The dairy sector is one of the fastest-growing industries, contributing significantly to the livelihood of dairy farmers. Among the available technologies, assisted reproductive techniques (ART) play a vital role in enhancing reproductive efficiency and accelerating production. In vitro embryo production (IVEP) associated with assisted reproductive techniques recently has shown significant progress in cattle. Instead of the significant progress in ART, the in vitro embryo production faces many challenges. In vitro developed embryos show fewer competencies as compared to naturally developed embryos because of scarcity of maternal signalling, oxidative stress and unavailability of balanced nutrients in culture media, which disrupt embryo development (Cagnone and Sirard, 2016). IVEP includes in vitro maturation (IVM) in vitro fertilization (IVF) and in vitro culture (IVC). Supplementing macro-molecules, growth factors, hormones and cytokines in culture media helps in achieving developmental competency under in vitro conditions. Previous studies indicate that growth factors exert regulatory effects on embryo development by acting through autocrine and paracrine pathways (Ahumada et al., 2013). It was reported that addition of estradiol, gonadotropins and granulosa cells in culture media helps in sheep oocytes maturation outside the follicle (LSS et al., 2022; Nia et al., 2025 and Hirao, 2025). In vitro maturation media (IVM) supplemented with epidermal growth factor (EGF) positively affects follicular fluid levels and stimulates meiosis in many species. Lonergan et al., (1996) revealed that epidermal growth factor (EGF) attributes to oocyte maturation and development likewise in cattle. Zhang et al., (2010) reported that locally derived fibroblast growth factor (FGF) massively affects oocyte maturation in IVM. Umdor et al., (2021) studied the supplementation of platelet-derived growth factor (PDGF) alone and in combination with FGF and found significant improvements in developmental competence. Recently, Pramanik et al., (2023) demonstrated that supplementation of leukemia inhibitory factor (LIF) throughout the culture media results in higher developmental competency of oocytes and embryos. Different combinations of growth factors positively affect blastocyst formation rate in cultured bovine embryos (Sirisathien et al., 2003). Bera et al., (2024) reported that different concentrations and combinations of PDGF+IGF1, EGF+FGF and T3+T4 significantly enhance cleavage rate and blastocyst formation rate. Among these growth factors, insulin-like growth factor-1 (IGF-1) is widely used because IGF-1 belongs to the insulin-like growth factor family and plays a key role in promoting cellular growth, cell division and specialization (Neirijnck et al., 2019). The objective of this study is to investigate the effect of insulin-like growth factor-1 (IGF-1) on developmental competence of cattle oocytes and embryos.

Materials and Supplement
 
All plastic ware used in this experimental study were purchased from Tarson products Pvt. Ltd. (Kolkata India). Chemicals, biochemical and mineral oil were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Syringe filters having pore size of 0.22 µm were sourced from Millipore Corp., Bedford, MA, USA. Sterile, disposable, non-toxic, non-pyrogenic plastic syringes and 19-guage hypodermic needles (Dispovan, Kolkata, India). All the experimental works have been completed by 2025 in Animal Biotechnology Lab., Eastern Regional Station, ICAR-National Dairy Research Institute, Kalyani, India.
 
Oocytes Aspiration, washing and In vitro maturation
 
Fresh cattle ovaries and oviducts were collected from the abattoir (Kolkata, India) in a thermos flask, containing saline (0.15 M NaCl) solution (35-38^oC) fortified with antibiotic (400 IU/ml penicillin) and carried to the laboratory within 2-3 hours. Ovaries were then trimmed for removal of adjacent tissues and washed 3-4 times in warm saline solution. Immature oocytes were aspirated from 3-8 mm follicle with the help of the 19-gauge hypodermic needle fitted with 5 ml disposable syringe containing 5 ml aspiration media (TCM-199, DPBS, 0.3% BSA and 60 µg/ml gentamycin solution). Collected oocytes were graded depending on the presence of cumulus cell layers covering. Total 855 immature oocytes were used for this experiment. Cumulus oocyte complexes (COCs) of Grade A (>3 layers) and B (2-3 layers) were given serial washing (4 to 5 times) in washing medium and then exposed to maturation medium (TCM-199 + 10% FBS + 5 µg/ml FSH-P + 0.33 mM sodium pyruvate + 50 µg/ml gentamicin sulfate). Approximately 25 to 30 COCs were kept in 100 ìl droplet of maturation medium supplemented with different concentration of insulin like growth factor-1 (0, 10, 25, 50, 100 ng/ml) covered with 2.5 ml to 3 ml sterile mineral oil in 35 mm petri dishes and kept for maturation in 5% CO₂ incubator for 24 hours at 38.5^oC with maximum humidity. Oocyte maturation was evaluated after 24 hours by examining cumulus cell layer expansion and the presence of a polar body.
 
Processing of sperm and In vitro fertilization with In vitro matured oocytes
 
The spermatozoa used for IVF throughout the study were from the same donor that had been tested for IVF earlier. The spermatozoa were prepared for fertilization as described earlier (Das et al., 2013). Briefly, two straws of frozen-thawed cattle semen were suspended in 1 ml of Working Bracket Oliphant (WBO) medium (Brackett and Oliphant, 1975) with 10 µg/ml heparin, 0.55 mM caffeine sodium benzoate, 7.1 mg sodium private and incubated for swim-up at 38.5^oC for 15 minutes. After incubation progressively motile sperm cells were taken by collecting 6 ml of WBO medium from the upper layer and centrifuged at 2500 rpm for 5 min. After that, the supernatant was removed and the pellet was dissolved in 1.2 ml of WBO medium and centrifuged at 2500 rpm for 5 min. Finally, the pellet was dissolved in l ml of Fertilization Bracket Oliphant (FBO). In the treatment group 100 µl droplet of FBO was supplemented with different concentration of insulin like growth factor-I (10 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml) with control group 0 ng/ml. The in vitro matured oocytes were washed twice with the FBO medium in the same maturation droplet and inseminated with 50 μl capacitated motile spermatozoa (1-2 million spermatozoa/ml) per droplet overlaid with pre-incubated light weight mineral oil of 35 mm culture dish and placed in 5% CO₂ incubator at 38.5^oC for 14-18 hrs with maximum humidity in air.
 
In vitro culture of early embryos
 
After completion of 14-18 hrs of incubation, the cumulus cells were separated by gentle pipetting in washing media and then presumptive zygotes were washed twice with modified Charles Rosenkrans 2 amino acid (mCR2aa) medium and cultured in 100 μl of mCR2aa medium supple--mented with different concentration of the Insulin like growth factor-1(10 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml). After 48 hrs the cleaved zygote/embryos were shifted in fresh 100 μl of mCR2aa blastocyst cultured medium in 5% CO₂ incubator at 38.5^oC with maximum humidity for 7 to 8 days.
 
Experimental design and statistical analysis of data
 
The experiment was conducted for six replicates in different concentrations of IGF-1 (10 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml) with control (0 ng/ml). Experimental data analyzed by using simple One Way ANOVA. Means were compared using Duncan multiple range test (IBM R Statistical Package for the social sciences R (SPSS version 23). Graphs were made by using GraphPad Prism version 8.4.3 (686) and the values were expressed as means±standard error.

Effect of the different concentration of IGF-1 on in vitro Maturation of oocytes
 
Supplementation of IGF-1 during in vitro maturation experiment resulted in a marked improvement as compared to the control group. Total 855 immature oocytes were used for this experiment. In control group (0ng/ml), the oocyte maturation rate was 90.30±0.84%. Upon supplementation of IGF-1 @ 10 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml, the maturation rates increased to 92.06± 0.66%, 92.51±1.07%, 96.44±0.09% and 93.50±0.62%, respectively. Notably, @ 50 ng/ml group shown the highest maturation percentage which was statistically significant (p<0.05) as compared to the control group (Table 1, Fig 1A). The increase in maturation may be hallmark to IGF’s role in enhancing mitochondrial function, energy metabolism and activation of key signaling pathways that promote both cytoplasmic and nuclear maturation and the up-regulation of IGF and activation of its receptor (IGF-1R) correlate with increased ATP production and heightened mitochondrial polarization in maturing oocytes (Biswas et al., 2023). These cellular changes are critical for the progression of meiosis and acquisition of developmental competence (Jung et al., 2023). These findings are consistent with reports in other species where IGF-1 supplementation facilitated the expansion of cumulus cells and enhanced nuclear maturation of oocytes by stimulating Ras/MAPK and PI3K/Akt pathways, leading to improve in vitro maturation outcomes (Sato et al., 2018; Li et al., 2016). Additionally, a dose-dependent effect was observed, with 50 ng/ml IGF-1 concentration have shown the positive effect on buffalo oocytes maturation (Badr and Zohery, 2011). Similar biphasic responses of IGF-1 have also been reported in mouse and buffalo oocytes, where optimal concentrations significantly improved maturation and developmental competence, whereas supra-optimal concentrations had no additional or negative effects (Palma et al., 1997).




 
Effect of the different concentration of the IGF-1 on the early embryonic developmental stages
 
From the present study it has been observed that different concentrations of IGF-1 affected embryonic development at various stages with notable differences. At the 2-cell stage with 50 ng/ml showed a significantly higher cleavage rate (85.35%±2.24) as compared to the control group (72.20%±2.74), while @ 25 ng/ml (79.07%±3.10) and @ 100 ng/ml (80.80%±2.22) groups showed lower rate without any significant differences. At the 4-cell embryonic stage @ 50 ng/ml IGF-1 resulted in a significantly higher percentage development (86.10%±1.90) than the control group (79.55%±2.64), whereas 100 ng/ml (79.83% ±3.70), 10 ng/ml (75.80% ±3.16) and 25 ng/ml (83.73% ±3.14) showed no significant differences. Though 8-cell stage did not exhibited any statistical difference (P>0.05) shown in Table 2, the 50 ng/ml IGF-1 showed the highest percentage of 8- cell stage embryo (64.00% ±3.67) and the lowest was recorded for the control group (51.48% ±3.72). At the 16-cell stage, 50 ng/ml IGF-1 supplementation increased developmental rate significantly (46.34% ±4.00) as compared to the control group (30.89% ±4.42), while other concentrations showing intermediate results. In the category of morula formation, 50 ng/ml (32.53% ±5.11) and 100 ng/ml (31.51% ±5.29) were showing significantly higher rate than the control group (17.93% ±3.71), while low concentration viz.10 ng/ml (17.02% ±3.04) and 25 ng/ml (20.34% ±2.68) did not show any significant difference. In the blastocyst development 50 ng/ml IGF-1 markedly improved (15.81% ± 1.07) as compared to the control group (6.01% ±1.24) and with 100 ng/ml (10.63% ±1.04). In the present study overall @ 50 ng/ml IGF-1 consistently promoted significantly higher (p<0.05) embryonic develop-ment at most of the stages, while other concentrations exhibited limited or intermediate effects (Table 2, Fig 1B to G). The beneficial effect of IGF-1 on embryo development is supported by prior studies, which reported increased cleavage rate, blastomere viability and blastocyst yield following IGF supplementation (Velazquez et al., 2008). Spanos et al. (2000) reported in human preimplantation embryos that IGF-1 increased the proportion of embryos reaching the blastocyst stage (from 49% to 74%) and significantly decreased apoptotic nuclei (from 16.3% to 8.7%). IGF-1 appears to exert its effect by promoting cell survival, cell division, reducing apoptosis and enhancing metabolic activity in early embryos (Sirard et al., 2006). This strongly suggests that IGF-1 acts as a survival factor during early embryonic development.

The present study demonstrated that supplementation with IGF-1 @ 50 ng/ml during in vitro maturation and culture of cattle oocytes and embryos enhanced the maturation of immature oocytes and promoted the developmental competence of early-stage embryos.

This work was supported by grants from ICAR-National Dairy Research Institute, Karnal. The authors acknowledge sincere thanks to the Director, Joint Director (Research), National Dairy Research Institute, Karnal and Head, Eastern Regional Station, National Dairy Research Institute, Kalyani for providing the necessary facilities to carry out the work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.