GUO Ri-hong, HE Pei-yuan, MAI Yan-long, DAI Zi-cun, CHEN Fang, SHI Zhen-dan

1 Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China

2 College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510620, P.R.China

3 Agricultural Development Service Center of Hekou Town, Yunfu 527126, P.R.China

Abstract The feasibility of a novel method to improve sow reproductive performance by combining inhibin immunization and hCG treatment was tested using in vivo and in vitro experiments. In the in vivo experiment, 106 sows were administered an inhibin immunogen on day 7 prior to weaning, and 56 non-treated sows served as the controls. Sows exhibiting oestrous behaviour on day 5 after weaning were artificially inseminated. On day 5 post-insemination, a subset of 50 inhibin-immunized sows received an injection of 1 000 IU human chorionic gonadotropin (hCG). Our results showed that pre-weaning immunization against inhibin marginally improved (P=0.068) total litter size and significantly increased (P=0.044) the live litter size. The overall differences in farrowing rates and live litter size tended toward significance (P=0.10) in the three groups, and the differences in total litter size were not significant (P=0.18). In the in vitro experiment, activin and hCG dose-dependently suppressed (P＜0.001) and stimulated (P＜0.001) progesterone (P4) secretion in cultured pig granulosa cells (GCs),respectively, and the suppression effect of activin was reversed (P＜0.001) by hCG. Activin suppressed P4 secretion mainly by downregulating (P＜0.001) the expression of StAR, Cyp11a1, and 3β-HSDII, whereas hCG prevented (P＜0.001) the suppression effects. These results indicate that the combination of pre-weaning immunization against inhibin and postinsemination hCG treatment provides a novel method for improving sow reproductive performance.

Keywords: sow, reproductive performance, inhibin, hCG, progesterone

1. Introduction

Immunization against inhibin is a well-documented method for improving the ovulation rate and litter size in animals, such as mice (Hasegawa et al. 2016; Takeo and Nakagata 2016), rats (Ishigame et al. 2004; Wang et al.2012), goats (Padilla et al. 2008; Wang et al. 2009), sheep(Naqvi et al. 2009; Dan et al. 2016), bovines (Mei et al.2009; Liu et al. 2013) and pigs (Wheaton et al. 1998).Immunization against inhibin typically increases the plasma concentration of follicle-stimulating hormone (FSH) (Medan et al. 2004; Li et al. 2011; Dan et al. 2016) and activin (Li et al. 2011; Bahareldin-Ali et al. 2015). On the other hand,immunoneutralization of inhibin bioactivity also directly enhances the proliferation of ovarian granulosa cells (GCs)and oestradiol (E2) secretion in cultured porcine (Cai et al.2015) and bovine GCs (Jimenez-Krassel et al. 2003).Thus, immunization against inhibin via the stimulation of FSH, the secretion of activin, and by the neutralization of inhibin bioactivity, enhances ovarian follicular development,E2 secretion, mature oocyte quality and early embryo development (Li et al. 2011; Liu et al. 2013; Yan et al.2015). With these benefits to reproductive performance by immunization against inhibin, it is therefore obvious that animal reproductive performance could also be improved using this technique.

Inhibin has been reported to regulate the proliferation of porcine granulosa cells (Cai et al. 2015; Li et al. 2018), and its polymorphisms are associated with follicle cysts in swine(Li et al. 2015, 2016; Wang et al. 2015), indicating that inhibin plays an important role in regulating porcine reproduction.Immunization against inhibin also increases the ovulation rate and litter size (Wheaton et al. 1998) in sows. However,detrimental effects on the farrowing rate have been observed in gilts that injected with a high dose of anti-inhibin antibody(Wheaton et al. 1998). In addition, our most recent investigation demonstrated that immunization against inhibin decreases plasma progesterone (P4) concentrations during the early stage of embryo development (Guo et al. 2020).These results indicate that immunization against inhibin may impair corpora luteal development and P4 secretion and potentially compromise embryonic implantation and the conception rate. Therefore, for the development of new techniques based on immunization against inhibin to maximize the reproductive performance of sows, the augmentation of luteal function appears to be essential.

Activin is secreted by small follicles (Knight and Glister 2001), it stimulates the development of in vitro-cultured rat follicles (Rabinovici et al. 1990) and human GCs (Rabinovici et al. 1990). To this extent, elevated plasma activin levels(Li et al. 2011; Bahareldin-Ali et al. 2015) partially account for the enhancement of follicular development and ovulation rate in inhibin-immunized animals. Studies on in vitrocultured luteinized human GCs have demonstrated that activin inhibits P4 production (Chang et al. 2014, 2015).Whether activin plays a conserved role in suppressing P4 secretion in porcine GCs has not been reported and remains to be resolved. Regardless, the suppressive effect of activin on P4 secretion in human GCs may explain the impaired corpora luteal development and reduced farrowing rate in animals following inhibin immunization.

hCG administration has been used to enhance lutea development and improve conception rates following artificial insemination or embryo transfer in cows (Hanlon et al. 2005), sheep (Khan et al. 2007), and pigs (Bolzan et al.2013; Seyfang et al. 2016). The enhancement effects of hCG on P4 secretion and farrowing rate could be used to overcome or alleviate the side effects of the increased activin levels following immunization against inhibin. However, this hypothesis needs to be tested by in vivo studies, and the effect of the combination of immunization against inhibin and hCG administration on P4 secretion in porcine GCs also needs to be studied using in vitro-cultured GCs.

Therefore, the objective of this study was to test the hypothesis that the combination of immunization against inhibin and hCG administration will improve reproductive performance by improving the farrowing rate and litter size in sows. These mechanisms were further examined in vitro by measuring P4 secretion in cultured luteinized porcine GCs that were treated with activin and hCG.

2. Materials and methods

2.1. Animals and experimental design

Animal experiments were conducted at the Yuanfeng Agricultural Co., Ltd. in Guangdong Province of China(21.85°N, 111.95°E) from November 2017 to March 2018. A completely randomized design was adopted for the present in vivo experiments, and a total of 162 Large White×Landrace mixed-parity lactating sows were assigned to three groups:the control group (Con, n=56), treated group 1 (T1, n=56),and treated group 2 (T2, n=50). Seven days prior to weaning, the Con pigs were injected with 1 mL of mineral oil adjuvant, and the T1 and T2 pigs were injected with 1 mL of inhibin immunogen (i.e., 1 mg mL-1of a recombinant porcine inhibin α-subunit protein in mineral oil adjuvant)prepared according to our previously published studies (Mei et al. 2009). Five to 6 days after weaning, the time when the inhibin antibody titre reached a peak (approximately 2 weeks after immunization according to our previous studies in cows, buffalos, and pigs), sows exhibiting signs of oestrus were artificially inseminated at the detection of oestrus and at 24 h intervals while they continued to exhibit oestrous. Sows in the T2 group were further treated with 1 000 IU hCG at 5 days after insemination. After pregnancies reached full term, the total and live litter sizes were recorded during the first 24 h. The design of the animal experiment is illustrated in Appendix A.

This experiment was approved by the Research Committee of Jiangsu Academy of Agricultural Sciences,China (Order No. 63 on July 8, 2014).

2.2. Culture of primary porcine GCs

The cell culture procedures are illustrated in Appendix B.In brief, primary GCs were isolated from healthy follicles(6-8 mm) as described in our previous studies (Cai et al.2015; Li et al. 2017). The cell density was adjusted to 2×106cells per well in a 6-well plate in 2 mL of culture medium(DMEM/F12 (319-085-CL, Wisent Corporation, Nanjing,China) with 10% FBS (10099141C, Gibco; Shanghai,China), 100 IU mL-1penicillin and 100 μg mL-1streptomycin(450-201-EL, Wisent Corporation). The cells were incubated in a humidified atmosphere containing 5% CO2at 37°C.The wells were washed two times with D-PBS (311-415-CL,Wisent Corporation) to remove unattached cells after 24-h of incubation. To induce luteinization and P4 secretion, GCs at a density of approximately 50% were treated with 10 μmol L-1forskolin (F3917, Sigma-Aldrich; St. Louis, MO, USA) for 2 days (Freimann et al. 2015). Subsequently, the luteinized GCs (LGCs) were cultured in DMEM/F12 medium with 2%FBS. Activin (338-GMP-050, R&D Systems; Minneapolis,MN, USA) (1, 10 and 100 ng mL-1according to Chang et al.(2015)) was then added to the medium to mimic the increase in activin in the serum in response to immunization against inhibin, and hCG (10, 100 and 200 IU mL-1, according to Qu et al. (2019)) was added to the medium to mimic hCG administration in the in vivo experiment. The cells were further cultured for an additional 2 days. Culture media were collected and stored frozen at -20°C until further analysis,and cells were collected for RNA and protein extraction. The in vitro experiments were performed using three biological replicates with two wells per replicate.

2.3. Measurement of P4 in the culture medium

P4 in the culture medium was measured using an ELISA Kit (Beijing North Institute of Biological Technology, Beijing,China) according to the manufacturer's instructions. The standard curve of the kit ranged from 0 to 30 ng mL-1, and the inter-assay and intra-assay coefficients of variation for these assays were less than 10%. Each sample was measured in triplicate.

2.4. Gene expression analysis

Total RNA from GCs was extracted using TRIzol Reagent(74104, Invitrogen, Shanghai, China) and reversetranscribed into cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (RR047A, TaKaRa, Dalian, China)according to the manufacturer's instructions. qPCR was performed on an ABI 7500 (Applied Biosystems, Shanghai,China) using a FastStart Universal SYBR Green Master Kit (04913850001, Roche, Shanghai, China). The primer sequences for StAR, 3β-HSDII, Cyp11a1, and β-actin were published in our previous study (Cai et al. 2015). Gene expression levels were calculated using the 2-ΔΔCtmethod and normalized to the expression levels of the β-actin internal housekeeping gene as described in our previous studies (Cai et al. 2015). Each sample was assayed in triplicate.

2.5. Western blot analysis

Total protein was extracted from the cells using radioimmunoprecipitation (RIPA) lysis buffer (P0013B,Beyotime Biotechnology, Haimen, China) with phosphatase inhibitor cocktail C (P1091, Beyotime Biotechnology). Then,the cell lysates were boiled in gel-loading buffer, and 30 μg of protein per lane was separated by SDS-PAGE on a 12%gel. The proteins were subsequently electro-transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore;Darmstadt, Germany). The membrane was blocked with 5% (w/v) bovine serum albumin for 1 h at room temperature,followed by incubation with primary antibodies against GAPDH (AF0911, 1:5 000; Affinity Biosciences, Changzhou,China), Cyp11a1 (DF6569, 1:1 000; Affinity Biosciences) and 3β-HSDII (DF6639, 1:1 000; Affinity Biosciences) overnight at 4°C. After being washed, the membrane was incubated with secondary antibodies for 1 h. Chemiluminescence was detected by an ECL Kit (35055, Pierce Chemical,Dallas, TX, USA) and visualized through Image Quant LAS 4000 (Fujifilm, Shanghai, China). The band intensity was quantified with ImageJ Software (NIH, Bethesda, MD, USA).

2.6. Statistical analysis

The experimental units of the in vivo and in vitro experiments were individual sows and each replicate of two wells,respectively. Data on litter size and live litter size from each group in the in vivo experiment, P4 data in culture medium from cells treated with activin or/and hCG, and gene and protein expression data of GCs were analysed using oneway analysis of variance (ANOVA), followed by Tukey's test for multiple comparisons. Data on the total litter size and live litter size from the treated sows (T1 and T2 were combined as one immunization group, named the treated group) and Con sows in the in vivo experiment, and the data from the P4 analysis in the culture medium from the control cells and cells subjected to forskolin induction were analysed using Student's t-test. The oestrus rate and farrowing rate data in the in vivo experiment were analysed using Chi-square analysis. All statistical analyses were performed using SPSS Statistics version 25.0 (SPSS Inc., Chicago, IL, USA).Statistical significance was set at P≤0.05.

3. Results

3.1. hCG treatment rescued the decrease in the farrowing rate caused by immunization against inhibin (in vivo experiment)

The reproductive performance of sows immunized against inhibin and treated with hCG is summarized in Table 1. The mean interval of weaning to oestrus was close to 5 days and did not appear to be influenced by immunization against inhibin 7 days prior to weaning. The percentage of sows exhibiting signs of oestrus in the treated sows was 93.40% (94.64% in T1 and 92.0% in T2 group) and was not significantly different (P=0.18) from that of Con sows (89.29%)(Table 1; Appendix C). After insemination and full-term pregnancy, the farrowing rate in Con, T1 and T2 sows was 92.00, 81.13 and 93.48%, respectively; however, the overall differences were not significant (P=0.10).

3.2. Immunization against inhibin increased the sow litter size (in vivo experiment)

The total litter size and live litter size of the Con sows was 10.22±0.38 and 9.96±0.37, respectively. Although both the total litter size and live litter size increased by nearly one piglet in T1 and T2 sows (Table 1), the increase in litter size and live litter size was not significant (P=0.18 for total litter size andP=0.10 for total litter size).

When T1 and T2 sows were combined as the treated group, its total litter size and live litter size was 10.99±0.24 and 10.79±0.23, respectively, the total litter size tended to increase (P=0.068), and live litter size increased significantly(P=0.044) compared with those of Con sows (Appendix C).

3.3. hCG reversed the repression effects on P4 secretion by activin in porcine LGCs (in vitro experiment)

The porcine LGCs treated with 10 μmol L-1forskolin were used asanin vitromodel. The concentration of P4 in the culture medium from primary LGCs was approximately 10 ng mL-1, and it increased (P＜0.001) approximately 180-fold to 1 763 ng mL-1after treatment with forskolin (Fig. 1-A).After treatment with forskolin, further treatment with activin and hCG dose-dependently inhibited and stimulated P4 secretion, respectively. A total of 10 and 100 ng mL-1activin reduced (P＜0.05) P4 secretion by approximately one-third and half (Fig. 1-B); 10, 100 and 200 IU mL-1hCG increased(P＜0.05) P4 secretion by approximately 2-, 3- and 3-fold,respectively (Fig. 1-C).

In the subsequent experiments to study the effects of the combination of immunization against inhibin and hCG administration on the function of porcine LGCs, 10 ng mL-1activin and 100 IU mL-1hCG were used. When LGCs were treated simultaneously with both activin and hCG,hCG removed the inhibitory effects of activin, and the concentration of P4 in the culture medium was not different(P=0.98) from that of hCG-treated cells (Fig. 1-D).

3.4. Regulation of the gene and protein expression of P4 synthetase by activin and hCG in porcine LGCs(in vitro experiment)

P4 synthesis in the ovary is mainly controlled by three steroidogenic enzymes: StAR, 3β-HSDII, and Cyp11a1. In response to treatment with forskolin, the expression ofStAR,3β-HSDII, andCyp11a1increased (P＜0.001) in cultured LGCsby approximately 8-, 4-, and 10-fold, respectively(Fig. 1-B). Activin (10 ng mL-1) alone downregulated(P＜0.01) the expression of all three genes, even to the level observed prior to forskolin treatment. hCG treatment (100 IU mL-1) alone upregulated (P＜0.01) the expression ofStARand3β-HSDIIbut not that ofCyp11a1(Fig. 2-A). Unlike the effect on P4 production, co-treatment with hCG only partially reversed the inhibitory effects of activin, with the expression ofStARandCyp11a1remaining low (P＜0.05), whereas3β-HSDIIexpression was unaltered (P＞0.05) in comparison to that of forskolin-treated cells (Fig. 2-A).

Forskolin increased (P＜0.05) the protein expression levels of Cyp11a1 and 3β-HSDII by 1.4 and 14.6 times,respectively (Fig. 2-B and C). Activin and hCG had similar effects on the protein expression of Cyp11a1 and 3β-HSDII as on P4 secretion, with activin downregulating (P＜0.05) and hCG upregulating (P＜0.05) their expression. hCG reversed the inhibitory effects of activin (Fig. 2-B and C).

Table 1 Summary of sow reproductive performance after pre-weaning inhibin immunization and post-insemination human chorionic gonadotropin (hCG) treatment (in vivo experiment)

Fig. 1 Effect of activin and human chorionic gonadotropin (hCG) on P4 secretion in cultured porcine luteinized GCs (LGCs)(in vitro experiment). A, P4 secretion after forskolin treatment. B, dose-dependent effects of activin (1, 10 and 100 ng mL-1) on P4 secretion from porcine LGCs. C, dose-dependent effects of hCG (10, 100 and 200 IU mL-1) on P4 secretion from porcine LGCs.D, the effect of the interaction between activin and hCG on P4 secretion from porcine LGCs. Primary pig GCs were treated with forskolin (10 μmol L-1) for 2 days to induce luteinization, activin or/and hCG at different concentrations. The data are shown as the mean±SE (n=3), and different letters at the top of each column indicate significant differences (P≤0.05).

4. Discussion

In the present study, we proposed and tested a novel method to improve sow reproductive performance by combining pre-weaning inhibin immunization and postinsemination hCG treatment. The in vivo and in vitro results indicated that immunization against inhibin combined with hCG treatment could constitute a new technique for improving reproductive performance in sows. In the in vivo experiments, immunization against inhibin improved the live litter size and tended to increase the total litter size but tended to reduce the farrowing rate. When immunization against inhibin was combined with a post-insemination hCG treatment, both litter size and farrowing rate tended to improve. The improvement in the farrowing rate is likely due to the increased conception rate following hCG treatment, which enhances the development and function of the corpus luteum. This finding is supported by the in vitro study showing that treatment with hCG corrected the luteal impairment effect of activin. Thus, this novel method holds promise for improving sow reproduction performance and is practical for the sow industry.

In the current study, the farrowing rate and litter size in the Con group were comparable to those observed on other commercial pig farms (Gadd 2011; Rensis and Kirkwood 2016). Here, a single injection of inhibin α-subunit immunogen significantly increased the live litter size by one piglet and tended to increase the total litter size by 0.78. The increase in litter size and live litter size of sows may result from enhanced follicle development after immunization against inhibin, as reported by Brown et al. (1990) and King et al. (1993). In sows, active immunization against inhibin has been shown to improve the ovulation rate by 35%(Brown et al. 1990) and 39% (King et al. 1993). Notably,the large variations in litter size and the limited number of sows used (only approximately 50 sows in each group)may obscure the effect of the treatments on the differences in immunization-induced ovulation rates and litter sizes in the present study. In addition, the elimination of excess embryos (up to 30 to 40%) during early gestation (Pomeroy 1960; Lambert et al. 1991) could reduce the number of conceived foetuses to normal levels. Thus, any increase in litter size following inhibin immunization would be limited.Nonetheless, the live litter size after immunization against inhibin was still significantly increased by one piglet, and this increase would be economically advantageous for practical sow production.

Our previous work in cows revealed that inhibin immunization improves the quality of early embryos, as reflected by the higher plasma concentrations of interferon-τ(Guo et al. 2020). Improved early embryo development has also been verified by in vitro studies on mature oocytes treated with anti-inhibin α-subunit antibodies (Li et al. 2011;Liu et al. 2013; Yan et al. 2015). However, immunization against inhibin still reduced the farrowing rate in this study,similar to the results of a previous study (Wheaton et al.1998). We hypothesized that inhibin immunization could impair the development of the corpus luteum, as previously demonstrated by the decreased P4 secretion in inhibinimmunized cows (Guo et al. 2020) and a concomitant reduction in conception and farrowing rates. To correct the P4 secretion, we treated sows with 1 000 IU of hCG at 5 days after insemination. This treatment resulted in a considerable improvement in the farrowing rate. It is possible that the hCG treatment caused a portion of sows with fewer embryos and corpora lutea to conceive and subsequently birth fewer piglets. This may be the reason that the numbers of total and live-born piglets in hCG-treated sows were (slightly but not significantly) lower than those observed in immunized sows.

Fig. 2 Regulation of the gene and protein expression of P4 synthetases by activin and human chorionic gonadotropin (hCG)in cultured porcine luteinized GCs (LGCs) (in vitro experiment). Effects of activin and hCG on the gene (A) and protein (B and C) expression of StAR, Cyp11a1, and 3β-HSDII. Primary pig GCs were treated with forskolin (10 μmol L-1) for 2 days to induce luteinization, and activin (10 ng mL-1) and hCG (100 IU mL-1) were then added. The data are shown as the mean±SE (n=3), and different letters at the top of each column indicate significant differences (P≤0.05).

Although the concentrations of hormones in the blood would have effectively illustrated the mechanism for the improved conception rate following hCG treatment, we did not collect blood samples from sows to reduce any possible stress that could block oestrus and adversely affect the conception rate. Instead, we used an in vitro model to elucidate these underlying mechanisms. As we theorized that the weakening of luteal function following inhibin immunization could be caused by high levels of plasma activin - as has been reported in dairy and water buffalo cows (Li et al. 2011; Bahareldin-Ali et al. 2015)- we investigated the effects of treatment with activin, hCG or both on P4 production and the mRNA and protein expression of P4 synthetases in cultured porcine GCs. Consistent with results previously reported in human LGCs (Chang et al.2014), treatment with activin significantly decreased P4 production. However, unlike the effects of activin on human LGCs (i.e., a reduction in the expression of StAR after activin treatment (Chang et al. 2015)), activin significantly reduced the expression of all three P4 synthetases measured. The protein levels of 3β-HSDII and Cyp11a1 were similarly regulated by activin, although to a lesser extent than the mRNA levels. Co-treatment with activin and hCG substantially upregulated the mRNA expression of the three synthetases, and the protein concentrations of 3β-HSDII and Cyp11a1 were also significantly upregulated. This leads us to believe that the upregulation of P4 synthetase expression in response to treatment with activin and hCG is responsible for the increase in the P4 concentrations observed in the culture medium. It should also be noted that co-treatment with activin and hCG resulted in P4 concentrations that were as high as those observed in response to treatment with hCG alone and much higher than the basal P4 production observed in the absence of activin. This indicates that treatment with hCG could completely negate the adverse effect of activin on P4 production and luteal function. Hence,in inhibin-immunized sows, the day 5 post-insemination hCG treatment should have completely restored luteal function and given rise to a conception rate higher than that of nonimmunized (Con) sows.

It should be noted that administering hCG on day 12(Bolzan et al. 2013; Seyfang et al. 2016), rather than during the first week of pregnancy (Stone et al. 1987), has been reported to improve P4 secretion and reduce embryo absorption in sows. Stone et al. (1987) proposed that P4 secretion in early pregnant gilts (i.e., the first week of pregnancy) is not constrained by luteotrophic factors despite the high LH/hCG receptor capacity of porcine luteal tissue owing to the antagonistic effects of other hormones from the pituitary and ovary. According to this theory, the combination of immunization against inhibin and hCG administration(increased activin level and reduced bioactivity of inhibin)could contribute to an increase in both P4 secretion and farrowing rate, which is supported by the results of the in vitro cell culture experiments presented in the current study. The combination of immunization against inhibin and hCG administration should be further studied in vivo.

5. Conclusion

In this study, we developed a new technique that combines pre-weaning inhibin immunization and post-insemination hCG treatment to improve litter size and conception rate in sows. Enhancing ovarian follicle development by immunization against inhibin helped to increase the number of embryos and therefore litter size. hCG treatment negates the damaging effect of activin on the function of the corpora lutea, thereby improving P4 production and the subsequent conception rate. This new technique may be applied to improve the reproductive performance of animals that suffer from ovarian insufficiencies owing to unfavourable conditions.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31802066).

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm