples, (2) samples from pregnancy alone, and (3) samples throughout the process of CL regression. Seven genes were found to be more stable in all states than 2 traditional reference genes (ACTB and GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4, YWHAE, and CDC42. When CL tissues from pregnant animals alone were analyzed, CST3, HIF1A, and CD63 were also identified as more stable than ACTB and GAPDH. Identification of these new reference genes will aid in accurate normalization of RT-qPCR results, contributing to proper interpretation of gene expression relevant to luteal physiology. Furthermore, our analysis sheds light on the effects of luteolysis and pregnancy on the stability of gene expression in the bovine CL.
