https://orcid.org/0000-0003-0868-7073
Abstract
This study aimed to investigate the effects of nano and organic selenium on ovarian activity, fertility rate, and progesterone hormone levels in Ossimi ewes. Thirty ewes were treated with intra-vaginal sponges impregnated with medroxyprogesterone acetate (40 mg) for 14 d and simultaneously assigned randomly to three groups. The 1st group ewe served as control (CG). Ewes in the 2nd and 3rd groups were treated orally with organic selenium 3 mg/ewe and Nano-selenium 0.2 mg/ewes during intra-vaginal sponges (IVS) treatment SeG and N-SeG, respectively. The ovarian activity was monitored using an ultrasonic device, and serum samples were harvested for progesterone measurement. The results revealed that ewes in SeG and N-SeG groups had a significantly (p < .05) higher number of small and large follicles after 48 h of IVS withdrawal than in the CG, respectively, on the left ovary. Also, the N-SeG group had a larger diameter of the Corpus luteum on both ovaries’ sides than CG, respectively, at the same time. However, the diameter of large follicles on both ovaries was significantly (p < .05) larger in the N-SeG after 72 h of IVS withdrawal than in SeG and CG. The ewes in N-SeG and SeG came in oestrus earlier (p < .05) than those in CG, and the oestrus durations were longer (p < .05) than that recorded in ewes of CG. The oestrus response and fertility rate were higher (p < .01) in N-SeG and SeG than in CG. Moreover, the progesterone concentrations were significantly higher (p < .05) in the N-SeG and SeG after 48 and 72 h of IVS withdrawal than in the CG. Also, the recorded serum progesterone concentrations in the included ewes non-significantly differed between groups during the first four months of pregnancy. It could be concluded that using nano and organic selenium can improve the quality of ovarian follicles and CL function and enhance the fertility rate in Ossimi ewes.
HIGHLIGHTS
Improvement of ovarian activity in ewes.
Improvement of ewes fertility rate.
Effect of Nano and organic selenium on Ossimi ewes
Introduction
Worldwide, sheep are a valuable livestock species due to their ability to convert forages and roughage feedstuffs unbefitting for human consumption into meat and milk, which are valuable sources of human dietary protein (Elshazly and Youngs Citation2019). In Egypt, sheep production is considered one of the chief livestock production sectors; it generates around 30% of all agricultural income (Elshazly and Youngs Citation2019). The Ossimi breed is one of Egypt’s most widespread local breeds, characterised by extended breeding season and low prolificacy (Emeash and Mostafa Citation2010; Gabr et al. Citation2016). Therefore, enhancing ewes’ reproductive performance will increase lamb yield, a crucial requirement for expanding Egypt’s sheep output (Mostafa and Farghal Citation2022).
It is known that Selenium (Se) maintains reproductive functions and enhances animal growth and metabolism (Rayman Citation2004), and significantly affects the oestrous response and fertility rate when supplementation during the oestrus cycle. However, Se supplementation to ewes before mating could increase fertility, prolificacy, and the twin’s number (Gabryszuk and Klewiec Citation2002; Abd-Elkareim et al. Citation2021). Moreover, Se promotes trophoblast migration and proliferation at the beginning of pregnancy by reducing mitochondrial oxidative stress; also, Se-treated trophoblasts exhibit increased viability, migration activity, and mitochondrial functional activity (Na et al. Citation2018).
Nanoparticles are particles with a diameter of 1–100 nm, with a large proportion of their atoms on the surface (Casabianca Citation2020). As a result, novel physical and chemical features, such as increased cellular uptake, reactivity, surface area and charge, and binding capabilities, have been accomplished, potentially opening a new platform for biosciences innovation, according to Feugang et al. (Citation2019) and Hashem and Sallam (Citation2020). Nanotechnology generates diagnostic and therapeutic agents in human medicine, but its applicability in animal health is still limited. For example, selenium nanoparticles have been employed as reactive oxygen species (ROS) scavengers in various investigations to protect against oxidative damage (Khalil et al. Citation2019). Previous studies have reported that the balance between ROS and antioxidants greatly influences the reproductive activities in female mammalian animals, such as endometrial changes in different luteal phases, folliculogenesis, ovulation, fertilisation, placental growth, embryogenesis, and implantation (Al-Gubory et al. Citation2010).
A previous study has demonstrated that Se deficiency contributes to many economically significant issues with livestock, such as reduced fertility, abortions, retained placentas, new-born weakness, decreased oocyte growth, the proliferation of theca and granulosa cells, as well as follicles, blood vessels, and stromal tissues in ovine foetal ovaries (Grazul-Bilska et al. Citation2011; Pecoraro et al. Citation2022). Furthermore, there are scarce studies on nano and organic selenium’s effect on ovarian activity in ewes. Therefore, the current study aimed to evaluate the effect of organic selenium and nano selenium on ovarian activity, progesterone concentration, and fertility rate in Ossimi ewes under Upper Egyptian conditions.
Materials and methods
Study location and experimental animals
This study was conducted at the Experimental Farm of Animal Production Department, Faculty of Agriculture, Al-Azaher University, Assuit, Egypt. This experiment was carried out from May 5 to Nov 20, 2021.
The present study included 30 healthy Ossimi adult ewes, showing normal clinical signs, aged between 3 and 3.5 years and with a body weight of 48 ± 2.5 Kg (mean ± SD). All animals were treated using intra-vaginal sponges (IVS) impregnated with 40 mg of medroxyprogesterone acetate (Pfizer manufactured, NV/SA, Puurs, Belgium); the IVS was withdrawn after 14 d. Animals were randomly divided into three equal groups (10/group), a control (CG), a nano selenium-treated group (N-SeG), and the organic selenium-treated group (SeG). Nano selenium-treated group received nano selenium orally (0.2 mg/kg body weight) during the IVS treatment (14 days). The nano selenium was obtained from Nano Green Company for Agricultural Development, Nubaria, EL-Behira, Egypt. Organic selenium-treated group received organic selenium orally (3 mg/kg body weight) during the time of the IVS treatment (14 d). The organic selenium was obtained from Electro Scient Chemical Company, Kasr El-Eieny, Cairo (Ali Citation2009; Musa et al. Citation2018).
All animals were housed in an outdoor shelter during the day and in a semi-open barn at night. The ewes were housed in open barns with sheds during the experimental periods. Animals were fed a daily farm ration, adjusted according to the National Research Council (NRC; 1985), and had free access to water. The chemical composition of the experimental diet is described in Table .
Table 1. Chemical composition of the experimental diet.
Oestrus and fertility parameter
Oestrus signs in ewes were checked twice a day, in the morning at 06:00 am and evening at 06:00 pm, 24 h after IVS withdrawal, using six healthy fertile rams (2–3 years old) for natural breeding. The ewe was considered in oestrus when it stood firmly to be mounted by the ram.
Onset oestrus is defined as the time (in hours) from the vaginal sponge withdrawal to the appearance of oestrus signs (Zonturlu et al. Citation2011).
The oestrus period: is defined as the sexual receptivity period and mating characterised by distinct behavioural symptoms of oestrus and estimated from the first to last signs of oestrus (Jarquin et al. Citation2014).
Oestrus response in ewes = Percentage of animals that come into heat after each treatment (Zeleke et al. Citation2005).
Fertility rate = Percentage of ewes conceived of the females that were mount (inseminated) (Zeleke et al. Citation2005).
Ultrasonography examination
Ovarian structures of ewes were monitored using a real-time ultrasonography, B-mode, diagnostic scanner equipped with a trans-rectal 5/7.5 MHz linear array transducer (Hitachi, EUB-405B, Japan). All follicles ≤2mm (small follicles), ≤4 mm (large follicles), corpora lutea diameter, and cross-sections of the uterus horn at days 16, and 17 of the oestrous cycle, i.e. 48, and 72 h after IVS withdrawal, respectively, was measured and mapped individually for each ewe, and ovulation occurs when large, expanding antral follicles are no longer visible (Ginther et al. Citation1997). And the amount of fluid accumulated in the uterus during oestrus was monitored. The transducer was equipped with a self-made connector to facilitate trans-rectal manipulation. The images of the ovaries and uteri were frozen on the monitor, and the ultrasound device’s built-in calliper was used to measure the structures’ maximum diameters.
On the 51st experimental day (representing day 35th after mating), the uterine content was scanned to determine pregnancy and the placentomes, chest, bipartite diameter, and orbit measurements in control and treated groups on the 60th–70th day after the mating. To prevent individual variance, the same operator performed all inspections.
Blood sampling
Blood samples were collected by venipuncture from the jugular vein into collection non-heparinized tubes and centrifuged at 4000 r.p.m x g for 15 min, and then serum was harvested and stored at −20 °C till assay. The blood samples were collected during the four days (24, 48, 72, and 96 h) after the intra-vaginal sponge’s removal and during the pregnancy months. The serum progesterone concentration (ug/dl) was assayed by ELISA kit (Enzyme immunoassay) to quantitatively determine progesterone concentration, Biocheck, Inc. Foster City, CCA 94404 USA). Serum urea concentration using assay kits supplied by the Diamond Chemical Company, Germany (Caraway and Watts Citation1987), Alanine transaminase (ALT) and Aspartate transaminase (AST) concentrations using assay kits supplied by Spectrum Chemical Company, Egypt, according to Young (Citation1997), and selenium concentration according with Norheim and Haugen (Citation2009), were determined at 0 d, 24, 48, 72 and 96 h of IVS removal.
Statistical analysis
Statistical Package for Social Sciences version 20 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The normal distribution of the data was confirmed using the Kolmogorov–Smirnov test. Data regarding the follicular population (small and large follicles), the corpora lutea diameter, uterine horns, measurements of the placentomes, chest, bipartitely diameter, orbit, and Serum Biochemical parameters were analysed using a one-way ANOVA according to the following General Linear Model: Yij = µ + Ti + Eij. Whereas Yij = experimental observation, µ = general mean, Ti = effect of treatments (i = control, nano, and organic selenium-treated groups), eij = experimental error. Oestrous response and fertility rates were statistically analysed using the Chi-square test. The results were expressed as means ± standard errors of the means (SEM). The p-values ≤ .05 were considered significant.
Results
Impacts of nano and organic selenium on ovarian activity
Data presented in Table revealed that the number of all follicular categories (Figure ) on the right ovary was slightly higher in the N-SeG than in SeG and CG; the difference in its number was not statistically significant. However, the right uterine horn diameter (Figure ) was significantly larger (p < .05) in N-SeG than in SeG and CG. In contrast, the number of small and large follicles in the N-SeG and SeG groups had larger (p < .05) than in the CG. Also, ewes treated with Nano-selenium had larger (p < .05) Corpus luteum diameter than CG and SeG in the left ovary after 48 h of the sponge’s removal.
Figure 1. Ultrasonograms of genital tracts of non-pregnant ewes; (A–D) Sagittal section in the uterine horn, and uterine with clear hypoechogenicity, and scanty amount of fluid. (E–H) Follicular phase, large sized follicle (≤4mm), and small sized follicles (≤2mm). (J) Luteal phase, Corpus luteum, (≤0.5 mm).
Table 2. The follicular population (small and large follicles), diameter of corpora lutea, and of the uterine horns in control and treated groups after 48 h of sponge removal (n = 30, Mean ± SE).
Table showed that ovarian activity in ewes after 72 h of sponge removal in large follicles diameter on both ovaries was significantly larger in the N-SeG than in SeG and CG. However, the diameters of CL on the right and left ovaries were larger (p < .05) in SeG and N-SeG than in CG, while the diameter of the uterine horn was larger in the N-SeG (p < .05) than in SeG and CG after 72 h of sponge removal.
Table 3. The follicular population (small and large follicles), diameter of corpora lutea, and of the uterine horns in control and treated groups after 72 h of sponge removal (n = 30, Mean ± SE).
Impacts of nano and organic selenium on the fertility rate and foetal measurement
As shown in Table , ewes treated with N-SeG and SeG came in oestrus earlier and remained longer in oestrus than in CG. However, the oestrus response was significantly higher (p < .01) in the N-SeG and SeG compared to CG, with values of 90% and 90% than 60%. Moreover, the ewes in the N-SeG and SeG showed higher (p < .01) fertility rates than in CG, with values of 80% and 80% than 50%, respectively (Table ).
Table 4. Oestrous duration, onset, response and fertility rate in control and treated groups (n = 30, Mean ± SE).
In pregnant ewes (Figure ), the difference in diameter of the placentomes after 60 d was insignificant between groups. Chest circumference was larger in N-SeG than CG, but the orbit diameter of the foetus did not differ in all groups. The differences in head diameters at the brain level (Biparital diameter) were insignificant between groups (Table ).
Figure 2. Ultrasonograms of genital tracts of pregnant ewes; (A–C) Pregnant ewe at 35th day, (D) beginning of ossification in the head; pregnant ewe at 60th–70th day, (E,F) all foetal parts are clear, umbilical cord; pregnant ewe at 60th–70th day, (G, H) placentome, (J) ribs shadow, heart location.
Table 5. The measurements of the placentomes, chest, bipartitely diameter, and orbit in control and treated groups at 60–70 d after mating (n = 6/group, Mean ± SE).
Impacts of nano and organic selenium on serum progesterone level and biochemical
Serum progesterone concentrations 24, 48, 72, and 96 h after the sponge’s removal were significantly higher (p < .05) in N-SeG than in CG. Also, there were non-significant differences between N-SeG and SeG after 24, 48, and 72 h of the sponge’s removal. While there were non-significant differences in the progesterone level during pregnancy among groups, except in the last month, the progesterone level was significantly higher in the N-SeG group than in SeG and CG (Table ).
Table 6. Progesterone concentration (ng/mL) in the control and treated groups during oestrous days and pregnancy months (n = 30 ewes. Mean ± SE).
There were non-significant differences among groups at 0 days for the Se concentrations. Serum Se concentration increased significantly after 24, 48, and 72 h of sponge removal in N-Seg and SeG compared with CG (Table ). No significant differences were recorded in the AST and ALT activities among groups after 24 h of sponge removal for liver function enzymes. While after 48 and 72 h, the ALT and AST activity was significantly higher in the N-SeG compared to CG. Also, AST activity was significantly higher in the N-SeG than in SeG and CG. However, no significant differences were found in the urea levels among groups after 24 and 48 h of sponge removal (Table ).
Table 7. Serum Biochemical parameters in the control and treated groups during oestrous days after 0, 24, 48, and 72 h of sponge removal (n = 30 ewes. Mean ± SE).
Discussion
Impacts of nano and organic selenium on ovarian activity
Our study evaluated the effect of oral Se supplementation in organic or nano-formulation on ovarian activity and fertility of Ossimi ewes. The oral route is economical, easily applied, and almost safe for different animal species (Turner et al. Citation2011; Hussein et al. Citation2022). In the current study, the ovarian follicular population, including the number of small and large-sized follicles and the follicular diameters, increased in the treated groups (N-SeG and SeG) compared to the control one. Therefore, this may be a possible mechanism to stimulate folliculogenesis by increasing FSH concentrations, as growing and mature follicles mark the onset of follicular maturation upon FSH and LH stimulation (Rabinovici and Jaffe Citation1990; Henderson et al. Citation2008). Interestingly, the diameters of the newly formed CL in both treated groups (SeG and N-SeG) were larger than that in the ovaries in CG after 48 h and 72 h from the IVS removal, which was also confirmed with the higher level of progesterone. Similar results were reported by Farahavar et al. (Citation2020), whereas, Progesterone is CL's principal secretory product and is required for the establishment and maintenance of pregnancy as well as the regulation of many reproductive functions; also, progesterone regulates the length of the oestrous cycle and the implantation of blastocysts. It also serves as a negative feedback mechanism in the hypothalamus, preventing subsequent follicular growth (Khanghah and Kor Citation2013; Ali et al. Citation2019). Moreover, Pecoraro et al. (Citation2022) reported that the Se promotes oocyte growth, the theca and granulosa cells proliferation, and follicles, blood vessels, and stromal tissues in ovine foetal ovaries.
Impacts of nano and organic selenium on the fertility rate and foetal measurement
The present study’s findings indicated that oral administration of nano and organic Se markedly improved the oestrous and fertility rates of ewes in terms of improved number and diameter of the ovarian follicles. Also, N-SeG and SeG administration significantly (p < 0.05) induced a higher oestrus response and fertility rate, and the ewes treated with nano and organic Se came in oestrus earlier and remained longer than CG (p < .05). These findings are comparable to those of Koyuncu and Yerlikaya (Citation2007) and Musa et al. (Citation2018), who found that administering Se to Merino sheep improved oestrous response and fertility. Se is a cofactor in the glutathione peroxidase enzyme system responsible for detoxifying extracellular free radicals (Smith and Akinbamijo Citation2000). On the other hand, Farahavar et al. (Citation2020) discovered that Se administration did not affect fertility, prolificacy, lambing rate, sex ratio, or viability rates in the first and second oestrus cycles compared to the control group.
However, the reproductive parameters of conception rate, lambing rate, and prolificacy were higher in Se-supplemented ewes than the control ewes. The conception rate was 56 vs. 68%, the lambing rate was 60 vs. 76%, and prolificacy was 1.07 vs. 1.12 (Sánchez et al. Citation2008). Also, administration of Se by intramuscular injection has considerable favourable effects on the incidence of oestrus, fertility, and prolificacy in ewes, as well as the live weight increases of lambs up to 60 d of age (Koyuncu and Yerlikaya Citation2007). After two Se injections, Gabryszuk and Klewiec (Citation2002) found that 3-year-old ewes had increased fertility, prolificacy, and body weight before mating and at lambing.
Increase of the uterine horn diameter (p < .05) in the N-SG after 48 and 72 h of sponge removal and chest circumference (p < .05) in N-SG as compared to CG at 60–70 d after mating may be due to the usage of Se nanoparticles as a ROS scavenger to protect against oxidative damage, (Khalil et al. Citation2019). Furthermore, the ROS balance and antioxidants significantly impact female mammalian reproductive functions as endometrial alterations in distinct luteal phases, folliculogenesis, ovulation, fertilisation, placental growth, embryogenesis, and implantation (Al-Gubory et al. Citation2010).
Impacts of nano and organic selenium on serum progesterone level and biochemical
The recorded serum progesterone concentrations in the included ewes non-significantly differed between groups during the first four months of pregnancy. On the other hand, the animals treated with Nano-selenium had higher progesterone concentrations during the last month of pregnancy than in the selenium and control groups. This study supports this result by the absence of significant effects on placental diameter after 60 d of pregnancy. According to Weems et al. (Citation2007), the CL must be maintained and secrete high amounts of progesterone until the developing placenta can take over progesterone production. On the other hand, Chakraborty et al. (Citation2017) discovered that the serum progesterone profile was 0.25, 0.27, and 0.36 ng/ml the day of oestrus in the animals treated with 1, 20, and 30 mg of IVF impregnated with progesterone for 14 d, moreover, At 60th day of pregnancy was 6.00, 7.01, and 7.59 ± 0.14 ng/ml, respectively. Also, Ayman et al. (Citation2015) reported that progesterone concentrations were 0.2, 0.4, and 1.2 ng/ml. After progesterone treatment, 24, 48, and 72 h switch from CL-dependent to placenta-dependent progesterone production happens as early as 55–90 d after pregnancy in sheep.
In addition, the biochemical measurements (activity of AST and ALT and urea level) ensured the safety and efficacy of the oral administration of nano and organic selenium on the health of the ewe. In addition, selenium is a form of antioxidant that helps the body get rid of the damaging effects of urea (Ucar et al. Citation2010), which may explain the urea levels stability in the present study within normal limits. The low activity of AST could be due to the role of Se in removing hydrogen peroxide, which leads to tissue breakdown by the action of glutathione peroxide enzyme, which is accompanied by a drop in the activity of AST and ALT enzymes in the blood serum (Haenlein and Anke Citation2011; Abdel Ghfar et al. Citation2022).
Conclusion
This study is one of the few reports to use nano versus organic Se to improve oestrous and ovarian activities of ewes under Egyptian conditions. Supplementation with nano selenium and organic selenium in clinically healthy ewes is beneficial in improving follicular population and CL function and could improve ewes’ oestrus responses and fertility rate. Moreover, the biochemical findings in the present study ensured the efficacy and safety of the oral administration of nano and organic selenium in ewes. Future studies into nano and organic selenium modification techniques could lead to the discovery of novel techniques for enhancing ovarian activity and fertility rates.
Ethics statement
All Institutional and National Guidelines for the care and use of animals were followed in accordance with the Egyptian Medical Research Ethics Committee (no. 14–126) and the Department of Animal Production, Faculty of Agriculture, Al-Azhar University, Assiut, Egypt.
Authors’ contributions
All the authors listed in this paper have contributed to the preparation and execution of this research. MEA, HAH: conceptualisation, methodology, investigation, visualisation, formal analysis, writing-original draft. MFA and SF: investigation, Data collection, formal analysis. MEA, MA: formal analysis, writing-original draft. All authors have read and agreed to the published version of the manuscript.
Acknowledgments
The authors would like to thank the staff members of Sheep Farm, Department of Animal Production, Faculty of Agriculture, Al-Azhar University, Assuit, Egypt, for their assistance in conducting the present experiments. Also, the authors extend their appreciation to the deanship of Scientific Research at King Khalid University for supporting this work under the grant number (R.G.P.2/345/44).
Disclosure statement
No potential conflict of interest was reported by the authors.
Data availability statement
Raw data were generated at the Experimental Sheep Farm belonging to the Faculty of Agriculture, Al-Azhar University, Assuit, Egypt. Data availability statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
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