Effects of gonadotropin-inhibitory hormone on testicular development and reproduction-related gene expression in roosters

Abstract Gonadotropin-inhibitory hormone (GnIH) plays a crucial role in regulating reproduction in the hypothalamus of poultry and has been intensely investigated since its discovery. This study aimed to assess the effects of GnIH on testicular development, as well as on reproduction-related hormone release and gene expression levels in roosters. The administration of exogenous GnIH resulted in a significant reduction in testis weight, testis volume and semen quality (p < 0.05). Additionally, exogenous GnIH significantly up-regulates the expression of GnIH, and down-regulates the expression of PRL (p < 0.05). GnIH application also decreased the GnRH, vasoactive intestinal peptide (VIP) and luteinizing hormone β subunit(LHβ)gene expression levels. Meanwhile, by neutralizing the effects of endogenous GnIH through immunization, testicular development on day 150 in roosters was significantly promoted. Compared to the control condition, GnIH immunization significantly down-regulated the expression of the VIP and PRL genes (p < 0.05). In conclusion, we found that exogenous GnIH treatment inhibited testicular development, reduces PRL gene expression, and suppressed reproductive performance in roosters. Conversely, GnIH immunization down-regulated VIP and PRL genes, activates the reproductive system, and promotes the reproductive activity and testicular development of roosters.


Introduction
Gonadotropin-inhibitory hormone (GnIH) acts as a key reproductive regulator in the avian hypothalamus.0][11][12][13] The results of these studies highlighted the pivotal role of GnIH, secreted by the hypothalamus, in the regulation of pituitary gonadotropins.GnIH is also a key regulator of seasonal reproduction in birds.Its expression levels remain high during the late stage of egg production in geese, which facilitates the transition from the breeding to the non-breeding period. 14Moreover, during the quail reproductive period, GnIH down-regulates plasma testosterone levels through a synergistic effect with melatonin, thereby influencing reproductive performance. 15,16GnIH is also directly involved in the regulation of germ cell proliferation and differentiation.A study on chickens suggested that GnIH and its receptor may have an autocrine/paracrine role in regulating the viability and maturation of ovarian granulosa cells, gonadal steroidogenesis, spermatocyte differentiation and spermatozoa maturation. 179][20] In the adult mouse, GnIH treatment was also found to induce dose-dependent histological changes during spermatogenesis, including a decrease in germ cell proliferation and the down-and up-regulation of survival markers and testicular apoptotic markers, respectively. 21Treatment with exogenous GnIH in sexually mature male birds can reduce blood testosterone concentrations and inhibit gonadotropin synthesis and release, and induce testicular cell apoptosis, leading to decreased semen viability. 10In immature male quail, continuous injection of GnIH decreases testosterone levels and testicular weight, leading to testicular cell apoptosis and marked reductions in seminiferous tubule diameter and numbers of spermatogenic cells.Injection of GnIH in mature male quails reduces pituitary LHb and FSHb mRNA levels, and it also decreases LH and testosterone concentrations in plasma in a dose-dependent manner.This indicates that exogenous GnIH application can suppress the synthesis and release of gonadotropins while also exerting a significant inhibitory effect on testicular development. 10Furthermore, studies on seasonal reproduction in the tree sparrow provide additional evidence of a negative correlation between GnIH and testicular development. 22mproving egg fertilization and hatching rates remains a major goal in the poultry breeding industry.The fertility of breeder roosters significantly impacts the fertilization rate, whereas testis development and semen quality are crucial factors in the production of breeding chickens.In this study, we employed exogenous GnIH administration and GnIH immunization to investigate the influence of GnIH on testis development, reproductive hormone release, and expression levels of reproduction-related genes in roosters, with the aim of establishing a theoretical foundation for enhancing breeding performance in poultry production.

Animals and materials
The study utilized Yueh-Chuan Yellow No. 5 roosters obtained from Guangdong Yueh-Chuan Breeding Co., Ltd, located in Guangdong, China.Animal experiments were carried out at the Zhongcun Animal Test Farm, which belongs to Zhongkai Agricultural Engineering College.Roosters were kept in individual cages on restricted feed and with free access to water and exposure to natural sunlight.The temperature was 25 ± 3 � C, with a relative humidity ranged from 75% to 85%.The GnIH peptide of chicken comprising 12 amino acids (SIRPSAYLPLRF; purity �98%) was synthesized by China Peptides Co., Ltd (Shanghai, China).Polyvinylpyrrolidone solution (30%; PVP-K30) was purchased from Dingguo Biology Co., Ltd (Beijing, China).Chicken GnIH immunogen conjugative hemocyanin (KLH), in order to facilitate the conjugative of immunogen and KLH, a cysteine residue (C) is added to the N terminal of amino acid sequence (SIRPSAYLPLRF-NH2), purity �95%.The immunogen sequence and conjugative were completed by China Polypeptide Co., Ltd (Shanghai, China).Normal saline was purchased from the pet hospital of South China Agricultural University.

Experiment 1: Effects of exogenous GnIH on the reproductive performance of roosters
Eighteen 191-d-old healthy adult roosters were randomly divided into three groups, as follows: Group P, in which the roosters were injected with 1 mL of GnIH (1 mg) dissolved in 30% PVP-K30 on day 1 (n ¼ 6); Group G, in which the roosters were injected with 1 mL of GnIH (1 mg) dissolved in saline (without PVP-K30) on days 1, 4 and 7 (n ¼ 6); and Group C, in which the animals were injected with 1 mL of saline on days 1, 4 and 7 (n ¼ 6).All injections were administered intramuscularly.Semen quality was assessed on the last day (day 10) of the experiment.Sperm collection training was carried out on the first day of the experiment and subsequently every other day.To ensure the absence of sperm contamination, feathers within approximately 1 cm of the cloaca were cut off before training, and all the sperm collection tools were sterilized.Semen quality was assessed according to semen volume, density and motility.Sperm motility refers to the percentage of sperm in the semen exhibiting forward movement.Hormone levels were determined by collecting blood samples, separating the serum, and storing it at −20 � C. Hypothalamic and pituitary tissue samples were collected after euthanasia and stored at −80 � C for the measurement of gene expression levels.Testicular samples were obtained for weight and volume determination.Testicular weight was measured using an electronic scale.Testicular volume was calculated using the formula 4/3 ab 2 , where a and b denote half of the long (length) and short (width) axes, respectively. 22

Experiment 2: Effects of active GnIH immunization on the reproductive performance of roosters
One hundred and twenty 50-d-old roosters of uniform weight and health were randomly assigned to a GnIH immunization group and a control group (n ¼ 60 per group), with six replicates per group and 10 animals per replicate.Following a 10-day feeding period, the formal trial commenced at 60 d of age and continued for 120 days until the roosters reached 180 d of age.The immunogen was administered intramuscularly on days 1, 21 and 41 (60, 80 and 100 d of age).Animals in the GnIH immunization group were injected with 1 mL of vaccine containing GnIH protein (0.2, 0.3 and 0.5 mg) emulsified with Freund's adjuvant (Sigma, St. Louis, MO), while those in the control group were injected with saline emulsified in adjuvant.Blood samples were collected from veins under the wings on days 0, 20, 40, 80, 100 and 120 (60, 80, 100, 140, 160 and 180 d of age; n ¼ 12), and the serum was separated and stored at −20 � C for the determination of antibody and hormone levels.Additionally, eight roosters were randomly selected from each group at 60, 90, 120, 150 and 180 d of age for the collection of testicular, hypothalamic, and pituitary samples following euthanasia.Hypothalamic and pituitary samples were snap-frozen and stored at −80 � C. Artificial sperm collection training (massage method) was performed on the roosters at 160 d of age.Once the conditioned reflexes had been established, sperm quality was assessed every 3 d, resulting in a total of five batches.

Ethics statement
This study was conducted following the recommendations in the Guide for the Care and Use of Laboratory Animals of Zhongkai University of Agriculture and Engineering (No. 2020071109).The study was approved by the university's Ethics Committee on 11 July 2020.

Measurement of antibody levels
Antibody titres were assessed using enzyme-linked immunosorbent assays (ELISAs) and were quantified through the measurement of optical density (OD) at 450 nm.The anti-GnIH antibody titre in plasma was also determined by ELISA.GnIH peptide was distributed in a 96-well microtiter plate (0.1 lg/well, 100 lL), and 100 lL of plasma sample (1:1600 dilution, 1% skimmed milk) was added to each well.The plate was incubated at room temperature to allow the binding of the anti-GnIH antibody to the encapsulated GnIH peptide.The antibody was labelled with horseradish peroxidase (HRP)-conjugated goat anti-chicken IgY antibody (Abcam, Cambridge, MA) and cross-bound to chicken immunoglobulin from blood samples.TMB-ELISA substrate solution (Thermo Scientific, Bremen, Germany) was added to each sample for visualization and 2 M H 2 SO 4 was added to stop the reaction.The anti-GnIH antibody titre was determined at an OD of 450 nm using an EON spectrophotometer (BioTek, Winooski, VT).The optimal concentration of the encapsulated antibody was determined to be 5 lg/mL, the optimal serum dilution was 1:6400, and the optimal dilution of the secondary antibody was 1:16,000.To reduce test bias, the same plate was used for the evaluation of the plasma samples of both immunized and control chickens.

Detection of hormone levels
The LH concentration in the chickens was determined using the radioimmunoassay (RIA) method.Chicken LH (USDA-cLH-K-3) was used as the control and USDA-cLH-I-3 as the radiolabeled ligand.Rabbit anti-chicken LH (USDA-AcLH-5) was used as the first antibody and donkey anti-rabbit serum as the secondary antibody.The assay's sensitivity was 0.02 ng/mL.Serial dilutions of samples obtained from the chickens showed an inhibition curve parallel to that of the standards.The plasma testosterone concentration was determined using a medical diagnostic RIA kit obtained from Beijing North Institute of Biotechnology Co., Ltd (Beijing, China).To minimize steroid-binding protein interference, 50 lL of plasma sample was added to 150 lL of 0.1 M phosphate-buffered saline (pH 7.4), followed by preheating at 70 � C in a water bath for 30 min to denature the steroid-binding protein and release the bound hormone.Subsequently, antibodies and 125I-labeled testosterone were added to the samples to determine the sensitivity.The coefficients of variation within the measurement range were found to be 0.2 and 0.5-10 ng/mL, respectively, both of which were less than 10%.All samples were evaluated using the same method.

Quantitative reverse transcription-PCR (qRT-PCR)
Total RNA was extracted from the hypothalamus and pituitary tissue with TRIzol reagent.Subsequently, reverse transcribed using a reverse transcription kit (Toyobo, Osaka, Japan).The synthesized cDNA was stored at −20 � C for the subsequent determination of the relative mRNA expression levels of GnRH, GnIH and vasoactive intestinal peptide (VIP) in the hypothalamus and LH and prolactin (PRL) in the pituitary by qPCR.The primers used for the amplification of GnRH, GnIH, PRL, VIP, LH and b-actin (used as the internal reference) were designed using NCBI Primer Blast with the mRNA sequences of the relevant chicken genes in GenBank serving as references (Table 1).All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.The qPCR reaction mixture (20 lL) included 10 lL of 2� SYBR Green PCR Master Mix, 0.5 lL of each forward and reverse primer (10 mmol/lL), 8 lL of ddH 2 O and 1 lL of cDNA.The PCR procedure comprised one cycle of predenaturation at 50 � C for 2 min and then at 95 � C for 10 min, followed by 40 cycles of 95 � C for 15 s and 72 � C for 1 min.

Data analysis
The qRT-PCR results were analysed using the 2 −DDCt method.The data were analysed using two-way ANOVA in SPSS version 19.0 (Chicago, IL).The LSD multiple comparison method was used to test for significant differences.The results are shown as means ± standard error of the mean.p < 0.05 indicated significant differences, p < 0.01 indicated extremely significant differences, and p > 0.05 indicating no significant differences.

Testicular development
Figure 1 illustrates that testicular weight and volume were significantly reduced after GnIH treatment (Groups G and P) compared to the control group (Group C) (p < 0.05).No significant differences in testicular volume and weight were observed between the different preparation methods (p > 0.05).

Semen quality
The semen volumes in Group G and Group P, which received exogenous GnIH administration, were significantly lower than that in Group C (p < 0.05).However, the semen volume in Group P was significantly higher than that in Group G, possibly due to different preparation methods.There was no difference in semen density among the three groups (p > 0.05).There was no significant  Roosters in Group P were injected with 1 mL of GnIH (1 mg) dissolved in 30% PVP-K30 on day 1; those in Group G were injected with 1 mL of GnIH (1 mg) dissolved in saline on days 1, 4 and 7; and those in Group C were injected with 1 mL of saline on days 1, 4, and 7. Data are presented as means ± S.E.M., n ¼ 6. � p < 0.05, �� p < 0.01, non-asterisk means the difference is not significant, p > 0.05.
difference in semen motility between Groups C and G.However, semen motility was significantly lower in Group P compared with Group C and G (p < 0.01) (Table 2).

Plasma hormone levels
After treatment, the serum LH concentration remained relatively stable across the three groups, ranging from 2 to 4 ng/mL, with no significant difference observed.The concentration of T in both group C and group P exhibited an increasing trend at d7 and d10.However, in group P, the concentration showed an initial increase at d4, followed by a slight decrease.Nonetheless, no significant difference was found among the groups (Figure 2).

Gene expression
The mRNA expression levels of reproductive axis-related genes in the hypothalamus of roosters were determined.The expression level of GnRH in groups G and P showed an increasing trend compared with group C. The expression levels of GnIH in Groups G and P were significant higher than in Group C, with Group G showing particularly significant levels (p < 0.01).The expression levels of VIP and LH in group C showed an increasing trend compared with those in group G and group P, but the differences were not statistically significant (p > 0.05).Meanwhile, PRL mRNA levels in Groups P and G were extremely significantly lower than those in Group C (p < 0.01) (Figure 3).

Antibody levels
The levels of anti-GnIH antibodies in the GnIH immunization group exhibited an immediate increase following the initial immunization at 60 d of age (experimental day 0).The levels of anti-GnIH antibody increased further after the second (80 d of age) and third (100 d of age) immunizations and peaked at 140 d of age (experimental day 80).Throughout the experimental period, the control group consistently exhibited low levels of anti-GnIH antibodies (p < 0.01) (Figure 4).

Testicular development
The testicular development of roosters in the immunization and control groups was assessed at 60, 90, 120, 150 and 180 d of age.Testicular development exhibited a similar trend in both groups, namely, testicular weight and volume increased rapidly with age and peaked at 120 d of age, after which the values for both parameters declined to some degree.Testicular weight and volume were both greater in the GnIH immunization group than in the control group  throughout the experiment, reaching significance at 150 d of age (p < 0.05) (Figure 5).

Semen quality
The semen quality of the two groups of roosters was compared (Table 3).No difference in semen volume was seen between the control group and the GnIH immunization group (p > 0.05).Additionally, semen density and motility in the GnIH immunization group were both greater relative to those of the control group, with an extremely significant difference being recorded in semen motility (p < 0.01).

Plasma hormone levels
Throughout the experiment, LH levels remained very low in roosters of both the GnIH immunization group and control group, although the immune group showed an increasing trend relative to the control group.Serum testosterone concentrations steadily increased in roosters of both groups between 60 and 120 d of age, when they peaked, and then subsequently decreased.There were no statistically significant differences in testosterone concentrations between the two groups (p > 0.05) (Figure 6).

Gene expression
The qRT-PCR analysis revealed significantly lower expression levels of VIP and PRL in the immunization group compared to the control group (p < 0.05).The expression levels of GnRH and GnIH were comparable between the two groups, while LH had a trend to increase by GnIH-immunization, although the difference was not significantly (p > 0.05) (Figure 7).

Discussion
GnIH is a key endocrine hormone in the hypothalamus, playing an important role in the regulation of poultry reproductive behaviour through binding to its specific receptor GPR147. 23,24In this study, we discovered that administrating exogenous GnIH to adult roosters resulted in significant inhibition of testicular development, including the loss of testicular weight and volume.These findings were consistent with those of previous reports, namely, that GnIH regulates poultry reproductive activity. 25Regarding the exogenous GnIH preparation methods, inhibition of testicular development was weaker when normal saline was used than when PVP-K30 was used (Group P).This difference could be attributed to the sustained release of the hormone when the latter method was utilized, thereby maintaining the effect of GnIH on testicular development.The experiment involved assessing semen quality in all three groups of roosters, and the results revealed that administration exogenous GnIH significantly inhibited spermatogenesis and sperm motility.This outcome is consistent with the finding that both GnIH treatment groups (Groups G and P)   exhibited negative effects on testicular development.
We further found that the GnIH gene expression was increased following exogenous GnIH treatment, which directly or indirectly enhanced the suppression of gonadotropin release and, consequently, inhibited gonadal development and spermatogenesis.This finding aligns with current understanding of GnIH-mediated regulation of avian reproduction. 8GnIH regulates animal reproductive activity by inhibiting gonadotropin secretion in the hypothalamus and pituitary gland, which are upstream of the reproductive axis.Several studies have demonstrated that peripheral injection of GnIH in adult avian inhibits gonadal activity, induces testicular apoptosis and reduces spermatogenic activity. 10,26In avian species, LH stimulates testosterone formation in interstitial cells, while FSH and testosterone stimulate testicular growth, differentiation, and spermatogenesis. 27In this study, the levels of LH and testosterone in the blood of roosters were consistent with the observed changes in testicular development and semen quality 28 and may account for the reduced development and maintenance of the gonads induced by GnIH through its inhibitory effects on gonadotropin synthesis and release.0][31] However, if PRL levels are too low, it can also hinder reproductive performance. 32,33revious studies have demonstrated that the highest concentration of PRL in birds aligns with gonadal regression. 29During the breeding season, PRL is present at levels that promote reproductive performance, whereas GnIH is present at a low level.When GnIH is administered, PRL concentrations fall to levels that exert an inhibitory effect on reproductive performance.The expression level of the LHb gene correlated with the decrease in blood LH levels, as well as the observed impaired testicular development and semen quality in this study.This finding could be attributed to the direct or indirect inhibition of pituitary LHb gene expression and the subsequent reduction in reproductive performance in adult males after GnIH treatment. 1,11,34These combined results indicate that the administration of exogenous GnIH can suppress reproductive activity and performance in animals by inhibiting the secretion of gonadotropins.
To investigate the long-term effects of GnIH in vivo and its role of in gonadal development and maintenance in roosters, we utilized active GnIH immunization to counteract the effects of endogenous GnIH.The presence of anti-GnIH antibodies stimulates the testicular development of roosters, as evidenced by the increase in testicular weight and volume, the improvement in semen quality.The anti-GnIH antibody neutralized endogenous GnIH and reduced its inhibitory effect, thereby promoting reproductive activity in the birds.The results of this study are consistent with numerous studies. 35,36In poultry production, frequent artificial sperm collection induces stress which, in the long term, reduces semen volume and quality in roosters.GnIH immunization can enhance semen volume and density, thereby improving breeding efficiency.Additionally, GnIH immunization can also be applied to roosters at the end of their service life, thereby prolonging their usefulness.Following immunization, the expression levels of VIP and PRL genes in the immune group were down-regulated, which was somewhat inconsistent with the results of experiment 1.This inconsistency may arise due to the differing mechanisms of active immune GnIH and exogenous injection GnIH in reproductive regulation.We hypothesized that after exogenous injection of GnIH, testicular degeneration leads to decreased LH and testosterone secretion capacity, 10 and down-regulated VIP and PRL gene expression.Immunization with GnIH leads to the down-regulation of GnIH, VIP and PRL expressions, resulting in up-regulated of pituitary gonadotropin gene expression and activation of the reproductive system. 14Previous studies have reported that administration exogenous PRL to chickens reduce hypothalamic GnRH and plasma LH concentrations, 37 as well as down-regulate LHb gene expression. 38These observations suggest that active immunization with GnIH, which inhibits VIP and PRL expression, 12 promotes testicular development and enhances reproductive performance in roosters.
In conclusion, our findings demonstrate that the administration of exogenous GnIH inhibited testicular development, reduces PRL gene expression, and suppressed reproductive performance in roosters.Conversely, immunization with GnIH down-regulated the expression of the PRL and VIP genes, activates the reproductive system, promoted testis development, and enhanced reproductive activity in the roosters.

Figure 1 .
Figure 1.The effect of exogenous GnIH treatment on testicular development in roosters.(A) Testicular weight and (B) testicular volume.Roosters in Group P were injected with 1 mL of GnIH (1 mg) dissolved in 30% PVP-K30 on day 1; those in Group G were injected with 1 mL of GnIH (1 mg) dissolved in saline on days 1, 4 and 7; and those in Group C were injected with 1 mL of saline on days 1, 4, and 7. Data are presented as means ± S.E.M., n ¼ 6. � p < 0.05, �� p < 0.01, non-asterisk means the difference is not significant, p > 0.05.

Figure 3 .
Figure 3.The effects of exogenous GnIH treatment on the expression of reproduction-related genes in roosters.(A) Relative mRNA expression levels of GNRH in the hypothalamus, (B) GnIH in the hypothalamus, (C) VIP in the hypothalamus, (D) PRL in the pituitary, and (E) LH b in the pituitary.Data are presented as means ± S.E.M., n ¼ 6. � p < 0.05, �� p < 0.01, non-asterisk means the difference is not significant, p > 0.05.

Figure 5 .
Figure 5.The effect of active GnIH immunization on the testicular development of roosters.(A) Testis weight and (B) testis volume.Data are presented as means ± S.E.M., n ¼ 6. � p < 0.05, �� p < 0.01, non-asterisk means the difference is not significant, p > 0.05.

Figure 6 .
Figure 6.The effect of active immunization against GnIH on the levels of reproductive Hormones of roosters.(A) Serum luteinizing hormone (LH) levels and (B) serum testosterone (T) levels.Data are presented as means ± S.E.M., n ¼ 6. a and b indicate differences (p < 0.05) among the time.Non-letter means the difference is not significant, p > 0.05.

Figure 7 .
Figure 7.The effects of active immunization against GnIH on the expression of reproductive axis-related genes in roosters.(A) Relative mRNA expression levels of GNRH in the hypothalamus, (B) GnIH in the hypothalamus, (C) VIP in the hypothalamus, (D) PRL in the pituitary and (E) LH in the pituitary.Data are presented as means ± S.E.M., n ¼ 6. � p < 0.05, �� p < 0.01, non-asterisk means the difference is not significant, p > 0.05.

Table 1 .
Primer sequences, melting temperature and amplicon length.

Table 2 .
Effect of exogenous GnIH treatment on semen quality in roosters.

Table 3 .
Effect of GnIH immunization on semen quality in roosters.