Oral Administration of Spirulina platensis at Early Gestation Modulates Litter Size and the Expression of Inhibin, Insulin, IGF-I, CO Q10, and BMP-15 in Ewes Induced for Twinning

Gestation in sheep necessitates the support of nutrients to avoid early embryonic mortalities. Therefore, this study investigates the effects of supplementing either L-arginine or Spirulina alga in the first trimester on the pregnancy rate, litter size, inhibin, insulin, IGF-I, CO Q10, and bone morphogenetic peptide 15 (BMP15) in maternal circulation. Animals were offered barley (500 g/head/day, 14% CP), alfalfa hay (1 kg/head/day, 12% CP), clean water, and balanced salt block licks as free choices. Forty Noemi and Najdi ewes were randomly allotted into three groups: control (C, n = 8), L-arginine (ARG, n = 16), and Spirulina (SP, n = 16). All females were implanted with CIDR for ten days. On days 8, 9, and 10, treated ewes were given a protocol comprised of human recombinant FSH at descending doses (50, 50, 40, 40, and 30, 30 IU, A.M, and P.M, respectively). At the fifth dose, animals were given an equivalent dose of hCG (240 IU). After CIDR withdrawal, ewes were exposed to fertile rams for mating. SP-ewes were orally given 50 ml (2%) Spirulina, and ARG-ewes were given 50 ml (35 mg/kg BW) L-arginine daily for 50 days postbreeding. Blood inhibin, insulin, IGF-I, CO Q10, and BMP15 were determined throughout gestation until parturition. The findings indicated that the conception rates were 25, 75, and 87.5% in C, ARG, and SP, respectively (P < 0.05). The percent of ewes giving birth to twins was 0, 25, and 50% in C, ARG, and SP, respectively (P < 0.05). The survival rates were 100, 81.8, and 83.3%, respectively. Birth weight was 5.6, 3.2, and 3.4 kg in C, ARG, and SP, respectively. Weaning weights were 28.3, 25.6, and 27.2 kg in C, ARG, and SP, respectively. BMP-15 was reduced (P < 0.05) in ARG than in C and SP. However, SP decreased (P < 0.05) inhibin more than in C and ARG. ARG and SP increased (P < 0.05) insulin than in C, whereas SP decreased (P < 0.05) IGF-I. SP increased CO Q10 compared with ARG. Ewes bearing twins revealed higher (P < 0.05) IGF-I (8.57 ng/ml) than those bearing singles (4.63 ng/ml); however, BMP-15 was higher in single (796.6 pg/ml) than in twin-bearing (387.5 pg/ml) ewes. In conclusion, providing early-gestating ewes with Spirulina enhances maternal health, productivity, and reproductive outcomes.


Introduction
Maximizing the productivity of the animal unit became a necessity in the current era of pandemics and political conficts. Tis target drew researchers' attention to animal production due to the infation of animal production supplies, particularly feedstufs, vitamins, minerals, vaccines, and other items. Lack of a balanced ration for an animal causes a decrease in its productivity and reductions in its productive lifespan and reproductive performance [1].
Sheep focks in the Arabian Gulf region constitute the primary source of meat since the inhabitants of this area consume mainly lamb and goat meat. L-arginine is an inducer for nitrous oxide production focused on various research aspects two decades ago [2]. Nutrients requirement during pregnancy have an indispensable role in neonatal health and the outcome of the ewe's production [3]. Several attempts have been conducted to maximize the productivity of farm animals using bioactive compounds like amino acids [2], probiotics [4], and microalgae [5]. Te work of Fuller Bazer and his colleagues was the pioneer to highlight the necessity of L-arginine for early embryonic implantation, survival, and development in small ruminants [6][7][8]. Subsequently, Zeitoun and his associates orally administered Larginine during the frst 56 days postmating in Najdi ewes and obtained a heavier birth weight by 35% than control lambs [9]. Tis fnding was later confrmed in mares, since Larginine supplementation in early pregnancy supported embryonic growth and the initiation of placentation [10].
Recently, research has focused on unconventional feed additives with valuable nutrients. Tese nutritious compounds include algae such as Spirulina and chlorella. Spirulina platensis was defned as flamentous cyanobacteria known as blue-green algae that act as single-cell proteins [11]. Spirulina contains (on a dry matter basis) a high percentage of protein (70%) and is rich in some amino acids like L-arginine (4.15%), glutamic acid (8.39%), lysine (3.03%), and methionine (1.15%), as well as vitamins such as A, B, E, and K [12]. Spirulina has been used as a diet additive in aquaculture [13,14], chicken [15], dairy cows [16,17], rabbits [18], and sheep [19]. Alghonaim and his group investigated the efects of Spirulina supplementation on the growth performance and digestibility parameters of sheep lambs [19].
Bone morphogenetic peptide-15 (BMP-15) is an oocytesecreted peptide that modulates folliculogenesis, oocyte health, and ovulation outcome in humans [20]. Tis peptide was targeted to be related to fecundity in ewes [21]. Besides, CO Q10 was recently focused on as a bio-indicator for the healthy oocyte [22,23]. CO Q10 was mentioned as a novel peptide that synthesized intercellular and played a vital role in the body as an electron transporter in the mitochondria [24]. Te use of CO Q10 as a supplement for alleviating reactive oxygen species and improving the health of oocytes in women was confrmed recently [25].
To the best of our knowledge, no one has attempted to test the role of Spirulina supplementation in pregnant ewes during various stages of gestation. Our previous studies demonstrated that supplementing pregnant ewes at an early stage of pregnancy with L-arginine maintained the survival of the newborn lambs [26,27]. Terefore, the objective of the present study was to explore whether supplementing a rich source of protein and antioxidants like Spirulina during early pregnancy in ewes would promote the implantation and subsequent survival of twin lambs, in addition to investigating the hormonal (i.e., inhibin and insulin) peptides (i.e., IGF-I and BMP-15) and antioxidant (CO Q10) expression in the ewes induced for twinning.

Animals and Location. Forty multiparous Noemi and
Najdi ewes (average BW 45 ± 3.2 kg; age 26 ± 3.5 months) were housed in semi-shaded pens in the experimental station, College of Agriculture and Veterinary Medicine, Qassim University, in the Central region of KSA. Animals were ofered barley (500 g/head/day), alfalfa hay (1 kg/head/ day), clean water, and balanced salt block licks as free choices. According to the NRC requirements, this ration provides about 192 g crude protein (CP) per head per day [28]. Animals were submitted to the regular vaccination regime. Te committee approved the animal handling and manipulation in this study of ethical animal care and welfare, Deanship of Scientifc Research, Qassim University (con-tract#7804-1-1-2019Q), KSA.

Experimental Designs.
Ewes were randomly allotted into three groups: control (C, n � 8) given the regular diet as mentioned above; ARG group ewes (n � 16) given the regular diet plus supplementation of L-arginine at a dose of 37.5 mg/kg BW/day for the 50 days following mating. SP ewes (n � 16) were given the regular diet plus 2.5 g/head/day Spirulina platensis powder dissolved in 50 ml distilled water for the frst 50 days postmating.

Twinning Protocol, Natural Mating, and Parturition.
All animals were exposed to intravaginal CIDR insertion for ten days; recombinant human FSH (GONAL-f, Serono, Bari, Italy) was administered (i.m.; AM and PM) in descending doses at 50, 50; 40, 40; and 30, 30 IU at days 8, 9, and 10 (a total dose of 240 IU, [26]). CIDR was removed on day 10 (AM; i.e., at the 5 th FSH dose), at which time an equivalent dose of hCG (240 IU, Pergnyl, Organon, Oss, Holland) was injected (i.m.). Ewes were subjected to fertile rams of the same breed for 72 hours. Te dates and times of the onset of estrus and mating were recorded for each ewe. Te estimated time elapsed between CIDR withdrawal and the onset of estrus was also recorded for each ewe. Ewes were examined for pregnancy by ultrasound machine (ALOKA SSD 500, Japan) with a 5 MHz endorectal linear array transducer 30 days postmating. Te newborn's number, sex, survival rate, and weight were recorded at parturition.

Blood Sampling and Peptide and Hormone
Determinations. Blood samples were collected via jugular venipuncture at days 0 (just before CIDR insertion), 8 (commencement of gonadotrophin administration), 10 (at CIDR withdrawal), at the onset of estrous and mating, days 7, 14, 21, and 28 postmating, and every other week thereafter until the day of delivery. Blood samples were collected in EDTA-Vacutainer ® tubes, subjected to centrifugation (3000 rpm/3 min./5°C), and plasma was harvested and stored frozen (−20°C) until analyzed.
Te study used commercial ELISA kits specifc to sheep (My BioSource, CA, USA). Sheep insulin was determined by sandwich ELISA kit (Cat. No. MBS705810) with standard 2 Veterinary Medicine International levels range 0-100 μ IU/mL, two μIU/mL sensitivity, and intra-and interassay CV of 8.6 and 11.2%, respectively. Sheep insulin-like growth factor-I (IGF-I) was determined in the samples by sandwich ELISA kits (Cat. No. MBS2122248). Te assay used standard concentrations ranging from 0-60 ng/mL, a sensitivity less than 0.35 ng/ mL, and intra-and interassay CV of 9.5 and 11.7%, respectively.
Sandwich ELISA kits were used to determine sheep inhibin (Cat. No. MBS743665). Te standard concentration range was 0-2500 pg/mL with a sensitivity is of 1.0 pg/mL, and the intra-and interassay C.V were 6.9 and 9.8%, respectively.
Sheep Co Q10 was determined by a competitive ELISA kit (Cat. No. MBS7252409). Te standard range was 0-100 ng/mL, the sensitivity was 1.0 ng/mL, and the intraand interassay CV were 8.5 and 9.3%, respectively.

Statistical
Analysis. Data of estrous, conception, and ofspring number, birth, and weaning weights were analyzed by ANOVA. Data of the hormones and peptides were analyzed by least square analysis of variance for the repeated measures [29]. Mean comparisons were conducted through Duncan's multiple range test (DMRT). Diferences were considered signifcant at P < 0.05. Data for discrete parameters were analyzed by the chi-square test according to SAS [29]. Table 1 illustrates that ewes given FSH expressed higher percentages of estrus compared with C ewes. Te time lapsed from CIDR removal until the onset of estrous was 56 ± 2.1, 36 ± 3.6, and 38 ± 4.3 hours (P � 0.03) in C, ARG, and SP, respectively. Conception rate was higher (P � 0.04) in SP and ARG than control. Te number (%) of ewes that gave birth to singles was 2 (25%), 8 (50%), and 6 (37.5%) (P � 0.04) in C, ARG, and SP, respectively. However, the number (%) of ewes that gave birth to twins was higher (P � 0.03) in SP than in ARG and C. Te failure of conception rate was higher in C than in ARG and SP (P � 0.03). Te ratio of gender (male: female) among treatments was 100% males, (10M : 6F) and (12M : 10F) in C, ARG, and SP, respectively. No signifcant efect was found due to the sex of the ofspring. Te mean lamb birth weight was higher (P � 0.03) in C than in ARG and SP. In C, ARG, and SP, the percent survival at birth was 100, 81.25, and 81.8%, respectively. However, survival at weaning was 100% among treatments. Te mean 90-day lamb weaning weight did not difer (P � 0.12) among treatments. Terefore, total kilograms of weaned lambs were higher in SP and ARG than in C. Te mean number of weaned lambs per each treated ewe was higher in SP and ARG than in C.
Te only signifcant correlation between the hormones and peptides was the positive correlation between IGF-I and inhibin (r � 0.71, P < 0.0001). However, there were nonsignifcant correlations among other metabolites.

Discussion
Inappropriate placental vascular development leads to insufciency of nutrients and gas exchange to the fetus (es) from the mother, resulting in intrauterine growth restriction (IUGR) or early embryonic death [30]. L-arginine was proven to stimulate nitrous oxide (NO) production, which enlarges the placental blood vessels and promotes angiogenesis. Tis function results in excess nutrients and oxygen provision to the fetuses in their frst trimester [31]. Administration of L-arginine at early gestation has previously been given to ewes bearing singles or twins and exhibited better implantation, fetal growth, development, and survival [26]. Whether the embryos at their early implantation and growth would require the amino acid L-Arginine per se or need a source of protein (i.e., containing L-arginine and other essential amino acids) for the synthesis of the enzymes and growth factors for a better uterine milieu and proper implantation was the focus of the current exploration.

Veterinary Medicine International
Spirulina platensis has been scrutinized as a food and feed source for humans and animals. Te main nutritious ingredients in Spirulina are; protein (∼60-70%) which is rich in sulfur-containing amino acids like methionine and cysteine; fats which possess a high percentage of polyunsaturated fatty acids (PUFA), like linoleic acid and c linolenic acid (GLA); and minerals like potassium, phosphorus, calcium, ferrous, copper, and zinc [32]. Also, Spirulina was shown to be a toxin-free microalga. It has been reported that supplementing diabetic mice with Spirulina improved the percentage of fetal implantation [33]. Moreover, El-Ratel and Gaber obtained a larger litter size (60% higher than control) from Spirulina-supplemented diet to rabbit does (300 mg/kg diet) [34]. Moreover, Tilapia and Zebra fsh provided with Spirulina increased egg production, vitellogenesis, hatching, and survival rates [35,36]. A few recent attempts have been made to test the efects of supplementation with Spirulina on the growth and immunity of dairy suckling calves [37] and sheep lambs [19]. Remote data, so far, have been published on the efects of microalgae (i.e., Spirulina) on the reproductive efciency of ruminants. Te current fndings confrm the surpass of Spirulina over Larginine on the total lamb outcome, leading to the concept that this alga contains high levels of nutrients that meet the pregnant ewes' requirements during their early gestation. Provision of the Spirulina in the current study resulted in approximately 4.3 times lambs' crops at weaning per treated ewe as obtained from control ewes and ∼1.5 folds higher than that given by L-arginine. Te antioxidant, immunestimulants, and growth properties of Spirulina alga must not be overlooked [19,[37][38][39]. Te high level of CO Q10 found in the SP-ewes in the current study would support the survival and higher implantation, postpartum growth rate, and high weaning weight. It has been previously stated that in hot climates, supplementation of rabbit diets at 300 mg Spirulina/kg diet improved the fetal developmental competence (in vivo and in vitro) and increased the litter size [34]. Te anabolic potency of Spirulina was later revealed in growing lambs raised under heat stress and provided with an 8 mg Spirulina/kg diet for 90 days [19]. Meanwhile, another biological explanation for adding Spirulina was suggested by a Korean research team, which monitored the efect of nanoparticles of Spirulina maxima on the in vitro development of porcine oocytes. Tey concluded that adding SP nanoparticles to the oocytes in the in vitro maturation media increased the preimplantation porcine and cloned embryo development by increasing glutathione [40]. From another point of view, providing the mother with a rich source of energy pre-and during early pregnancy would augment fertilization, early cleavage, and early embryonic implantation and development [41]. Previous studies supported the concept that providing pregnant ewes [42] or young, growing lambs [38] with Spirulina enhanced ofspring birth weight and lamb growth rate, respectively. Ewes given Spirulina in the current study revealed higher (P < 0.05) levels of insulin, CO Q10, and BMP15; however, the same ewes revealed lower levels of IFG-I and inhibin than control ewes. Tere found a strong positive correlation between inhibin and IGF-I, the relationship that explains the lower inhibin in peripheral blood is accompanied by lower IGF-I, which leads to higher healthy ovulated oocytes [43]. In control pregnant ewes, there was a decrease in IGF-I in the last 30-45 days near delivery; that means this growth factor might have played its role in fetal muscle development during early and mid-gestation [44][45][46]. Conversely, the current data revealed that Spirulina and L-arginine elevated maternal insulin levels above those found in control ewes. During pregnancy, the demand for insulin increases due to the interference between pregnancy hormones and insulin sensitivity, leading to a reduction in insulin sensitivity [47]. Te increased insulin in the circulation of the ARG and SP ewes in the current study could facilitate fetal metabolism and provide a source of energy for the twins, ensuring their ongoing growth, development, and survival. Furthermore, Lunesu and his colleagues demonstrated that ewes exposed to diets difering in the quantity and quality of carbohydrates evoke insulin sensitivity during intrauterine and postnatal lives [48]. It has been confrmed that BMP-15 and GDF9 are major determinants of ovulation rate and litter size (i e., fecundity), and that these genes exist in prolifc sheep breeds. Te current concern is whether or not these growth factors are inherent to the prolifc sheep or might exist in the ewes induced for twin/multiple births. Te trend of high levels of BMP-15 in SP and LG ewes accounted for 1.6 and 1.4, more than control, respectively. Tis fnding confrms the hypothesis of the relationship between BMP-15 and ovulation rate in spontaneous or induced ovulation. In this respect, Davis pointed out the existence of four mutants of the BMP-15 genes [21]. Te current fnding that the high concentration of BMP-15 coincided with the higher ovulation rate in SP ewes was previously stated by Davis and his associates [49]. Tey found that crossing Booroola Merino with Inverdale resulted in daughters having active ovaries and high ovulation rates, averaging 4.4 oocytes [49]. Te two mutations, BMP-15 and BMPR-1B, increased ovulation by 44% and 90%, respectively. A recent study suggested a possible interaction between BMP15 and FecB/GDF on increasing ovulation rates in Luzhong mutton sheep in China [50]. CO Q10 has been shown to possess three main functions on the cellular level, namely, (i) transfer of reducing equivalents in the electron transport chain, (ii) generation of superoxide anion radical, O2 * −, and (iii) quenching of free radicals [51]. It has been proved that supplementation of CO Q10 revealed a potent antioxidant function leading to reduced free radicals in the exercised subjects [52]. In a study on mice, supplementing females sufering from preeclampsia with CO Q10 alleviated the adverse symptoms, improved health, and increased the number of pups born surviving [53]. Te capability of CO Q10 as an antioxidant for improving human oocyte quality via its benefcial impact on the follicular level has been confrmed [54]. Te current fnding exhibited the excellence of Spirulina over L-arginine in elevating CO Q10 in maternal circulation. Tis observation would augment the excess growth of fetuses in SP compared to ARG-treated ewes.
In conclusion, providing pregnant ewes during their early stage of gestation with 2.5 g Spirulina platensis per a head per day for the frst 50 days postbreeding would Veterinary Medicine International enhance the twin-bearing maternal health, fertilization, implantation, and pre-and postnatal survival of fetuses in estrous-synchronized/induced twinning Najdi and Noemi ewes. Also, supplementing pregnant ewes with the said dose of Spirulina kept the hormones, fecundity peptide, and the antioxidant CO Q10 at levels sufcient to maintain the ofspring's survival and postnatal growth. Further studies using a larger sample size of ewes and testing the provision of Spirulina alga and its micronutrients during the three trimesters of gestation are warranted.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author, upon reasonable request.

Ethical Approval
Tis study was approved by the Animal Care and Welfare Committee, Deanship of Scientifc Research, Qassim University, Kingdom of Saudi Arabia (7804-1-1-2019Q).

Conflicts of Interest
Te authors declare that they have no conficts of interest.