Incorporation of fresh leaves of wormwood (Artemisia herba alba) and/or rosemary (Rosmarinus officinalis) in the diet of rams: Effect on testicular function, sexual behavior, and blood parameters

Abstract This study aimed to investigate the effect of wormwood and rosemary supplementation on some reproductive traits of Barbarine rams. The experiment lasted 2 months. Twenty‐four adult rams were divided into four groups (n = 6) balanced for the weight (53.3 ± 1.2 kg body weight [BW] ± SD). All rams received 1200 g of straw and 600 g of barley. Control rams (C) without aromatic medicinal plant (AMP), while experimental rams received 20 g of fresh rosemary leaves (R), 20 g of fresh wormwood leaves (A), and 10 g of fresh rosemary leaves plus 10 g of fresh wormwood leaves (RA). The results revealed that the live weight of all rams increased (p < .05) in the RA group compared to the C, A, and R groups. Scrotal circumference increased in the R rams when compared to the controls rams (p < .05). For sperm parameters we showed that the A rams had higher sperm concentrations (p < .05). But, the sperm volume decreased in the R rams (p > .05). However, when the rams received rosemary plus wormwood, their sperm volume increased (p > .05). The sperm mass motility was higher for the A, R and AR rams in comparison to the C rams (p = .05). On the other hand, biochemical analysis of the seminal fluid showed no effect of diets on calcium and total proteins concentration. But the measurement of glucose and seminal insulin showed a decrease (p < .05) in these two biochemical markers in group A rams and a decrease (p < .05) in insulin without modification of the glucose concentration in R rams. Blood glucose and insulin decreased in the animals on AMP diet compared to the other groups (p < .05) while aspartate aminotransferase (AST) increased (p < .05). Rosemary leaves (R and RA groups) increased (p < .05) plasma cortisol compared to the other groups. It can be concluded that the addition of Rosmarinus officinalis and/or Artemisia herba alba in ram diet can have a positive effect on the reproductive function by increasing the concentration and motility of sperm, plasma testosterone, and sexual behavior.


| INTRODUC TI ON
Reproductive performance is an important parameter affecting flock profitability (Pardos et al., 2008), in which the reproductive capacity of the rams plays an important role. In fact, 50% of the reproductive potential of a flock is provided by the ram (MacLaren, 1988). Food supplementation with plants rich in active molecules can present a natural and ecological opportunity to improve the reproductive health of farm animals.
However, there is little research that has focused on their effect on reproductive performance, mainly in male reproduction. In female performances (Laadraoui et al., 2018) showed the administration of 80 and 150 mg/kg/day of methanol extract of Artemisia herba alba during the entire period of gestation decreases the fertility of Swiss mice. However, (Selmi et al., 2016) showed that essential oil has a positive effect on male mice reproduction. Regarding Rosmarinus officinalis (Elmi et al., 2017) showed a total spermicidal effect on human spermatozoa at 0.8 mg mL −1 compared to Artemisia herba alba.
However, (Touazi et al., 2018) showed that small concentrations of Rosmarinus officinalis essential oil increase the fertility of cocks' spermatozoa in vitro and after artificial insemination. Moreover, Rosmarinus officinalis affects some hormones and cellules on the testes. It decreased the testosterone level in rats. However, it increased the number of spermatogonia, spermatocyte, Leydig cell and spermatid (Heidari-Vala et al., 2013).
To our knowledge, this is the first in vivo study that has looked into the possible effects of Artemisia herba alba and Rosmarinus officinalis fresh leaves supplementation on semen quality, biochemical profiles of semen, testicular size, sexual behavior, serum testosterone, and some blood parameters.

| Location and animals
The trial was carried out in the sheep research farm of the National Institute of Agricultural Research in Tunisia (INRAT). Twenty-four adult rams of the Barbarine breed (mean live weight 53.3 ± 1.2 kg at the start of the experiment) were selected from the farm flock on the basis of their ability to ejaculate in an artificial vagina. The animals were reared under similar conditions and were continuously exposed to natural photoperiod. Prior to the experiment, the animals were treated against internal parasites using Oxfendazol (Medivet, Soliman, Tunisia) at a conventional dose of 5 mg/kg body weight (BW) and vaccinated against enterotoxaemia.

| Experimental protocol
The experiment lasted 2 months (March 15-May 15). The animals were divided into four groups balanced for live weight and were placed in individual boxes (2m 2 each) inside a barn, well ventilated with wide windows on all sides so that animals had continuous exposure to natural daylight. Each of the 24 rams was fed on a daily basis a diet composed of a mixture of 1200 g (1044 g dry matter (DM)) of wheat straw and 600 g (552 g DM) of barley grains, which was offered into two equal meals (at 08:00 and 19:00 h). Rams in treatment C (control) received a diet without aromatic and medicinal plants (AMP), while experimental rams received 20 g (8 g DM) of fresh Rosemary (R), 20 g (5.8 g DM) of fresh Artemisia (A) and rams received 10 g (4 g DM) of fresh Rosemary officinalis plus 10 g (2.9 g DM) of fresh Artemisia herba alba (RA) ( Table 1). The experiment lasted 60 days. All animals were subjected to an adaptation period that lasted 15 days. Abbreviation: g DM, grams of dry matter.

| Feed and water intake
Feed and water intake were daily assessed for each individual ram by measuring the quantity of feed refusals.

| Body weight and testicular circumference
Animal live weight was measured before the distribution of the morning meal. Testicular circumference was measured using a caliber on the same occasions.

| Blood parameters
Blood samples were taken every 15 days in the morning before feeding and watering. Blood was withdrawn from the jugular vein using heparinized 10 mL vacuum tubes, centrifuged for 15 min at 3000 g, and the recovered plasma was aliquoted and then stored at −20°C before it was assayed, in the Department of Veterinary Medicine at the University of Sassari, Italy, for biochemical analyses: • Glucose, total proteins, urea, non-esterified fatty acid (NEFA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) was determined using commercial kits (ALCYON (Italy)-
• Plasma testosterone in ovine was determined using DRG

| Semen parameters
Collection of semen was done using rams that were put individually in the collection room in presence of a teaser female that was previously inducted into estrus. Estrus was induced by inserting a progestogen-impregnated vaginal sponge for 6-7 days followed by daily injections of 250 μg of estradiol benzoate for three consecutive days.
Semen traits were assessed every 15 days at the same day of blood sampling. One ejaculate per ram was collected to assess for sperm volume, motility, concentration, and for some biochemical parameters of seminal plasma.
Ejaculates are recovered in glass tubes (4 mL) graduated to the nearest 0.1 mm. This allowed direct determination of the ejaculate volume without considering the frothy part on the top.
Concentration (the number of spermatozoa/mL) was determined using a spectrophotometer calibrated to measure sheep sperm concentration at 550 nm (Accucell R; IMV, Paris, France). Four μl of fresh semen were diluted in 3996 μL of physiological saline solution. Mass activity (wave motion or motility score) in undiluted semen was assessed by examining a drop of semen under a warm stage phase contrast microscope at 40× magnification (score, 0-5).
Each ejaculate was then centrifuged for 15 min at 1500 g and recovered seminal plasma was aliquoted and stored at −80°C be-

| Sexual behavior and testosterone level
Sexual behavior was assessed with rams ordered in a random sequence and exposed to two ewes in estrus for a 15-min period in a test pen of 6 m 2 . The test facility was constructed to eliminate outside distractions and to prohibit the rams being tested from seeing any sheep other than the teaser ewes. Libido was evaluated by observing mating sexual approaches included vulva sniffing, flehmen, lateral approaches, and mount attempts without ejaculation.
Latency to first reaction (time separating introduction of the rams in the pen and observation of the first of any of the behavior traits) and total activity time was also recorded (Kilgour & Whale, 1980).

| Statistical analysis
The data obtained are presented as means ± SD. For all traits that were measured for AMP rams and the corresponding measures for C animals, measurements/ram was averaged, and statistical analysis was based on these means. The data statistical analysis was performed according to the MIXED models' procedure (SAS version 9.1; SAS Inst., Cary, NC, USA, 2005). Sources of variation included treatment and measurement time. The random variable was rams' diets.
Levels of statistical significance were set at p < .05.

| Diet composition, daily feed intake
Analysis of the chemical composition of the different components of the diet showed that the highest percentage of crude protein was recorded in Artemisia herba alba and that the leaves of Rosmarinus officinalis were richer in water compared to the leaves of Artemisia herba alba ( Table 2).
Daily control of feedstuff intake showed that there is no difference between the different groups. In fact, the incorporation of the fresh leaves of Artemisia herba alba and/or Rosmarinus officinalis did not affect the feeding behavior of the animals ( Table 3).

| Bodyweight and scrotal circumference changes
The evolution of the bodyweight of the rams in relation to the diet is presented in Figure 1. The initial weight in the entire group was 53.3 ± 1.2 kg. All groups have shown an increase in live weight during the experiment period as compared to the initial one. At the end of the trial, the highest value was recorded in the R group and the lowest value in the AR group. However, all these differences between groups were not significant (p > .05). Only at the end of the experiment, an average difference of 3 kg was recorded between the R rams and the RA rams (p < .05).

| Semen characteristics
All the rams adapted to the semen collection procedures. For experimental rams, changes throughout the experimental phase for the volume of the ejaculate, sperm concentration, and mass activity score are shown in

| Sexual behavior and testosterone level
Sexual behavior tests showed an improvement in the precopulatory behavior studied as well as the libido score in animals TA B L E 2 Chemical composition of the different foods of the diet (n = 8). Semen calcium (mg/dL) 6.18 ± 1.8 a 6.6 ± 1.8 a 6.8 ± 1.6 a 6.8 ± 1.9 a <.05 <.01 <.05

Rosmarinus officinalis
Note: a, b, and c indicate the significant differences between the groups at the threshold of 5 or 1%.
supplemented by the leaves of the two aromatic plants (p < .05).
The improvement was greater in the group that received Artemisia.
These behavioral observations were confirmed by the increase in plasma testosterone concentration recorded in these same rams (p < .05) (Table 4).

| Blood parameters
The analysis of some blood biochemical markers to study the effect of supplementation of rams with these two aromatic plants showed that the parameters measured remained in the range of the sheep species and had no effect on the general health of the animals.
However, a significant decrease in glucose in the RA group and insulin in the R group was recorded. On the other hand, the study of the hepatic function showed an increase in the AST enzyme in RA rams and no effect on the AST enzyme. Plasma cortisol analysis showed a significant increase (p < .01) in animals that consume rosemary leaves (R and RA groups) ( Table 5).

| DISCUSS ION
Raising sheep in good environmental and nutritional conditions has always been the concern of breeders throughout the world. This concern is justified by the search for a better quality of meat, sheep milk, and also the reproductive capacity of both males and females and the health of their progenies (Martin & Walkden-Brown, 1995;Prache & Nozières-Petit, 2015).
In recent decades, changes have been observed in the procedure of breeding throughout the world and in Tunisia. Indeed, a majority of breeders opt most often nowadays for a sedentary mode and the flocks of sheep are fed either freely in local natural pastures, or with a commercial feed or by combining these two types (Retaillé, 2003;Sicard, 2012;Yabrir et al., 2015). However, due to climate change, with the scarcity of rainfall and the aridity of land, the diet of sheep is largely impacted, which can lead to physiological and behavioral disturbances affecting, in particular, the reproductive system of sheep and their ability to generate offsprings, which is a loss for both the farmers and consumers (Belkadi, 2019;Belkhiri et al., 2019;Pottier et al., 2007).
In order to improve the diet of their sheep flocks when green pastures are scarce, farmers have recourse to commercial feed supplements (Rekik et al., 2007), which are usually quite expensive and not always easily available.

F I G U R E 1 Effect of incorporating Artemisia herba alba and
Rosmarinus officinalis in the diet of rams on bodyweight evolution. Testosterone (ng/mL) 5.91 ± 2.47 a 6.21 ± 2.13 a 9.54 ± 2.92 b 9.85 ± 1.95 b <.05 Note: a, b, and c indicate the significant differences between the groups at the threshold of 5 or 1%. It has been reported in several studies that the incorporation of plants species in sheep's diet can have an impact on different growth parameters, both muscular and skeletal, as well as on energy metabolisms, certain endocrine pathways, but also on the reproductive function and sexual performance of rams (Aouadi et al., 2014;Baazaoui et al., 2017;Hanafy et al., 2009;Odhaib et al., 2018;Yagoubi et al., 2022).
Considering results from our study, rams diet supplementation with Artemisia herba alba or Rosmarinus officinalis did not lead to significant changes in blood metabolites, energy and protein pa- Our results have shown an increase of plasma cortisol level in the supplemented rams. This could be due to the increase of their sexual activity. Indeed, some studies have shown that cortisol levels can be significantly affected by certain sexual practices as it has been reported that sex between men increases plasma corticosterone levels (Borg et al., 1992). Cortisol levels were highest during mating, mounting, and intromission (Borg et al., 1991).
Originally, it was thought that cortisol secretion was mainly due to a reaction to a stressful situation (fear, physical or psychological aggression, climatic aggression, microbial infections, etc.) in order to allow the organism to stay awake and to adapt metabolically to an increased need for energy (Selye, 1956). Over the decades, several studies have shown that cortisol can be secreted by the adrenal glands even in situations of well-being (intense sports practices, positive emotions in love, etc.). Other studies have reported an increase in plasma corticosterone levels after mating in many mammal species such as stallions, pigs and bulls (Borg et al., 1991;Rabb et al., 1989;Selye, 1956), and rats and mice (Belkadi,  Note: a, b, and c indicate the significant differences between the groups at the threshold of 5 or 1%. (Bonilla-Jaime et al., 2006), authors have shown that corticosterone levels may enhance up to two-fold after male exposure to physical or no physical contact with females either in estrous phase or in nonreceptive phase.
Furthermore, it has been shown that during sexual attraction in human courtship, the hypothalamic-pituitary-adrenal axis is stimulated in conjunction with the hypothalamic-pituitary-testicular axis in men when they see a woman with an attractive appearance (van der Meij et al., 2010). This could be explained by the probable existence of active nervous connections between different hypothalamic nuclei.

The incorporation of plants rich in active molecules, such as
Artemisia herba alba and/or Rosmarinus officinalis in the diet of rams, affects their physiology parameters including reproductive activity.
The addition of a small quantity of these plants to the daily diet of rams showed an improvement in some reproductive parameters including scrotal circumference, sperm concentration, testosterone level, and sexual behavior in addition to some biochemical changes in blood and sperm. There are few reports on how Artemisia herba alba and/or Rosmarinus officinalis affects reproduction function (15, 18, 62) many of which targeted in vitro study (Ahmed et al., 2022;Malo et al., 2011;Motlagh et al., 2014) or female reproduction (Akpa et al., 2012;Smeti et al., 2015). To our knowledge, this study is the first to investigate the effects of Artemisia herba alba and/or Rosmarinus officinalis on rams' reproductive capacity. On the basis of the study findings, some interesting features are being discussed below.
Body performance presents an important parameter for judging the reproductive capacity of a ram (Akpa et al., 2012). In the present study, it has been emphasized that at the end of the experiment we did not record differences in food intake and live weight of all rams increased. However, scrotal circumference was significantly higher in R rams when compared to controls and other experimental groups. In this context, (Khataibeh & Daradka, 2007) showed that intra-gastric administration of Artemisia herba alba caused an increase in BW and in the weight of the testes, epididymides, seminal vesicle, ventral prostate, and vas deferens. However, the ingestion of Rosmarinus officinalis did not affect the BW and testes weight, but decreased the weights of epididymides, ventral prostates, seminal vesicles, and preputial glands (Nusier et al., 2007).
Sperm quality is the main factor that limit male reproductive efficiency (Osadchuk & Osadchuk, 2020). Semen traits were affected by the imposed treatment. Indeed, A rams had higher sperm concentrations than C, R and AR animals. However, when rams received  (Cuevas et al., 2013) was affected by Rosmarinus officinalis and Artemisia herba alba, which partly explains the plant effect on sperm motility. In contrast to sperm protein, we showed an effect of treatment on the energy metabolism of sperm. In fact, we recorded a decrease in sperm glucose and insulin in A rams because of the positive correlation between them (Elsamanoudy et al., 2016). As shown in the literature, the sugar composition of seminal plasma has been correlated with fertility, mainly because of its importance for sperm energy production (Garner, 2001), and glucose is a source of energy for spermatozoa (Leahy et al., 2019). However, in the present study, the dramatic drop of sperm glucose concentration in rams that received Artemisia herba alba separately did not affect the sperm quality parameters. On the contrary, rams that have Blood testosterone level is an important indicator of fertility and reproductive activity (Apfelbeck et al., 2016;Preston et al., 2012).
This steroid hormone promotes sexual behavior and cognitions in order to facilitate mate acquisition or mating directly (Roney et al., 2003). Testosterone levels increase after male sexual activity (Dabbs & Mohammed, 1992).
Indeed, the increase in this parameter shows an increase in the reproductive activity of the animals (Apfelbeck et al., 2016;Preston et al., 2012). In our study, testosterone concentration doubled in rams that received Artemisia herba alba in the diet (p < .05), which shows an improvement in the reproductive activity of rams. This result is confirmed by sexual behavior tests which showed an improvement in most of the pre-copulatory behavior (p < .01) and libido score (p < .05) in rams that received Artemisia herba alba. Contrary to our study (Khataibeh & Daradka, 2007) showed a negative effect of Artemisia herba alba on the hormonal secretion of follicle-stimulating hormone (FSH) and blood testosterone in rats.

| CON CLUS ION
It emerges from our present study that dietary supplementation of rams with Artemisia herba alba or with Rosmarinus officinalis has positive effects on their reproductive activity shown by the enhancement of their libido score, testicular circumference, sexual behavior, and fertility as a consequence of the improvement of the spermatic quality as well as the hormonal status regulating the testicular function. This diet could represent a beneficial nutritional potential for the improvement of breeding conditions and development of sheep breeds in Tunisia.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and feed legislation, if appropriate.