Abstract

Environmental pollutants and lifestyle severely threaten human and animal health, leading to disturbances of various functions, including infertility. So, exploring a safe treatment that could effectively reverse infertility remains a challenge. The current study was intended to explore the fertility-enhancing effect of Juglans Regia oil in two successive generations of rats; F₀ and F₁. J. Regia oil was initially tested for in vitro antioxidant assay via ROS and DPPH, followed by in vivo toxicity testing. In the fertility assessment, eighteen pairs of male and female rats (n=36, 1:1, F₀ generation) were divided into three groups and dosed with 1 mL/kg and 2 mL/kg daily of J. Regia oil and saline, respectively, up to pre-cohabitation, cohabitation, gestation and lactation periods. The reproductive performance, including body weight, live birth index, fertility index, and litter size, was assessed. Hormonal and antioxidant markers of F₁ generations were assessed with the histopathological evaluation of male and female organs. The oil of J. Regia showed great antioxidant potential (P < 0.05) in DPPH (1,1-diphenyl-2-picrylhydrazyl) and ROS (Reactive Oxygen Species) methods (P<0.05). The continued exposure of the F₀ and F₁ generations to J. Regia oil did not affect body weight, fertility index, litter size, and survival index. We have found pronounced fertility outcomes in both genders of F₀ and F₁ generations with J. Regia 2 mL/kg/day in comparison to the control. Results showed that J. Regia significantly increased (P < 0.05) luteinizing hormone (LH), plasma testosterone, follicular stimulating hormone (FSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in both generations. Histology of both generations reveals improved spermatogenesis and folliculogenesis with enhanced architecture. Altogether, the present results suggest that J. Regia improved fertility in both male and female rats by improving hormonal activities and oxidative stress.

Keywords:
fertility; sex hormones; walnut; two-generation study; oxidative stress

Resumo

Os poluentes ambientais e o estilo de vida ameaçam gravemente a saúde humana e animal, levando a distúrbios de diversas funções, incluindo a infertilidade. Assim, explorar um tratamento seguro que possa reverter eficazmente a infertilidade continua sendo um desafio. O presente estudo teve como objetivo explorar o efeito do aumento da fertilidade do óleo de Juglans regia em duas gerações sucessivas de ratos: F0 e F1. O óleo de J. regia foi inicialmente testado para ensaio antioxidante in vitro via ERO e DPPH, seguido por testes de toxicidade in vivo. Na avaliação da fertilidade, 18 pares de ratos machos e fêmeas (n = 36; 1:1; geração F0) foram divididos em três grupos e dosados com 1 mL/kg e 2 mL/kg diariamente de óleo e solução salina de J. regia, respectivamente, até os períodos de pré-coabitação, coabitação, gestação e lactação. Foi avaliado o desempenho reprodutivo, incluindo peso corporal, índice de nascidos vivos, índice de fertilidade e tamanho da ninhada. Marcadores hormonais e antioxidantes das gerações F1 foram mensurados por meio da avaliação histopatológica de órgãos masculinos e femininos. O óleo de J. regia apresentou grande potencial antioxidante (P < 0,05) nos métodos DPPH (1,1-difenil-2-picril-hidrazil) e ERO (Espécies Reativas de Oxigênio) (P < 0,05). A exposição contínua das gerações F0 e F1 ao óleo de J. regia não afetou o peso corporal, o índice de fertilidade, o tamanho da ninhada e o índice de sobrevivência. Encontramos resultados relevantes de fertilidade em ambos os sexos das gerações F0 e F1 com 2 mL/kg/dia de J. regia em comparação com o controle. Os resultados mostraram que J. regia aumentou significativamente (P < 0,05) as atividades do hormônio luteinizante (LH), testosterona plasmática, hormônio foliculoestimulante (FSH), glutationa peroxidase (GPx) e superóxido dismutase (SOD) em ambas as gerações. A histologia de ambas as gerações revela espermatogênese e foliculogênese melhoradas com arquitetura aprimorada. De modo geral, os presentes resultados sugerem que J. regia melhorou a fertilidade em ratos machos e fêmeas, as atividades hormonais e o estresse oxidativo.

Palavras-chave:
fertilidade; hormônios sexuais; nozes; estudo de duas gerações; estresse oxidativo

1. Introduction

Infertility can be defined as the failure to accomplish a pregnancy clinically after 12 or more months of regular unsafe sexual intercourse (Szamatowicz and Szamatowicz, 2020SZAMATOWICZ, M. and SZAMATOWICZ, J., 2020. Proven and unproven methods for diagnosis and treatment of infertility. Advances in Medical Sciences, vol. 65, no. 1, pp. 93-96. http://dx.doi.org/10.1016/j.advms.2019.12.008. PMid:31923772.
http://dx.doi.org/10.1016/j.advms.2019.1... ). It has been estimated that around 15% of couples are infertile globally (Sun et al., 2019SUN, H., GONG, T.-T., JIANG, Y.-T., ZHANG, S., ZHAO, Y.-H. and WU, Q.-J., 2019. Global, regional, and national prevalence and disability-adjusted life-years for infertility in 195 countries and territories, 1990-2017: results from a global burden of disease study. Aging, vol. 11, no. 23, pp. 10952-10991. http://dx.doi.org/10.18632/aging.102497. PMid:31790362.
http://dx.doi.org/10.18632/aging.102497... ). Within all infertility issues, human sterility attributed to male fertility issues reports for 45-50% (Kumar and Singh, 2015KUMAR, N. and SINGH, A.K., 2015. Trends of male factor infertility, an important cause of infertility: a review of literature. Journal of Human Reproductive Sciences, vol. 8, no. 4, pp. 191-196. http://dx.doi.org/10.4103/0974-1208.170370. PMid:26752853.
http://dx.doi.org/10.4103/0974-1208.1703... ), and female accounts for 46.6% of polycystic ovarian syndrome cases (PCOS), constituting the principal cause (Deshpande and Gupta, 2019DESHPANDE, P.S. and GUPTA, A.S., 2019. Causes and prevalence of factors causing infertility in a public health facility. Journal of Human Reproductive Sciences, vol. 12, no. 4, pp. 287-293. http://dx.doi.org/10.4103/jhrs.JHRS_140_18. PMid:32038077.
http://dx.doi.org/10.4103/jhrs.JHRS_140_... ). To date, factors underlying the dysfunction of the reproductive system are partly understood. The use of recent drugs to improve fertility dysfunction is insignificant in achieving higher pregnancy outcomes (Ye et al., 2020YE, R.J., YANG, J.M., HAI, D.M., LIU, N., MA, L., LAN, X.B., NIU, J.G., ZHENG, P. and YU, J.Q., 2020. Interplay between male reproductive system dysfunction and the therapeutic effect of flavonoids. Fitoterapia, vol. 147, pp. 104756. http://dx.doi.org/10.1016/j.fitote.2020.104756. PMid:33069836.
http://dx.doi.org/10.1016/j.fitote.2020.... ). So, the future focus will be on formulating more viable treatment choices to prevent or treat dysfunctions of the reproductive system to improve fertility.

For several years, locally grown plants in place of supplements have been used globally to vitalize, energize, and enhance male sexual functions. Several plants extract have now been identified for improving fertility and other sexual functions, including Parkia Biglobosa (Obeten et al., 2022OBETEN, K.E., ISAAC, U.E., ARCHIBONG, V.B., OSHIE, P.A., ETUKUDO, E., ONANUGA, I.O., OCHIENG, J.J. and LEMUEL, A.M., 2022. Histomorphology and sperm profile of animal models administered with aqueous seed extract of Parkia biglobosa. Journal of Cell and Animal Biology, vol. 15, no. 1, pp. 1-8. http://dx.doi.org/10.5897/JCAB2022.0471.
http://dx.doi.org/10.5897/JCAB2022.0471... ), Eugenia Uniflora (Nkpurukwe et al., 2022NKPURUKWE, C.I., OBIANDU, C., OWHORJI, B.I., TAMUNO-OPUBO, A., OBIANDU, A.C. and OKARI, K., 2022. Improvement in some reproductive parameters of male wistar rats administered with leaf extract of Eugenia uniflora. Sch J App Med Sci, vol. 10, no. 5, pp. 793-797. http://dx.doi.org/10.36347/sjams.2022.v10i05.020.
http://dx.doi.org/10.36347/sjams.2022.v1... ), Bryonia Laciniosa (Sud and Sud, 2017SUD, K. and SUD, S.J.E.J.B., 2017. A scientific review on Shivlingi Beej (Bryonia laciniosa): a mystical ethno-medicine for infertility. European Journal of Biomedical and Pharmaceutical Sciences, vol. 4, no. 8, pp. 1098-1102.), Terminalia Catappa (Oyeniran et al., 2021OYENIRAN, O.H., ADEMILUYI, A.O. and OBOH, G., 2021. Comparative study of the phenolic profile, antioxidant properties, and inhibitory effects of Moringa (Moringa oleifera Lam.) and Almond (Terminalia catappa Linn.) leaves on acetylcholinesterase and monoamine oxidase activities in the head region of Fruitfly (Drosophila melanogaster Meigen) in vitro. Journal of Food Biochemistry, vol. 45, no. 3, pp. e13401. http://dx.doi.org/10.1111/jfbc.13401. PMid:32691858.
http://dx.doi.org/10.1111/jfbc.13401... ), Tribulus Terrestris (Zheleva-Dimitrova et al., 2012ZHELEVA-DIMITROVA, D., OBRESHKOVA, D. and NEDIALKOV, P.J., 2012. Antioxidant activity of tribulus terrestris: a natural product in infertility therapy. International Journal of Pharmacy and Pharmaceutical Sciences, vol. 4, no. 4, pp. 508-511.) and Lepidium Meyenii (Tafuri et al., 2021TAFURI, S., COCCHIA, N., VASSETTI, A., CAROTENUTO, D., ESPOSITO, L., MARUCCIO, L., AVALLONE, L. and CIANI, F., 2021. Lepidium meyenii (Maca) in male reproduction. Natural Product Research, vol. 35, no. 22, pp. 4550-4559. http://dx.doi.org/10.1080/14786419.2019.1698572. PMid:31805775.
http://dx.doi.org/10.1080/14786419.2019.... ). The active constituents of these plants, including terpenoids, phenols, alkaloids, and saponins, may have a role in restoring fertility (Ogidi et al., 2019OGIDI, O.I., GEORGE, D.G. and ESIE, N.G., 2019. Ethnopharmacological properties of Vernonia amygdalina (Bitter Leave) medicinal plant. Journal of Medicinal Plants Studies, vol. 7, no. 2, pp. 175-181.). Polyphenols, especially flavonoids, are recognized to have estrogenic (Akbaribazm et al., 2021AKBARIBAZM, M., GOODARZI, N. and RAHIMI, M., 2021. Female infertility and herbal medicine: an overview of the new findings. Food Science & Nutrition, vol. 9, no. 10, pp. 5869-5882. http://dx.doi.org/10.1002/fsn3.2523. PMid:34646552.
http://dx.doi.org/10.1002/fsn3.2523... ) or androgenic capabilities (Abaho et al., 2022ABAHO, I., MASEMBE, C., AKOLL, P. and JONES, C.L.W., 2022. The use of plant extracts to control tilapia reproduction: current status and future perspectives. Journal of the World Aquaculture Society, vol. 53, no. 3, pp. 593-619. http://dx.doi.org/10.1111/jwas.12863.
http://dx.doi.org/10.1111/jwas.12863... ) that have a crucial role in maintaining fertility. Such findings pointed out that flavonoids can be crucial in recovering spermatozoa and spermatogenesis (Ye et al., 2020YE, R.J., YANG, J.M., HAI, D.M., LIU, N., MA, L., LAN, X.B., NIU, J.G., ZHENG, P. and YU, J.Q., 2020. Interplay between male reproductive system dysfunction and the therapeutic effect of flavonoids. Fitoterapia, vol. 147, pp. 104756. http://dx.doi.org/10.1016/j.fitote.2020.104756. PMid:33069836.
http://dx.doi.org/10.1016/j.fitote.2020.... ).

One of the flavonoid-rich fruit is walnut (Juglans Regia L.) of family Juglandaceae. It contains many bioactive compounds like serotonin and melatonin, also rich in magnesium and other minerals and polyunsaturated fatty acid (Tapia et al., 2013TAPIA, M.I., SÁNCHEZ-MORGADO, J.R., GARCÍA-PARRA, J., RAMÍREZ, R., HERNÁNDEZ, T. and GONZÁLEZ-GÓMEZ, D., 2013. Comparative study of the nutritional and bioactive compounds content of four walnut (Juglans regia L.) cultivars. Journal of Food Composition and Analysis, vol. 31, no. 2, pp. 232-237. http://dx.doi.org/10.1016/j.jfca.2013.06.004.
http://dx.doi.org/10.1016/j.jfca.2013.06... ). A study conducted on essential oils taken from the leaves of J. Regia were analyzed by both GC-FID and GC-MS methods and exhibited a total of 38 compounds. High antioxidant activity was found in essential oil like polyunsaturated omega three and omega six fatty acids (Rather et al., 2012RATHER, M.A., DAR, B.A., DAR, M.Y., WANI, B.A., SHAH, W.A., BHAT, B.A., GANAI, B.A., BHAT, K.A., ANAND, R. and QURISHI, M.A., 2012. Chemical composition, antioxidant and antibacterial activities of the leaf essential oil of Juglans regia L. and its constituents. Phytomedicine, vol. 19, no. 13, pp. 1185-1190. http://dx.doi.org/10.1016/j.phymed.2012.07.018. PMid:22951389.
http://dx.doi.org/10.1016/j.phymed.2012.... ). Antioxidant potential and phenolic content were assessed in oils, kernels and bagasse pellets of various J. Regia cultivars.

Adelakun et al. (2019b)ADELAKUN, S.A., UKWENYA, V.O., OGUNLADE, B.S., A, J. and G, A., 2019b. Nitrite-induced testicular toxicity in rats: therapeutic potential of walnut oil. JBRA Assisted Reproduction, vol. 23, no. 1, pp. 15-23. PMid:30106544. studied the reproductive capacity of J. Regia by conducting an experiment on rats. The findings of this research proposed that male rats showed increased reproductive capacity as a result of taking oil by increasing sperm count. Ikwuka et al. (2021)IKWUKA, D.C., ANYAEHIE, B.U., NWOBODO, E., UMEGBOLU, E.I. and NWORGU, C.C., 2021. Ameliorative effects of African walnut on nicotine-induced reproductive toxicity in rat model. International Journal of Health Sciences, vol. 15, no. 1, pp. 3-8. PMid:33456436. also support this finding that the frequent use of J. Regia boosts male fertility in Wistar rats. This improvement in sperm count and motility may be due to the presence of glycosides, saponins, PUFs and alkaloids that have antioxidant potential. Moreover, saponins may also improve sperm parameters, as reported earlier. Similarly, Masterson et al. (2020)MASTERSON, J.M., KIM, H.H. and ROBBINS, W.A., 2020. Walnuts improve semen quality in infertile men: a randomized control dietary intervention trial. Fertility and Sterility, vol. 114, no. 3, pp. e23-e24. http://dx.doi.org/10.1016/j.fertnstert.2020.08.092.
http://dx.doi.org/10.1016/j.fertnstert.2... found that J. Regia improved the semen quality and sperm motility.

Reproductive hormones are essential in the development and regular functions of the reproductive system. FSH and Testosterone are important for the fulfillment of reproductive abilities in males (Walker and Cheng, 2005WALKER, W.H. and CHENG, J., 2005. FSH and testosterone signaling in Sertoli cells. Reproduction, vol. 130, no. 1, pp. 15-28. http://dx.doi.org/10.1530/rep.1.00358. PMid:15985628.
http://dx.doi.org/10.1530/rep.1.00358... ). Studies showed that sex hormones like FSH, LH, and testosterone levels are associated with spermatogenesis (Khaki et al., 2009KHAKI, A., FATHIAZAD, F., NOURI, M., KHAKI, A.A., KHAMENEHI, H.J. and HAMADEH, M., 2009. Evaluation of androgenic activity of Allium cepa on spermatogenesis in the rat. Folia Morphologica, vol. 68, no. 1, pp. 45-51. PMid:19384830.). Similarly, in females, the causes of infertility have been linked to disturbances in hormones, including FSH, LH, Estradiol, etc. LH plays a crucial role in the menstrual cycle and functions in conjunction with FSH. The FSH/LH production is governed by estrogen level. The increase in estrogen level makes the pituitary gland to start making additional LH and to halt FSH production. The LH shift makes the egg release from the ovary by a process named as ovulation (Ali and Hisham, 2016ALI, A. and HISHAM, Q., 2016. The role of FSH, LH and prolactin hormones in female infertility. International Journal of PharmTech Research, vol. 6, no. 5, pp. 109-118.). Sudha and Reddy reported that infertility/ anovulation may be caused by hormonal imbalance, as balance in hormones is necessary to produce adequate follicles required for ovule development (Sudha and Reddy, 2013SUDHA, G. and REDDY, K., 2013. Causes of female infertility: a crosssectional study. International Journal of Latest Research in Science and Technology, vol. 2, no. 6, pp. 119-123.). Plant-derived polyphenols that influence endocrine/hormonal activities in humans have received great attraction for researchers because of their possible beneficial effects with no adverse effect.

J. Regia presented its exclusive actions by augmenting fertility and decreasing fertility-related problems, including hormonal imbalance. Assessment of body weight and blood parameters provides information regarding an animal's health and reproductive effects (Aly et al., 2009ALY, H.A., DOMÈNECH, Ò. and ABDEL-NAIM, A.B., 2009. Aroclor 1254 impairs spermatogenesis and induces oxidative stress in rat testicular mitochondria. Food and Chemical Toxicology, vol. 47, no. 8, pp. 1733-1738. http://dx.doi.org/10.1016/j.fct.2009.03.019. PMid:19306909.
http://dx.doi.org/10.1016/j.fct.2009.03.... ). However, no research has been executed to explore the beneficial role of J. Regia on reproduction in two generations. Therefore, a two-generational study was established to explore the beneficial and protective use of J. Regia oil on fertility and reproductive hormones in experimental animals.

2. Materials and Methods

2.1. Chemicals

All chemicals used in this study were of analytical grades including Ferric Chloride, Petroleum ether, Ethanol (96%), Formalin, Potassium hydroxide, Glacial acetic acid, Sulphuric acid, Sodium Hydroxide, Mayer’s and Dragendoff’s reagents, HCl, Tetramethylbenzidine (TMB), HRP (Horseradish peroxidase), FBS (Fetal bovine serum), Etoposide, DPPH, DMEM (Dulbecco’s Modified Eagle Medium), Gallic acid and Folin-Ciocalteu reagent.

2.2. Identification and extraction of plant materials

The walnuts were purchased from a local market in Quetta (Baluchistan), Pakistan, along with their plants, and were identified at the KU Herbarium and Botanical Garden, University of Karachi. The specimens were identified as Juglans Regia L. of Juglandaceae, G.H. No. 95591. The fruits were then manually cracked, shelled, and subjected to expeller pressing to obtain oils. This method for acquiring oil from oleaginous seeds is the simplest and oldest method with no use of chemicals. The superior quality oil is extracted, and it utilizes low operation costs than solvent extraction.

2.3. Preliminary phytochemical screening

A small quantity of samples was taken for the phytochemical tests by methods in accordance with little modifications (Abdillah et al., 2015ABDILLAH, S., TAMBUNAN, R.M., FARIDA, Y., SANDHIUTAMI, N.M.D. and DEWI, R.M., 2015. Phytochemical screening and antimalarial activity of some plants traditionally used in Indonesia. Asian Pacific Journal of Tropical Disease, vol. 5, no. 6, pp. 454-457. http://dx.doi.org/10.1016/S2222-1808(15)60814-3.
http://dx.doi.org/10.1016/S2222-1808(15)... ; Alshammaa, 2016ALSHAMMAA, D., 2016. Preliminary screening and phytochemical profile of Mangifera indica leave’s extracts, cultivated in Iraq. International Journal of Current Microbiology and Applied Sciences, vol. 5, no. 9, pp. 163-173. http://dx.doi.org/10.20546/ijcmas.2016.509.018.
http://dx.doi.org/10.20546/ijcmas.2016.5... ; Esienanwan et al., 2020ESIENANWAN, E.E., LUAVESE, P.A., EZINNE, C.C., ADEPOJU, I.O. and GODWIN, O., 2020. Comparative qualitative phytochemical analysis of oil, juice and dry forms of garlic (Allium sativum) and different varieties of onions (Allium cepa) consumed in Makurdi metropolis. International Journal of Plant Physiology and Biochemistry, vol. 12, no. 1, pp. 9-16. http://dx.doi.org/10.5897/IJPPB2019.0285.
http://dx.doi.org/10.5897/IJPPB2019.0285... ).

2.4. Antioxidant assays

2.4.1. Antioxidant assay by ROS

Primary cortical cells were used for the determination of Reactive Oxygen Species (ROS) generation. The method was adopted with some modifications (Kamiloglu et al., 2020KAMILOGLU, S., SARI, G., OZDAL, T. and CAPANOGLU, E., 2020. Guidelines for cell viability assays. Food Frontiers, vol. 1, no. 3, pp. 332-349. http://dx.doi.org/10.1002/fft2.44.
http://dx.doi.org/10.1002/fft2.44... ; Vargas et al., 2014VARGAS, F.S., SOARES, D.G., RIBEIRO, A.P., HEBLING, J. and SOUZA COSTA, C.A., 2014. Protective effect of alpha-tocopherol isomer from vitamin E against the H2O2 induced toxicity on dental pulp cells. BioMed Research International, vol. 2014, pp. 895049. PMid:24587995.).

2.4.2. Antioxidant assay by DPPH

An upgraded DPPH assay (1,1-diphenyl-2-picrylhydrazyl) was selected for the evaluation of tested compounds. The DPPH radicals diminish at 517 nm of absorbance in the presence of antioxidant potential. The test compounds at different concentrations (10, 20, 100, 200 and 300 μg/mL) were added to 0.1mM DPPH (3 mL) and allowed to react. The mixture was swirled and incubated at room temperature for 30 min. A microplate reader (SkanIt Software 5.0 of RE, ver. 5.0.0.42) was utilized to assess the absorbance at 517 nm. The test compound reducing power was calculated with the absorbance of ascorbic acid, which served as control (Arif et al., 2022ARIF, A., MAJEED, S., SARFARAZ, M., LODHI, M., KHATIAN, N., AZIZ, A. and MAJEED, H.K., 2022. Evaluation of anti-inflammatory, antioxidant and xanthine oxidase inhibitory potential of N-(2-hydroxy phenyl). Acetamide., vol. 5, no. 2, pp. 309-318. http://dx.doi.org/10.31580/pjmls.v5i2.2528.
http://dx.doi.org/10.31580/pjmls.v5i2.25... ).

Radical Scavenging Activity was equated in percentage via Equation 1.

2.5. Brine shrimp toxicity testing

Toxicity of A. Cepa was evaluated to rule out % Mortality with the help of the Brine shrimp method (Artemia Saline). Etoposide served as a positive control in this method (Meyer et al., 1982MEYER, B., FERRIGNI, N., PUTNAM, J., JACOBSEN, L., NICHOLS, D. and MCLAUGHLIN, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica, vol. 45, no. 5, pp. 31-34. http://dx.doi.org/10.1055/s-2007-971236. PMid:17396775.
http://dx.doi.org/10.1055/s-2007-971236... ).

2.6. Acute oral toxicity

The study on the acute oral toxicity of J. Regia was executed according to the OECD guidelines (OECD, 1987ORGANISATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 1987. OECD guideline for the testing of chemicals: acute oral toxicity. Paris: OECD.). Rats of both genders were used and were supplied with distilled water and feed adlibitum. Twenty rats were randomly grouped into four groups (n=5). A single oral dose was given; J. Regia was administered to rats (n=5) at a dose of (100,1000 and 2000 mg/kg bw) in comparison to the control. The rats were examined for toxicity signs like behavioral changes and mortality for 48 hours (Oyewusi et al., 2015OYEWUSI, J., SABA, A. and OLUKUNLE, J., 2015. Phytochemical, elemental and acute toxic effects of methanol extract of onion (Allium cepa) bulbs in Wistar albino rats experimentaly pre-exposed and rested for a week. Vom Journal of Veterinary Science, vol. 10, pp. 65-74.; Saidu et al., 2007SAIDU, Y., BILBIS, L., LAWAL, M., ISEZUO, S., HASSAN, S. and ABBAS, A., 2007. Acute and sub-chronic toxicity studies of crude aqueous extract of Albizzia chevalieri harms (Leguminosae). Asian Journal of Biochemistry, vol. 2, no. 4, pp. 224-236. http://dx.doi.org/10.3923/ajb.2007.224.236.
http://dx.doi.org/10.3923/ajb.2007.224.2... ).

2.7. Animal ethical approval

The study was performed under the approval of the Board for Advanced Studies and Research dated 28-03-2019 (reference No 05071/pharm), University of Karachi and Pharmacology Departmental Research Committee, the for use of animals according to the National Institute of Health guidelines (NIH guidelines Islamabad, Pakistan) (NRC, 2011NATIONAL RESEARCH COUNCIL – NRC, 2011. Guide for the care and use of laboratory animals. Washington, DC: National Academies Press.).

2.8. Experimental animals

Healthy and sexually mature Wistar rats of both sexes were equally used for the experimental fertility study. The animals were procured from the animal house, H.E.J, University of Karachi, and were kept in plastic cages having proper ventilation under standard temperature conditions (25 to 30 ^oC) with 12 h light and dark cycle. The experimental animals were placed in an animal house Department of Pharmacology, University of Karachi. Male rats aged 12 weeks (140-160 g) and female rats aged 14 weeks (160 g to 180 g) were used and given an acclimatization period for a week. The experimental protocol fulfills the guidelines on the appropriate use and care of laboratory animals (NRC, 2011NATIONAL RESEARCH COUNCIL – NRC, 2011. Guide for the care and use of laboratory animals. Washington, DC: National Academies Press.), and the experimental protocol and design was permitted by the Board of Advanced Studies and Research (BASR), University of Karachi, ASRB/No./05071/Pharm {Resolution No. 10(P)07}.

2.9. Experimental protocol

The study was conducted for a total duration of seven months. A modified method of Vohra and Khera (2016)VOHRA, P. and KHERA, K.S., 2016. Effect of imidacloprid on reproduction of female albino rats in three generation study. Journal of Veterinary Science & Technology, vol. 7, no. 4, pp. 2-7. http://dx.doi.org/10.4172/2157-7579.1000340.
http://dx.doi.org/10.4172/2157-7579.1000... was used. Eighteen pairs of rats (n=36, F₀ generation) were grouped into six experimental groups (n=6) randomly; three males and three females in each group, and were accordingly treated. Group I served as control and administered distilled water daily; Group II animals were given a low dose of J. Regia oil, 1 mL/Kg/day; Group III animals were given J. Regia oil at a high dose of 2 mL/Kg/day.

All animals were dosed 30 days by gavage before mating then male animals in each group were singly paired with female animals for seven days. Female rats were subjected to the soiled bedding of an adult male rat to align their estrus cycle (female animals were fertile and receptive during this period) (Ain et al., 2022AIN, N.U., KHAN, R.A., MIRZA, T. and FAYYAZ, T.B., 2022. The effects of Ficus carica on male and female reproductive capabilities in rats. Evidence-Based Complementary and Alternative Medicine, vol. 2022, pp. 1799431. http://dx.doi.org/10.1155/2022/1799431. PMid:36317103.
http://dx.doi.org/10.1155/2022/1799431... ). The vaginal plug was noticed to confirm the pregnancy of each female rat. Body weight was weekly recorded before mating, during mating, and in gestational periods. All F₀ and F₁ female parental animals were monitored twice daily for any toxicity signs. F₀ and F₁ animals were treated with J. Regia oil throughout the pre-cohabitation (30 days), cohabitation (21 days), gestation (21 days), and lactation periods.

The expected female rats were permitted to carry the pregnancy to term. Afterwards fertility indices (fertility index, the number of pups, survival index) were estimated. Offspring relating to both generations were counted and observed for any abnormality on postnatal days 0, 4,7, 14 and 21 days.

The same doses of the J. Regia oil (as of the F₀ generation) were given to F₁ off-springs during their growth, adulthood, and up to breeding. Pregnant female animals of the F₁ generation were continuously given the J. Regia oil during their pre-cohabitation, cohabitation period, gestation and lactation until the F₂ generation pups were weaned. Afterwards, when pups were delivered, all F₁ animals were sacrificed and their blood samples and reproductive organs were taken for biochemical and histopathological assessment. The same protocol for the F₀ generation was followed till the birth of the F₂ generation (Vohra and Khera, 2016VOHRA, P. and KHERA, K.S., 2016. Effect of imidacloprid on reproduction of female albino rats in three generation study. Journal of Veterinary Science & Technology, vol. 7, no. 4, pp. 2-7. http://dx.doi.org/10.4172/2157-7579.1000340.
http://dx.doi.org/10.4172/2157-7579.1000... ).

2.10. Reproductive performance of F₀ and F₁ rats

The reproductive parameters studied were mentioned in the form of ratios, weights, and indices that analyze all stages starting from conception till weaning. These fertility parameters were computed as follows (Equations 2 to CHIANG, C. and FLAWS, J.A., 2019. Subchronic exposure to di (2-ethylhexyl) phthalate and diisononyl phthalate during adulthood has immediate and long-term reproductive consequences in female mice. Toxicological Sciences, vol. 168, no. 2, pp. 620-631. http://dx.doi.org/10.1093/toxsci/kfz013. PMid:30649530.
http://dx.doi.org/10.1093/toxsci/kfz013... NARAYANA, K., PRASHANTHI, N., NAYANATARA, A., KUMAR, H.H., ABHILASH, K. and BAIRY, K.L., 2005. Effects of methyl parathion (o, o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis. Mutation Research, vol. 588, no. 1, pp. 28-34. http://dx.doi.org/10.1016/j.mrgentox.2005.08.012. PMid:16226487.
http://dx.doi.org/10.1016/j.mrgentox.200... 6):

2.11. Blood parameter assessment

All F₁ generation male and female animals were sacrificed and their blood samples were collected by heart puncture and kept in B-Ject gel clot activator vacuum tubes under aseptic conditions. Samples were centrifuged in Eppendorf machine at 4000 rpm for 10 min. The separated serum was kept at -80 ^oC for hormonal and biochemical testing. All samples were analyzed for hormonal estimation (FSH, LH, testosterone, and estradiol (Alam, 2019ALAM, J.M., 2019. Anti-Müllerian Hormone (AMH): predictor of follicular function, fertility status and onset of menopause. Chemistry Research Journal, vol. 4, no. 5, pp. 76-80.; Sultana et al., 2018SULTANA, I., ALAM, J.M., ALI, H. and NOUREEN, S., 2018. Analysis and comparison of total, bio-available and free testosterone levels in various age groups of men. Chemistry Research Journal, vol. 3, no. 2, pp. 181-186.) via ELISA Roche Diagnostics, Basil). SOD and Glutathione (GPx) were estimated by the kit acquired from Glory Science Co., Ltd. (Glory Science Co.2022#45).

2.12. Histopathological evaluation

The rats were sacrificed by cutting off the diaphragm. Ovaries and testes of the selected rats of F₁ generation were identified with the help of a histopathologist; ovaries were dissected out from the surrounding fats, and measured weight. The separation of the horns of the uterus from the vagina was done by cutting the uppermost point of the cervix than preserved in 10% formalin, and later treated through usual processes and embedded in paraffin. Similarly, testes were removed from the scrotum, dissected from adherent tissues, weighed then fixed in 10% formalin, and later treated through usual processes and embedded in paraffin. Blocks of paraffin were cut serially and longitudinally at 4 μm thickness, followed by Hematoxylin-Eosin staining. Stained slides were analyzed and compared in groups (Ellenburg et al., 2020ELLENBURG, J.L., KOLETTIS, P., DRWIEGA, J.C., POSEY, A.M., GOLDBERG, M., MEHRAD, M., GIANNICO, G. and GORDETSKY, J., 2020. Formalin versus bouin solution for testis biopsies: which is the better fixative? Clinical Pathology, vol. 13, pp. X19897262. http://dx.doi.org/10.1177/2632010X19897262. PMid:31922127.
http://dx.doi.org/10.1177/2632010X198972... ).

2.13. Statistical analysis

Statistical analysis was implemented using the SPSS software (version 20.0). Multiple groups were compared by one-way analysis of variance applying the Bonferroni post-hoc test. The results were regarded to be statistically significant if the value lies between P < 0.05 and highly significant when P < 0.01.

3. Results

3.1. % yield of J. Regia oil

The percentage yield of J. Regia oil was 47.62% from 8 Kg of fruits.

3.2. Preliminary phytochemical screening

The presence of J. Regia constituents was mentioned in Table 1. The qualitative phytochemical analysis reveals that Juglans Regia possesses flavonoids, saponins and phenols.

3.3. Antioxidant assays

3.3.1. Antioxidant assay of J. Regia by ROS

The DCF-DA method was employed to determine the ROS production and % Viability of J. Regia oil on primary cortical neuronal cells and was found nontoxic and significantly prevented H₂O₂-induced oxidative damage. % Cell viability in neuronal cells after incubation using indicated concentrations of J. Regia is shown in Figure 1, which showed protective efficacy against H₂O₂-induced toxicity on neuronal cells and had no cytotoxic effect in neuronal cells at concentrations up to a dose of 100 (µg/mL).

3.3.2. Antioxidant assay of J. Regia by DPPH

The % inhibition of J. Regia oil at different concentrations is shown in Table 2. The oil was observed with radical scavenging potential by DPPH utilizing Ascorbic Acid as a reference standard. Among various concentrations, J. Regia exhibited the highest activity at 100 μg/mL and 300 μg/mL i.e., 74.65 ± 1.24% and 74.24 ± 4.94%, respectively; while Ascorbic acid exhibited the strongest scavenging activity of 88.14 ± 1.28% at 300 μg/mL.

3.4. Brine shrimp lethality testing

The J. Regia oil was assessed for brine shrimp lethality studies, and the results were mentioned in Table 3. No significant toxicity to brine shrimp Artemia salina was found with J. Regia oil at different concentrations, and significant results represented the oil is biologically safe. The % mortality of J. Regia at 1000 μg/mL concentration is 20% compared to standard Etoposide, which shows 100% mortality at 1000 μg/mL, indicating oil is significant and safe for use.

3.5. Acute oral toxicity

Regarding the safety of the J. Regia plant, acute toxicity showed that it is safe at low concentrations and lethal at higher doses (2000 mg/kg). Moreover, some dose-dependent behavioral changes were also observed in high-dose treated groups, including depression, clustering, folding and sleeping, unsteady gait, and loss of appetite. The results regarding the acute toxicity (LD₅₀) of J. Regia by using the Karber Method are mentioned below (as shown in Table 4). The arithmetic method of Karber was utilized for calculation.

3.6. Fertility assessment

The two-generation fecundity study was conducted for seven months that showed no congenital abnormalities in any of the pups of all the groups of F₀ and F₁ generations. The body weights of F₀ and F₁ animals are mentioned in Table 5, representing insignificant differences (P>0.05) among both generations and control.

The number of pups was counted in each generation and noticed for congenital abnormalities. In all groups, no congenital abnormality was observed. The number of pups increased significantly in the first and second generations compared to the control group (df 5, 30; F 3.37; P < 0.05).

Post Hoc analysis through the Bonferroni test reveals that high-dose administration of Juglans Regia produced statistically significant differences in the number of pups in both generations as compared to low-dose group and control group rats, as shown in Table 6.

3.7. Reproductive performance

The reproductive performance was assessed by observing several parameters, including live birth index, fertility index, and litter size. Insignificant changes were found in all treated groups' live birth, fertility, and survival indexes (P > 0.05). All these parameters were improved in all treated groups, just like the control group. However, a significant increase in litter size was noticed in the treated group in both generations (P < 0.05) when compared to the control group as shown in Table 7.

3.8. Hormonal parameters

FSH, LH, Estradiol and Testosterone levels were tested in the plasma of both control and treated male and female rats. In higher doses, statistically significant raised values of FSH and LH were found in female rats (df 5,30; F 32.38; P < 0.05) (df 5,30; F 105.2; P < 0.05), respectively, when compared to the control. Similarly, statistically significant differences in estradiol (df 5,30; F 101.8; P < 0.05) and testosterone (df 5,30; F 81.49; P < 0.05) were observed in male rats after administration of high doses of J. Regia as compared to control. A significant decrease was found in Estradiol in female animals (df 5,30; F 60.05; P < 0.05) as compared to the control (as shown in Figure 2).

3.9. Oxidative parameters

SOD levels in treated rats were found to be statistically highly significant in male rats (df 5,30; F 25.02; P < 0.01) and female rats (df 5,30; F 6.28; P < 0.01) with high dose treatment as compared to control. Additionally, statistically significant results in male (df 5,30; F 27.4; P < 0.05) and female (df 5,30; F 13.13; P < 0.05) were also observed on GPx levels with high dose treatment as compared to control (as shown in Figure 3).

3.10. Histopathological evaluation

Figure 4 displays the effect of J. Regia on histopathological parameters. Control ovaries (a) showed the external cortex surrounded by an intact capsule having a profuse stroma. A lot of blood vessels parted by unattached connective tissues in the medulla were also observed. The histological ovarian architecture of treated low-dose (b) animals was similar to controls. Similarly, high-dose J. Regia treatment slide (c) showed Graafian follicle and zona pellucida along with preantral, small antral, and large antral follicles and corpus luteum of different stages were observed.

Microscopic examination of the control group testis figure (a) showed an architecture of tubular seminiferous epithelia normally present with occasional Sertoli cells and spermatogenic cells at the interstitial area and base of the tubule having negligible connective tissues at the tubular surroundings. Microscopic examination of the low-dose treated testis (b) showed normal seminiferous tubules similar to the control group. Similarly, testis (c) treated with a high dose of J. Regia showed normal architecture of tubular seminiferous epithelia along with Sertoli cells and spermatogenic cells having minimal connective tissues at the tubular surrounding.

4. Discussion

J. Regia’s therapeutic benefits have been recognized since ancient civilizations. In traditional medicine, walnuts were used to treat diabetes, cancer, inflammation, diarrhea, prostate disorders and cardiovascular disorders (Zhang et al., 2021ZHANG, Y.Y., NI, Z.J., ELAM, E., ZHANG, F., THAKUR, K., WANG, S., ZHANG, J.G. and WEI, Z.J., 2021. Juglone, a novel activator of ferroptosis, induces cell death in endometrial carcinoma Ishikawa cells. Food & Function, vol. 12, no. 11, pp. 4947-4959. http://dx.doi.org/10.1039/D1FO00790D. PMid:34100505.
http://dx.doi.org/10.1039/D1FO00790D... ). Walnut fruit contains polyphenols, fatty acids, cardiac glycosides, carbohydrates, steroids, minerals, tannins, proteins, dietary fiber, melatonin, α-tocopherol, plant sterols, folate, vitamin E, vitamin C, and vitamin A family compounds (Rusu et al., 2020RUSU, M.E., FIZESAN, I., POP, A., MOCAN, A., GHELDIU, A.M., BABOTA, M., VODNAR, D.C., JURJ, A., BERINDAN-NEAGOE, I., VLASE, L. and POPA, D.S., 2020. Walnut (Juglans regia L.) Septum: assessment of bioactive molecules and in vitro biological effects. Molecules, vol. 25, no. 9, pp. 2187. http://dx.doi.org/10.3390/molecules25092187. PMid:32392837.
http://dx.doi.org/10.3390/molecules25092... ).

In the current study, J. Regia oil represented the presence of flavonoids, saponins and phenols. Phenolic compounds are usually associated with antiatherogenic, antioxidant, anti-inflammatory, and anticancer properties. Slatnar et al. (2015)SLATNAR, A., MIKULIC-PETKOVSEK, M., STAMPAR, F., VEBERIC, R. and SOLAR, A., 2015. Identification and quantification of phenolic compounds in kernels, oil and bagasse pellets of common walnut (Juglans regia L.). Food Research International, vol. 67, pp. 255-263. http://dx.doi.org/10.1016/j.foodres.2014.11.016.
http://dx.doi.org/10.1016/j.foodres.2014... reported high content of walnut phenolic content in the skin of J. Regia that covers the kernel. Recent studies suggest the presence of polyphenols, squalene and tocopherols strongly correlated to the antioxidant capacity of J. Regia oil (Gao et al., 2019GAO, P., CAO, Y., LIU, R., JIN, Q. and WANG, X., 2019. Phytochemical content, minor‐constituent compositions, and antioxidant capacity of screw‐pressed walnut oil obtained from roasted kernels. European Journal of Lipid Science and Technology, vol. 121, no. 1, pp. 1800292. http://dx.doi.org/10.1002/ejlt.201800292.
http://dx.doi.org/10.1002/ejlt.201800292... ; Jahanban-Esfahlan et al., 2019JAHANBAN-ESFAHLAN, A., OSTADRAHIMI, A., TABIBIAZAR, M. and AMAROWICZ, R., 2019. A comparative review on the extraction, antioxidant content and antioxidant potential of different parts of walnut (Juglans regia L.) fruit and tree. Molecules, vol. 24, no. 11, pp. 2133. http://dx.doi.org/10.3390/molecules24112133. PMid:31195762.
http://dx.doi.org/10.3390/molecules24112... ).

The antioxidant capacity of various oils can be determined accurately and rapidly using DPPH testing. In the current study, J. Regia showed 74.65% % inhibition at 100 µg/mL, that showed its highest free radical scavenging activity. Gao et al. (2022)GAO, P., DING, Y., CHEN, Z., ZHOU, Z., ZHONG, W., HU, C., HE, D. and WANG, X., 2022. Characteristics and antioxidant activity of walnut oil using various pretreatment and processing technologies. Foods, vol. 11, no. 12, pp. 1698. http://dx.doi.org/10.3390/foods11121698. PMid:35741896.
http://dx.doi.org/10.3390/foods11121698... documented the antioxidant role of J. Regia oil through DPPH and reported that it is due to the presence of δ-tocopherol and TPC in J. Regia oil. Our findings concluded that J. Regia oil exhibited greater antioxidant capacity, and this antioxidant potential is due to the occurrence of phenolic compounds, tocopherols and according to some recently reported data also, melatonin, an indoleamine that exhibits high antioxidant capacity (Arranz et al., 2008ARRANZ, S., CERT, R., PÉREZ-JIMÉNEZ, J., CERT, A. and SAURA-CALIXTO, F., 2008. Comparison between free radical scavenging capacity and oxidative stability of nut oils. Food Chemistry, vol. 110, no. 4, pp. 985-990. http://dx.doi.org/10.1016/j.foodchem.2008.03.021. PMid:26047290.
http://dx.doi.org/10.1016/j.foodchem.200... ; Pycia et al., 2019PYCIA, K., KAPUSTA, I., JAWORSKA, G. and JANKOWSKA, A., 2019. Antioxidant properties, profile of polyphenolic compounds and tocopherol content in various walnut (Juglans regia L.) varieties. European Food Research and Technology, vol. 245, no. 3, pp. 607-616. http://dx.doi.org/10.1007/s00217-018-3184-3.
http://dx.doi.org/10.1007/s00217-018-318... ).

The ROS production and % viability testing of J. Regia oil was tested by the DCF-DA method. It was found non-toxic in comparison to H₂O₂ and increased neuronal cell viability by 77.42% at 100 µg/mL. Laubertová et al. (2015LAUBERTOVÁ, L., KOŇARIKOVÁ, K., GBELCOVÁ, H., ĎURAČKOVÁ, Z. and ŽITŇANOVÁ, I., 2015. Effect of walnut oil on hyperglycemia-induced oxidative stress and pro-inflammatory cytokines production. European Journal of Nutrition, vol. 54, no. 2, pp. 291-299. http://dx.doi.org/10.1007/s00394-014-0710-3. PMid:24817646.
http://dx.doi.org/10.1007/s00394-014-071... ) reported J. Regia oil can increase SOD activity, can enhance the antioxidant capacity of cells, decrease ROS activity and can prevent the release of cytokines depending on oil concentration. Based on our results, we supposed that J. Regia oil has the antioxidant ability to neutralize reactive oxygen intermediates and may contribute to progressing endogenous antioxidant defenses.

Toxicity studies are experimental screening procedures used to affirm the safety of any drug or herbal product with animal models. The critical criterion for toxicological evaluation is Mortality (Liu et al., 2019LIU, M., YANG, S., YANG, J., LEE, Y., KOU, J. and WANG, C., 2019. Neuroprotective and memory-enhancing effects of antioxidant peptide from walnut (Juglans regia L.) protein hydrolysates. Natural Product Communications, vol. 14, no. 7, pp. 80. http://dx.doi.org/10.1177/1934578X19865838.
http://dx.doi.org/10.1177/1934578X198658... ). In the Brine shrimp lethality test, the % mortality of J. Regia oil was found to be 20% at a maximum concentration that shows it is safe at low concentrations and lethal at higher doses. Similarly, the result of its oral acute toxicity test revealed that it has a high safety range when administered orally. The results attained in our study of acute oral toxicity revealed no mortality at the evaluated dose of 1000 mg/kg BW. Thus, it is proposed that the oral lethal dose (LD50) of J. Regia oil is up to 1000 mg/kg BW. The animals were noticed to be normal, having no changes within their eyes, fur, skin and mucous membranes. Neither any noticeable toxicity signs were observed, including tremors, coma, convulsions, nor any behavioral change was found.

J. Regia oil is a rich source of polyunsaturated fatty acids (PUFs) required for many physiological mechanisms and fertility (Panth et al., 2016PANTH, N., PAUDEL, K.R. and KARKI, R., 2016. Phytochemical profile and biological activity of Juglans regia. Journal of Integrative Medicine, vol. 14, no. 5, pp. 359-373. http://dx.doi.org/10.1016/S2095-4964(16)60274-1. PMid:27641607.
http://dx.doi.org/10.1016/S2095-4964(16)... ). PUFs are used as an energy source during oocyte maturation and embryo development before implantation (Wathes et al., 2007WATHES, D.C., ABAYASEKARA, D.R. and AITKEN, R.J., 2007. Polyunsaturated fatty acids in male and female reproduction. Biology of Reproduction, vol. 77, no. 2, pp. 190-201. http://dx.doi.org/10.1095/biolreprod.107.060558. PMid:17442851.
http://dx.doi.org/10.1095/biolreprod.107... ). Robbin and colleagues reported that 75 g of walnuts per day added to a diet enhanced sperm motility, vitality, and morphology in a group of healthy young men as compared to the control group and considered improved semen quality and sperm motility associated with an increase in blood serum omega-6 and omega-3 PUFs (Robbins et al., 2012ROBBINS, W.A., XUN, L., FITZGERALD, L.Z., ESGUERRA, S., HENNING, S.M. and CARPENTER, C.L., 2012. Walnuts improve semen quality in men consuming a Western-style diet: randomized control dietary intervention trial. Biology of Reproduction, vol. 87, no. 4, pp. 101-108. http://dx.doi.org/10.1095/biolreprod.112.101634. PMid:22895856.
http://dx.doi.org/10.1095/biolreprod.112... ). Current data demonstrated an increased number of pups production in both F₁ and F₂ generations with both doses of J. Regia oil. This indicated that its oil presented beneficial actions by augmenting the fertility power of the female and male rats. J. Regia contains a significant amount of polyunsaturated fatty acids such as linoleic acid, oleic acid and linolenic acid (Arranz et al., 2008ARRANZ, S., CERT, R., PÉREZ-JIMÉNEZ, J., CERT, A. and SAURA-CALIXTO, F., 2008. Comparison between free radical scavenging capacity and oxidative stability of nut oils. Food Chemistry, vol. 110, no. 4, pp. 985-990. http://dx.doi.org/10.1016/j.foodchem.2008.03.021. PMid:26047290.
http://dx.doi.org/10.1016/j.foodchem.200... ). Thus preventing the conversion of testosterone to dihydrotestosterone by inhibiting 5-α reductase and this way restores the plasma testosterone level in males and estrogen in postmenopausal women (Assi et al., 2022ASSI, M.A., MOHAMMED, M.H. and ABDULKAHALEQ, L.A., 2022. Evaluate the effect of alcoholic extraction of walnut on gonadal hormones of treated rats. Caspian Journal of Environmental Sciences, vol. 20, no. 1, pp. 145-153.; Bostani et al., 2014BOSTANI, M., AQABABA, H., HOSSEINI, S.E. and ASHTIYANI, S.C., 2014. A study on the effects of walnut oil on plasma levels of testosterone pre and post puberty in male rats. American Journal of Ethnomedicine, vol. 1, no. 4, pp. 266-275.). However, it seems that enhanced testosterone levels using J. Regia oil were because of its effect on Leydig cells, as well as its intrusion with the testosterone biosynthesis was probably due to the prostaglandin synthesis stimulation (Assi et al., 2022ASSI, M.A., MOHAMMED, M.H. and ABDULKAHALEQ, L.A., 2022. Evaluate the effect of alcoholic extraction of walnut on gonadal hormones of treated rats. Caspian Journal of Environmental Sciences, vol. 20, no. 1, pp. 145-153.). We observed that rats in both F₁ and F₂ generations represented fertility-enhancing effects by increasing litter size. Similarly, F₂ off-springs showed improved live birth index in the high dose group compared to the control group. No effects were observed on the survival index.

Reproductive hormones are essential in the development and regular functions of the reproductive system. FSH and Testosterone are important for fulfilling reproductive abilities in males (Walker and Cheng, 2005WALKER, W.H. and CHENG, J., 2005. FSH and testosterone signaling in Sertoli cells. Reproduction, vol. 130, no. 1, pp. 15-28. http://dx.doi.org/10.1530/rep.1.00358. PMid:15985628.
http://dx.doi.org/10.1530/rep.1.00358... ). Studies showed that sex hormones like FSH, LH, and testosterone are associated with spermatogenesis (Khaki et al., 2009KHAKI, A., FATHIAZAD, F., NOURI, M., KHAKI, A.A., KHAMENEHI, H.J. and HAMADEH, M., 2009. Evaluation of androgenic activity of Allium cepa on spermatogenesis in the rat. Folia Morphologica, vol. 68, no. 1, pp. 45-51. PMid:19384830.). FSH, LH, testosterone, and Estradiol levels were estimated in both male and female animals. Significantly raised levels of LH and FSH were observed by J. Regia oil low and high doses in female rats. Similarly, Testosterone levels were found to be significantly raised in male rats, depicting its potential as a fertility-enhancing agent. These results are in agreement with previous results that explained that J. Regia significantly improves sex hormones, especially LH and FSH that enhance conception and testicular growth (Ghorbani et al., 2014GHORBANI, R., MOKHTARI, T., KHAZAEI, M., SALAHSHOOR, M., JALILI, C. and BAKHTIARI, M., 2014. The effect of walnut on the weight, blood glucose and sex hormones of diabetic male rats. International Journal of Morphology, vol. 32, no. 3, pp. 833-838. http://dx.doi.org/10.4067/S0717-95022014000300015.
http://dx.doi.org/10.4067/S0717-95022014... ). Additionally, Estradiol levels were significantly decreased and maintained as in the control group. Since FSH/LH surge is required for ovulation, that is triggered by LH releasing hormones and depends on estradiol levels (Jeje et al., 2021JEJE, S.O., AKPAN, E.E., KUNLE-ALABI, O.T., AKINDELE, O.O. and RAJI, Y., 2021. Protective role of Allium cepa Linn (onion) juice on maternal dexamethasone induced alterations in reproductive functions of female offspring of Wistar rats. Current Research in Physiology, vol. 4, pp. 145-154. http://dx.doi.org/10.1016/j.crphys.2021.06.001. PMid:34746834.
http://dx.doi.org/10.1016/j.crphys.2021.... ). Our results suggest that J. Regia oil in higher doses significantly improved FSH levels by balancing the estradiol and improving fertility, probably due to the increased pituitary sensitivity to gonadotropin-releasing hormone (Lienou et al., 2012LIENOU, L.L., TELEFO, B.P., BALE, B., YEMELE, D., TAGNE, R.S., GOKA, S.C., LEMFACK, C.M., MOUOKEU, C. and MOUNDIPA, P.F., 2012. Effect of the aqueous extract of Senecio biafrae (Oliv. & Hiern) J. Moore on sexual maturation of immature female rat. BMC Complementary and Alternative Medicine, vol. 12, no. 1, pp. 36. http://dx.doi.org/10.1186/1472-6882-12-36. PMid:22482701.
http://dx.doi.org/10.1186/1472-6882-12-3... ). Our findings are in accordance with a study that explained that J. Regia successfully raised sex hormones in rats (Bostani et al., 2014BOSTANI, M., AQABABA, H., HOSSEINI, S.E. and ASHTIYANI, S.C., 2014. A study on the effects of walnut oil on plasma levels of testosterone pre and post puberty in male rats. American Journal of Ethnomedicine, vol. 1, no. 4, pp. 266-275.). J. Regia contains α-linolenic acid, which is converted to arachidonic acid, to make prostaglandins that play an important role in ovulation and testicular steroidogenesis (Adelakun et al., 2019aADELAKUN, S.A., UKWENYA, V.O., OGUNLADE, B.S., A, J. and G, A., 2019a. Nitrite-induced testicular toxicity in rats: therapeutic potential of walnut oil. JBRA Assisted Reproduction, vol. 23, no. 1, pp. 15-23. PMid:30106544.). Robbins et al. (2012ROBBINS, W.A., XUN, L., FITZGERALD, L.Z., ESGUERRA, S., HENNING, S.M. and CARPENTER, C.L., 2012. Walnuts improve semen quality in men consuming a Western-style diet: randomized control dietary intervention trial. Biology of Reproduction, vol. 87, no. 4, pp. 101-108. http://dx.doi.org/10.1095/biolreprod.112.101634. PMid:22895856.
http://dx.doi.org/10.1095/biolreprod.112... ) explained that J. Regia, if added to the diet, improved sperm vitality, motility, and morphology.

Oxidative stress plays a crucial role in the pathogenesis of numerous inflammatory diseases and reproductive performances (Alahmar, 2019ALAHMAR, A.T., 2019. Role of oxidative stress in male infertility: an updated review. Journal of Human Reproductive Sciences, vol. 12, no. 1, pp. 4-18. http://dx.doi.org/10.4103/jhrs.JHRS_150_18. PMid:31007461.
http://dx.doi.org/10.4103/jhrs.JHRS_150_... ). Reactive oxygen species in physiological processes may behave as key signaling molecules, but excessive levels may also mediate pathological processes involving reproduction. Glutathione and Superoxide dismutase (SOD) are physiological antioxidants, the master detoxifiers and maestro of the immune system (Ozougwu, 2016OZOUGWU, J.C., 2016. The role of reactive oxygen species and antioxidants in oxidative stress. International Journal of Research, vol. 1, no. 8, pp. 1-8.). ROS affects multiple pathological and physiological processes in the ovaries, from oocyte maturation to fertilization leading to infertility (Lu et al., 2018LU, J., WANG, Z., CAO, J., CHEN, Y. and DONG, Y., 2018. A novel and compact review on the role of oxidative stress in female reproduction. Reproductive Biology and Endocrinology, vol. 16, no. 1, pp. 80. http://dx.doi.org/10.1186/s12958-018-0391-5. PMid:30126412.
http://dx.doi.org/10.1186/s12958-018-039... ). Glutathione and SOD shield eggs from damage caused by oxidative stress during folliculogenesis in females as well as they improve sperm count, motility and spermatozoa maturation in males (Mannucci et al., 2022MANNUCCI, A., ARGENTO, F.R., FINI, E., COCCIA, M.E., TADDEI, N., BECATTI, M. and FIORILLO, C., 2022. The impact of oxidative stress in male infertility. Frontiers in Molecular Biosciences, vol. 8, pp. 799294. http://dx.doi.org/10.3389/fmolb.2021.799294. PMid:35071326.
http://dx.doi.org/10.3389/fmolb.2021.799... ). The current study represented improved glutathione and SOD levels after both low and high doses of J. Regia oil consumption in rats that confirmed the antioxidant potential of J. Regia oil. These results are in-accordance with Miao et al. (2020)MIAO, F., SHAN, C., SHAH, S.A.H., AKHTAR, R.W., GENG, S., NING, D. and WANG, X., 2020. The protective effect of walnut oil on lipopolysaccharide–induced acute intestinal injury in mice. Food Science & Nutrition, vol. 9, no. 2, pp. 711-718. http://dx.doi.org/10.1002/fsn3.2035. PMid:33598156.
http://dx.doi.org/10.1002/fsn3.2035... that exhibited J. Regia oil significantly improved the antioxidant capacity by restoring glutathione and SOD levels and reducing the release of inflammatory factors from LPS‐induced intestinal injury.

5. Conclusion

This study has revealed that J. Regia effectively promotes conception and fertility in both males and females by enhancing ovulation and spermatogenesis. Thus, the results of this study render access to a safer natural alternative to overcome fertility issues. Plant products for the treatment of infertility issues will be more acceptable for economic reasons and side effects than chemical agents.

5.1. Limitations and areas for future research

Two major limitations in the current study could be addressed in future research. First, the study focused mainly on the bi-generational study model and only the microscopic structure of the ovaries and testes of the experimental animals. If more resources had been available, we would have studied the correlation of the Juglans Regia oil on fertility by conducting infertility models, i.e., female and male infertility models. Furthermore, due to a limited budget, we restrict towards F₂ generation; in case of more facilities and financial resources, we must go for further F₃ and F₄ generation to scrutinize mirror images of study results. Secondly, further genomic markers will be evaluated for the exact molecular mechanisms.

Acknowledgements

The authors are thankful to the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, for providing essential facilities for this research work.

References

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