Effect of letrozole on reproductive indices of aged broiler breeder roosters

The importance of androgen-estrogen balance has been postulated to be more than complete levels of estrogen or androgen for an optimum reproductive function in males. In aged broiler breeder males, the low fertility is influenced by greater plasma and testicular estradiol as well as a low synthesis of plasma and testicular testosterone concentrations. Since aromatase is the main enzyme that converts the androgens into estrogens; therefore, it has been hypothesized that using the aromatase inhibitors could increase endogenous testosterone production without an associated increase in circulating estrogens. It is hypothesized that these steroid hormone changes that occur during post-peak fertility can affect the expression of several genes whose activity may be associated with steroid hormone changes, including Foxj1, PVRL3, and LPR2 genes. Estradiol plays a vital role in the proliferation and differentiation of the epithelial cells, which cause activation of the Foxj1 gene in these cells. The knocked-out mice for the Fox j1 gene (epithelium lacked ciliated cells) informing the importance of Foxj1 in estradiol-dependent epithelial cell differentiation. In this study, the effects of an aromatase inhibitor, letrozole, were evaluated on reproductive indices of broiler breeder roosters at short-term (three weeks) and during an interval (three days of use and two days of a stop).

Eighteen 55-weeks-old roosters were randomly divided into three groups receiving letrozole T0, T1, and T2 (0, 0.015, and 0.03 mg/kg of body weight, respectively). Deranged semen was collected via a syringe from the distal cauda of the vas deferens from each rooster as a single sample (n=6). Seminal traits, steroid hormone concentrations, the relative abundance of Pvrl3, Foxj1, and Lpr2 mRNA, and in vitro fertility were assessed at the end of the trial. Testes were carefully removed and the epididymal region was dissected free of extraneous tissues and weighed to determine the gonadal/somatic index [testis weight (g)/body weight (kg)]. Total RNA was extracted from the tissues using the TRI® reagent (Ambion, USA) according to the manufacturer’s protocol. The nucleotide sequences of all candidate genes of the roosters (Gallus gallus) were obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/). The primer was designed using Primer3Plus online software and assessed using OligoCalc and PrimerBLAST. Beta-actin was considered an internal control. Quantification of all transcripts was performed using QuantiFast Eva Green PCR Kit (HOT FIRE Pol® EvaGreen ® qPCR Mix Plus) in a 20 μL reaction volume containing 3 μL single-strand cDNA, 7 μL of the master mix, 0.75 μL of either forward and reverse primers, and 8.5 μL of distilled H2O in 20 μL by a Rotor-Gene 6000 Real-Time PCR software (Corbett Research, Sydney, Australia). The data were tested for normality using the Shapiro-Wilk test. Single and repeated measurement data were analyzed with the GLM and Mixed procedures of SAS software. Rooster's testicular data and body weight were considered as covariates in the statistical models. Duncan’s multiple range test was used to compare means. Sperm quality characteristics and in vivo fertility were analyzed with the same models.

The results showed that gonadal somatic index (GSI), sperm total and forward motility, viability, membrane integrity, semen concentration, and fertility rate were improved for letrozole groups compared with the control group (P<0.001). Lipid peroxidation and sperm abnormalities measurements in the T1 and T2 groups were lower compared to the control group. The epithelium thickness and tubular diameter of seminiferous, and the epididymal and testes sperm count increased in the T1 and T2 groups. A concentration of testosterone was statistically increased in T2 group and testicular concentration testosterone was lower in both letrozole groups compared to the control group. FSH concentration was significantly influenced by letrozole (P<0.05). Plasma concentration of estrogen decreased in the T1 and T2 groups and the lowest one was in T2 group, also testicular concentration was lower compared with the control group. The ratio of estrogen to testosterone was significantly influenced by T1 group. An increase in relative mRNA levels was observed for a PVRL3 gene (related to spermiation) in testes, and a decrease of Foxj1 gene (related to ciliogenesis) and LPR2 in epididymal (related to endocytosis) in the T1 and T2 groups.

Based on the results of this study, using aromatase inhibitor improved reproductive indices in aged broiler breeder roosters. It is suggested that increasing testosterone alone cannot improve reproductive performance and a balance in the estradiol/testosterone ratio is necessary to achieve optimal fertility.