All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Kasetsart University, Bangkok, Thailand (Approval No. ACKU04660). The study was conducted in accordance with the National Research Council of Thailand Guidelines for the Care and Use of Animals for Scientific Research and complied with internationally accepted principles for the ethical use of animals in research. The study was designed, conducted, and reported in accordance with the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure transparent and reproducible reporting of animal experiments.

Before enrollment, all animals underwent comprehensive veterinary examinations, and only clinically healthy animals free from systemic disease, lameness, or reproductive disorders were included. Throughout the study, animals were maintained under routine veterinary supervision, including regular health assessments, vaccination, parasite control, and appropriate husbandry practices. Feed and clean drinking water were provided ad libitum, and animal health and welfare were monitored daily.

Blood collection was performed by experienced veterinarians using standard aseptic procedures to minimize stress and discomfort. Skeletal muscle biopsies were conducted under local infiltration anesthesia with 2% lidocaine using sterile techniques. Following tissue collection, hemostasis was achieved with gentle pressure; the biopsy site was disinfected with povidone-iodine; a topical antibiotic spray was applied; and animals were monitored daily for wound healing, appetite, locomotion, and signs of pain, inflammation, or infection. No biopsy-related complications or adverse welfare events were observed.

The principles of Replacement, Reduction, and Refinement (3Rs) were incorporated throughout the study by using the minimum number of animals necessary to achieve the scientific objectives, minimizing invasive procedures wherever possible, and ensuring that all procedures were performed by trained personnel to reduce pain, stress, and unnecessary animal suffering.

The study was conducted from September 2018 to May 2019 at the dairy research facilities of Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom Province, Thailand. Both experiments were performed under standard commercial dairy management conditions with routine veterinary supervision, including vaccination, parasite control, and regular health monitoring.

This study consisted of two independent randomized controlled experiments designed to evaluate the effects of FPP supplementation on endocrine, molecular, and reproductive responses in tropical crossbred dairy cattle. Experiment 1 evaluated changes in circulating IGF-1 concentrations and skeletal muscle IGF-1 mRNA expression, whereas Experiment 2 investigated the effect of FPP supplementation on the interval from calving to first postpartum estrus.

Before enrollment, all animals underwent comprehensive clinical examinations, and only clinically healthy animals without evidence of systemic illness, lameness, or reproductive disorders were included. Throughout the study, animals remained under continuous veterinary supervision with routine health assessments, vaccination, parasite control, and monitoring to ensure that concurrent diseases did not influence the experimental outcomes.

Experiment 1: Ten clinically healthy crossbred Holstein–Friesian dairy heifers (87.5%-93.75% Bos taurus × Bos indicus), aged 16-18 months and weighing 256-307 kg (mean ± Standard error of the mean [SEM]: control = 279.6 ± 6.81 kg; FPP = 279.4 ± 8.84 kg), were housed under identical management conditions and randomly allocated in a completely randomized design to either a control group receiving the basal diet or an FPP-supplemented group (30 g/head/day; n = 5/group). The basal diet consisted of concentrate and roughage formulated to meet nutrient requirements.

The FPP used in this study was a commercial feed additive (Lianol® Dairy; Huvepharma, Antwerp, Belgium), produced through controlled microbial fermentation of potato-processing by-products. The fermentation process hydrolyzes potato proteins into highly digestible protein fractions and low-molecular-weight bioactive peptides while generating essential amino acids and indole metabolites that may support hepatic metabolic function. Previous studies in monogastric species have demonstrated improvements in endocrine function, circulating IGF-1 concentrations, growth, metabolic recovery, and survival following FPP supplementation. However, these mechanisms have not been confirmed in dairy cattle. The supplementation level of 30 g/head/day was selected based on the manufacturer's recommended inclusion rate (25 g/head/day) and previous studies demonstrating biological responses at 25-50 g/head/day [41]. This dosage enabled evaluation of endocrine and reproductive responses without altering the nutritional composition of the basal diet. Animals received the experimental diets for 45 days and were fed twice daily with unrestricted access to clean drinking water.

Experiment 2: Twenty-four transition dairy cows (parity 1-2), aged 2.8-5.2 years and having body condition scores of 3.0-3.25 (5-point scale), were enrolled from 3 weeks before the expected calving date until 6 weeks postpartum. Only cows that experienced normal parturition were retained in the experiment. Animals were balanced for parity and body condition score before random allocation to either a control group or an FPP-supplemented group (30 g/head/day; n = 12/group).

Diets were formulated to meet the nutrient requirements of transition and early-lactation dairy cows. FPP was administered daily by top-dressing onto the concentrate ration. Feed was offered twice daily, and the basal ration was formulated to provide approximately 3% of body weight as dry matter intake according to the nutritional requirements of growing heifers, transition cows, and lactating dairy cows (Table 1). All animals consumed the entire FPP supplement throughout the experimental period.

Experiment 1: Approximately 5 mL of blood was collected from the jugular vein into plain vacuum tubes on days 0, 15, 30, and 45 before the morning feeding (07:00-08:00 h) to minimize postprandial variation. Samples were allowed to clot and centrifuged at 3,000 × g for 15 min within 1 h of collection to obtain serum, which was stored at −80°C until analysis. Serum IGF-1 concentrations were measured after approximately 2 weeks of storage. Body weight was recorded at 15-day intervals throughout the experiment.

On day 45, skeletal muscle biopsy samples were obtained aseptically from the semitendinosus muscle using sterile Tru-cut biopsy needles (14-gauge, 15 cm; TSK Laboratory, Tochigi, Japan) following local infiltration with 2% lidocaine (Lignospan®; Septodont, Saint-Maur-des-Fossés, France). Approximately 50 mg of tissue was collected, immediately snap-frozen in liquid nitrogen, and stored at −80°C until molecular analysis.

Following biopsy collection, gentle pressure was applied to achieve hemostasis; the biopsy site was disinfected with povidone-iodine; a topical antibiotic spray was applied; and animals were monitored daily for wound healing, locomotion, appetite, and signs of inflammation or discomfort. No biopsy-related complications were observed.

Experiment 2: Estrous activity was monitored continuously using an automated pedometer system (Afifarm®; Afimilk Ltd., Kibbutz Afikim, Israel). Estrous alerts generated from increased locomotor activity were confirmed by transrectal ultrasonography through identification of a dominant follicle and the absence of a corpus luteum, thereby minimizing false-positive estrus detection under tropical production conditions.

All experimental procedures, sample collection, laboratory analyses, and estrus confirmation were performed by the same trained investigators in accordance with standardized operating procedures to minimize operator-related variation.

Serum IGF-1 concentrations were measured using a bovine-specific enzyme-linked immunosorbent assay kit (CUSABIO Technology LLC, Houston, TX, USA; Catalog No. CSB-E08893b) according to the manufacturer's instructions. The assay sensitivity was 0.94 ng/mL with a detection range of 0.75-60 ng/mL. All samples were analyzed in duplicate. The intra- and inter-assay coefficients of variation were <15%. Concentrations were calculated from a seven-point standard curve generated using four-parameter logistic regression. Manufacturer validation data demonstrated acceptable recovery and assay parallelism for bovine serum.

Total RNA was extracted from skeletal muscle using TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA) followed by purification with the Direct-zol RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA). RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and only RNA samples with A260/A280 ratios between 1.9 and 2.1 were used.

Complementary DNA was synthesized using a commercial reverse-transcription kit according to the manufacturer's protocol. Quantitative real-time polymerase chain reaction (PCR) was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR Green chemistry and the primers listed in Table 2. The amplification protocol consisted of initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melt-curve analysis confirmed amplification specificity. No-template controls were included in every run to verify the absence of contamination and genomic DNA amplification.

Relative IGF-1 gene expression was normalized against the reference gene beta-actin (ACTB) and calculated using the 2^−ΔΔCq method. All reactions were performed in triplicate. Amplification efficiencies for both target and reference genes ranged from 90% to 110%, with correlation coefficients (R²) >0.90. Single-peak melt curves confirmed amplicon specificity, and ACTB expression remained stable across all experimental groups.

Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Data normality was evaluated using the Shapiro-Wilk test, whereas homogeneity of variance was assessed using Levene's test.

Serum IGF-1 concentrations were analyzed using two-way repeated-measures analysis of variance with treatment, time, and their interaction included as fixed effects. When significant treatment × time interactions were detected, Bonferroni-adjusted post hoc comparisons were performed.

Differences in skeletal muscle IGF-1 gene expression and the interval from calving to first postpartum estrus were analyzed using independent-samples t-tests. Results are presented as mean ± SEM. Statistical significance was established at p < 0.05. Effect sizes were expressed as Cohen's d, and 95% confidence intervals were reported for the principal comparisons.

Because serum IGF-1 concentrations were not normally distributed at the later sampling points (Shapiro-Wilk test, p < 0.05), between-group comparisons at each sampling day were additionally evaluated using the Mann-Whitney U test as a complementary non-parametric analysis.

To evaluate whether the nonsignificant difference in the calving-to-first postpartum estrus interval was due to insufficient sample size, a post hoc power analysis was performed. Based on the observed effect size (Cohen's d = 0.80), 12 cows per group, and α = 0.05, the achieved statistical power was approximately 46%, indicating that approximately 26 cows per group would be required to achieve 80% statistical power for detecting a difference of similar magnitude.

Throughout both experimental periods, no adverse health events attributable to FPP supplementation were observed. All animals remained clinically healthy throughout their respective study periods. In Experiment 1, mean body weight at the end of the 45-day supplementation period did not differ significantly between the control and FPP groups (312 ± 8.00 kg vs. 315 ± 9.91 kg, respectively; p > 0.05), indicating that FPP supplementation had no adverse effect on growth performance or general health status during the study.

Relative skeletal muscle IGF-1 mRNA expression measured on day 45 is presented in Figure 1.

Quantitative real-time PCR analysis normalized to the reference gene ACTB demonstrated no significant difference in skeletal muscle IGF-1 mRNA expression between the control and FPP groups (0.82 ± 0.07 vs. 0.84 ± 0.14, respectively; p = 0.90).

Amplification efficiencies for both the target and reference genes were within the acceptable range (90%-110%), and melt-curve analysis confirmed the presence of a single specific amplification product. Triplicate reactions demonstrated minimal technical variation, confirming the reliability and reproducibility of the gene expression analysis.

Changes in serum IGF-1 concentrations during the supplementation period are presented in Figure 2. Serum IGF-1 concentrations did not differ significantly between the control and FPP groups at baseline (day 0; p = 0.056) or after 15 days of supplementation (day 15; p = 0.151; Mann-Whitney U test).

However, significant differences were observed during the later stages of supplementation. On day 30, cows receiving FPP supplementation exhibited significantly higher serum IGF-1 concentrations than those in the control group (17.97 ± 4.93 ng/mL vs. 7.82 ± 0.87 ng/mL, respectively; p = 0.016). This difference persisted on day 45, with the FPP group maintaining significantly higher circulating IGF-1 concentrations than the control group (18.45 ± 4.13 ng/mL vs. 7.72 ± 0.75 ng/mL; p = 0.016).

The observed between-group differences corresponded to large effect sizes, with Cohen's d values of 1.28 and 1.61 on days 30 and 45, respectively.

Within-group analysis demonstrated a progressive increase in circulating IGF-1 concentrations throughout the supplementation period in the FPP group, whereas concentrations remained relatively stable in the control group.

The interval from calving to first postpartum estrus is presented in Figure 3.

All 24 enrolled dairy cows completed the reproductive monitoring period from 3 weeks prepartum to 6 weeks postpartum. No severe postpartum disorders were observed during the study, and body condition scores remained within the predetermined range (2.75-3.25) for all animals.

Cows receiving FPP supplementation exhibited a numerically shorter interval from calving to first postpartum estrus than control cows (43.83 ± 2.40 days vs. 50.00 ± 2.06 days, respectively). However, the difference did not reach statistical significance (p = 0.06). The estimated mean difference between groups was 6.17 days (95% confidence interval: −0.39 to 12.73 days), corresponding to a large effect size (Cohen's d = 0.80).

Although statistical significance was not achieved, the magnitude of the observed effect suggests a biologically relevant improvement in postpartum reproductive recovery following FPP supplementation.

The present study demonstrated that dietary FPP supplementation increased circulating IGF-1 concentrations in dairy cattle, whereas no significant changes were observed in skeletal muscle IGF-1 mRNA expression or the interval from calving to first postpartum estrus. These findings indicate that FPP primarily modulates systemic endocrine regulation rather than local tissue-specific gene expression and may contribute to metabolic adaptations associated with postpartum reproductive recovery.

In ruminants, circulating IGF-1 is synthesized predominantly in the liver in response to growth hormone (GH) stimulation and represents the principal endocrine component of the somatotropic axis [16, 17]. During early lactation, dairy cows commonly experience hepatic GH resistance characterized by reduced GH receptor (GHR) expression and impaired downstream signaling despite elevated circulating GH concentrations [10, 15, 17]. This uncoupling of the GH-IGF-1 axis is closely associated with NEB, reduced hepatic IGF-1 synthesis, and delayed resumption of ovarian cyclicity [2, 15].

The progressive increase in circulating IGF-1 observed after approximately 30 days of supplementation suggests gradual restoration of hepatic endocrine function rather than an immediate metabolic response. Fermentation improves protein digestibility, increases the availability of essential amino acids, and generates bioactive peptides that may enhance hepatic metabolism and endocrine signaling [36–38, 40]. Adequate amino acid availability has been reported to promote GHR expression and activate the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling pathway, which regulates hepatic IGF-1 transcription [17, 36–38]. Improved nutritional status may also increase circulating insulin concentrations, facilitating recoupling of the somatotropic axis [11, 17, 36]. Nevertheless, direct evidence that FPP-derived metabolites, including indole-3-acetic acid, activate hepatic GH signaling or the JAK2/STAT5 pathway in dairy cattle is currently unavailable [40, 41, 43, 45]. Therefore, the present findings are more appropriately interpreted as evidence of improved nutritional status rather than direct endocrine stimulation by FPP, and the precise molecular mechanisms remain to be established [17, 26–28].

Beyond hepatic synthesis, the biological activity of circulating IGF-1 is regulated by IGFBPs, particularly IGFBP-2 and IGFBP-3 [20, 28]. IGFBP-3 stabilizes circulating IGF-1 by forming a ternary complex with the acid-labile subunit, whereas IGFBP-2 is generally increased during catabolic conditions and reduces IGF-1 bioavailability [20, 26–28]. Although IGFBPs were not measured in the present study, the increased circulating IGF-1 observed after FPP supplementation may reflect both enhanced hepatic synthesis and a more favorable IGFBP profile, thereby improving IGF-1 stability and endocrine activity.

The delayed endocrine response observed in this study is consistent with previous reports demonstrating that improvements in the somatotropic axis occur gradually as animals transition from a catabolic to a more anabolic state [17, 36, 37]. Moreover, the evaluated supplementation level (30 g/head/day) falls within the biologically effective range previously reported for fermented concentrated potato protein (25-50 g/head/day) [41], indicating that commercially practical inclusion rates can improve endocrine status under tropical production conditions. However, because only a single supplementation level was evaluated, future dose-response studies are warranted.

Despite the increase in circulating IGF-1, skeletal muscle IGF-1 mRNA expression remained unchanged following FPP supplementation. This finding is consistent with previous studies demonstrating that circulating IGF-1 is regulated primarily by hepatic synthesis, whereas skeletal muscle IGF-1 acts predominantly via autocrine and paracrine mechanisms that control muscle growth and protein turnover [16, 18–20]. Consequently, the absence of changes in skeletal muscle IGF-1 expression does not contradict the endocrine response; rather, it highlights tissue-specific regulation of the IGF system.

Furthermore, mRNA abundance does not necessarily reflect protein abundance or biological activity, as post-transcriptional regulation, translational efficiency, and protein turnover can independently influence local signaling [18–20]. Therefore, future investigations should quantify skeletal muscle IGF-1 protein concentrations, along with downstream signaling molecules such as phosphorylated AKT and the mammalian target of rapamycin, to clarify local biological responses.

Consistent with previous studies in Holstein cows supplemented with fermented concentrated potato protein [41], the present study demonstrated a delayed increase in circulating IGF-1 after approximately 30 days of supplementation. However, to our knowledge, this is the first study to simultaneously evaluate systemic endocrine responses and skeletal muscle IGF-1 gene expression in tropical crossbred dairy cattle, providing novel evidence that FPP primarily influences systemic endocrine regulation rather than local IGF-1 expression in skeletal muscle under tropical production conditions.

Although FPP supplementation did not significantly shorten the interval from calving to first postpartum estrus, supplemented cows exhibited a biologically meaningful numerical reduction of approximately 6 days. Even modest reductions in the postpartum anestrous interval can improve reproductive efficiency and economic performance in dairy herds [2, 5, 6]. The concurrent increase in circulating IGF-1 together with the shorter postpartum estrus interval is consistent with the established role of IGF-1 in promoting granulosa cell proliferation, estradiol synthesis, follicular responsiveness to gonadotropins, follicular development, and ovulation [23–25]. IGF-1 may also influence reproductive function centrally by stimulating hypothalamic kisspeptin neurons and activating the HPG axis [33–35].

Because endocrine measurements and reproductive outcomes were obtained from separate experimental cohorts, a direct correlation between circulating IGF-1 concentration and reproductive performance could not be established. Nevertheless, the present findings suggest that elevated circulating IGF-1 can occur independently of skeletal muscle IGF-1 expression, supporting a predominantly systemic mechanism of action [16, 18–20, 38].

No adverse effects on voluntary feed intake or milk production were observed, indicating that supplementation with 30 g/head/day was well tolerated under the experimental conditions, although these parameters were recorded descriptively and were not statistically analyzed. Furthermore, as a value-added agro-industrial by-product, FPP may represent a sustainable nutritional strategy that reduces dependence on imported protein sources while improving metabolic adaptation in tropical dairy production systems where feed quality is frequently variable [40].

A major strength of this study is the integrated evaluation of endocrine, molecular, and reproductive responses to FPP supplementation, providing novel mechanistic insight into the regulation of the somatotropic axis in tropical crossbred dairy cattle.

Nevertheless, several limitations should be acknowledged. The study was conducted under tropical field conditions, where seasonal variation, heat stress, and other environmental factors could not be completely controlled. The relatively small sample size in Experiment 1 may have reduced statistical power to detect subtle changes in skeletal muscle IGF-1 expression. In addition, important components of the somatotropic axis, including hepatic GHR expression, circulating insulin, IGFBPs, non-esterified fatty acids, β-hydroxybutyrate, glucose, and inflammatory biomarkers, were not measured. Feed intake and milk production were monitored but were not predefined study outcomes and therefore were not statistically analyzed.

Reproductive performance was evaluated only until first postpartum estrus, whereas longer-term fertility indicators, including conception rate at first service, pregnancy rate, services per conception, days open, and calving interval, were not investigated. Furthermore, endocrine measurements and reproductive outcomes were obtained from separate cohorts, preventing direct within-animal association analyses. Finally, a post hoc sensitivity analysis indicated that the reproductive experiment had approximately 46% statistical power, suggesting that approximately 26 cows per treatment group would be required to detect the observed effect size with 80% power.

Future studies should include larger, multi-herd populations, evaluate multiple supplementation levels, integrate endocrine and reproductive measurements in the same animals, quantify hepatic signaling pathways and metabolic biomarkers, determine long-term reproductive performance, and assess the economic feasibility of FPP supplementation in commercial tropical dairy production systems.