**Dietary Supplementation with Goji Berries (***Lycium barbarum)* **Modulates the Microbiota of Digestive Tract and Caecal Metabolites in Rabbits**

**Paola Cremonesi 1,†, Giulio Curone 2, Filippo Biscarini 1,†, Elisa Cotozzolo 3, Laura Menchetti 4,\*, Federica Riva 2, Maria Laura Marongiu 5, Bianca Castiglioni 1, Olimpia Barbato 6, Albana Munga 7, Marta Castrica 8, Daniele Vigo 2, Majlind Sulce 7, Alda Quattrone 6, Stella Agradi 2,\* and Gabriele Brecchia <sup>2</sup>**


**Simple Summary:** The microbial community that inhabits specific areas of the body, developing a symbiotic relationship with the host, is termed the microbiota. The intestinal microbiota plays a pivotal role in different physiological processes and is influenced by many factors, including nutrition. Goji berries are a popular nutraceutical product that have been proposed as a dietary supplement in some livestock species, including rabbits, but their effects on the composition of the microbiota have never been investigated. This study evaluated the effects of Goji berry supplementation on the microbiota of different digestive tracts (stomach, duodenum, jejunum, ileum, caecum and colon) of the rabbit, using a modern method of analysis. Our results suggest that Goji berries could modulate the microbiota of the rabbit's digestive tract increasing the growth of beneficial bacteria, such as Ruminococcaceae, Lachnospiraceae, Lactobacillaceae, and particularly, the genus *Lactobacillus*. These findings suggest that Goji berries could be used to produce innovative feeds for rabbits, although further studies are necessary to evaluate their impact on productive performance, gut immune system maturation, as well as resistance to gastrointestinal disorders.

**Abstract:** Goji berries show health benefits, although the possible mechanisms of action, including compositional changes in the gut microbiome, are still not fully understood. The aim of this study was to evaluate the effect of Goji berry supplementation on microbiota composition and metabolites in the digestive tracts of rabbits. Twenty-eight New Zealand White rabbits were fed with a commercial feed (control group, C; *n* = 14) or the same diet supplemented with 3% of Goji berries (Goji group, G; *n* = 14), from weaning (35 days old) until slaughter (90 days old). At slaughter, samples from the content of the gastrointestinal tracts were collected and analyzed by Next Generation 16S rRNA Gene Sequencing to evaluate the microbial composition. Ammonia and lactic acid were also quantified in caecum. Results showed differences in microbiota composition between the groups for two phyla (Cyanobacteria and Euryarchaeota), two classes (Methanobacteria and Bacilli), five orders, fourteen

**Citation:** Cremonesi, P.; Curone, G.; Biscarini, F.; Cotozzolo, E.; Menchetti, L.; Riva, F.; Marongiu, M.L.; Castiglioni, B.; Barbato, O.; Munga, A.; et al. Dietary Supplementation with Goji Berries (*Lycium barbarum)* Modulates the Microbiota of Digestive Tract and Caecal Metabolites in Rabbits. *Animals* **2022**, *12*, 121. https://doi.org/ 10.3390/ani12010121

Academic Editors: Iveta Plachá, Monika Pogány Simonová and Andrea Lauková

Received: 29 November 2021 Accepted: 3 January 2022 Published: 5 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

families, and forty-five genera. Ruminococcaceae (*p* < 0.05) and Lachnospiraceae (*p* < 0.01) were more abundant in G than in C group. Lactobacillaceae also showed differences between the two groups, with *Lactobacillus* as the predominant genus (*p* = 0.002). Finally, Goji berry supplementation stimulated lactic acid fermentation (*p* < 0.05). Thus, Goji berry supplementation could modulate gastrointestinal microbiota composition and caecal fermentation.

**Keywords:** Goji fruit; intestinal bacterial community; caecum; lactic acid; ammonium; rabbit

#### **1. Introduction**

Goji berries, the fruits of the *Lycium barbarum* plant, are often used in traditional Chinese medicine for their nutritional and therapeutic properties, and are also widespread as supplementation in Western diets [1,2]. Their health benefits are associated with biologically active compounds, including polysaccharides, carotenoids, polyphenols, amino acids, ascorbic acid, and unsaturated fatty acids [3], although their mechanisms of action are still not fully understood. Recent evidence has shown that the fruit could modulate the gut microbiota and thus have a role in the prevention and treatment of several gastrointestinal diseases in mice [4,5], rats [6] and humans [7]. Recently, Goji berries have also been proposed as a dietary supplement for some livestock species, with the dual aim of improving productive performance and product quality [8–10]. In rabbits, Goji berry supplementation seems to improve reproductive [11] and productive performances, [12] energy metabolism [13], and meat quality [14,15] in a dose-dependent manner, but its effects on gut microbiota have not yet been investigated.

The microbiota represents a complex ecosystem of microorganisms which inhabits specific niches of the body and plays important roles in physiological processes developing symbiotic relationships with the host [16]. The intestinal microbiota is involved in the digestion and absorption of nutrients, maturation and stimulation of the immune system, as well as protection against pathogenic infections [17]. The bacterial microbiota composition along the gastrointestinal tract of adult rabbits fed with a commercial diet has recently been characterized [18]. This study showed interesting differences among the various sections of the digestive system in bacterial richness and diversity [18]. Within the same species, however, bacterial community composition of the gastrointestinal tract can be influenced by several factors, including nutrition [13]. Goji supplementation could therefore induce favorable changes in the intestinal microbiota of the rabbit with beneficial effects on health and productive performance, as seen in other animal species and humans [4–10].

The rabbit is a very interesting species because it can be a pet, livestock or animal model. Both in pet and farmed rabbits, the digestive system is a common site of diseases that are often associated with changes in intestinal microbiota [19,20]. In particular, the peri-weaning period is the most critical physiological phase as the diet transition induces changes in the gut microbiota increasing sensitivity to gastrointestinal pathogens [21]. Antibiotics are commonly used to control intestinal infections; however, according to recommendations of the European Union, this practice should be reduced [22,23]. An innovative strategy to limit the incidence of gastrointestinal disorders could be the use of specific feeds for pet and farmed rabbits integrated with nutraceutical products such, as *Lycium barbarum* fruit, to favor the growth of a beneficial gut microbiota. Understanding the effects of Goji berry supplementation on the intestinal microbiota can therefore have important implications for the health of rabbits. The rabbit could be also considered as an animal model for diet-induced changes in gut microbiota, as it has already been used for studies exploring the effect of nutrition on productive [24–26], reproductive [27,28], and immunological traits [29,30].

The aim of this study was to investigate the effect of Goji berry supplementation on the microbiota composition of the different tracts of the digestive system (stomach, duodenum, jejunum, ileum, caecum, and colon) in the rabbit. For this purpose, the microbiota of all the

sections of the digestive apparatus was analyzed using Next Generation 16S rRNA Gene Sequencing. In addition, metabolites from bacterial fermentation in the caecum (lactic acid and ammonia) were also quantified.

#### **2. Materials and Methods**

#### *2.1. Animals and Samples Collection*

The experimental trial was conducted in the facilities of the Faculty of Veterinary Medicine of the Agricultural University of Tirana, Tirana, Albania.

The rabbits were maintained under the supervision of a responsible veterinarian and in accordance with the Directive 2010/63/EU regarding the protection of animals kept for farming purposes. The lowest number of rabbits necessary to obtain reliable results was used for the trial.

According to dietary treatment, 28 New Zealand White male rabbits were randomly assigned into two groups from weaning (35 days of age) until slaughter (90 days of age): control group (*n* = 14 animals, C), fed with a commercial pellet, and Goji group (*n* = 14 animals, G), fed with the same feed of the C group supplemented with 3% of Goji berries (Gianluca Bazzica, Foligno, Italy) before pelleting (Table 1). At weaning the average body weight was 875 ± 115 g and 893 ± 135 in C and G groups, respectively. Feeds from the same batches were previously used in other experiments [11,13–15].


**Table 1.** Feed formulation and chemical composition (as fed) of control group and Goji group diet.

<sup>1</sup> Per kg diet: vitamin A 11,000 IU; vitamin D3 2000 IU; vitamin B1 2.5 mg; vitamin B2 4 mg; vitamin B6 1.25 mg; vitamin B12 0.01 mg; alpha-tocopherol acetate 50 mg; biotine 0.06 mg; vitamin K 2.5 mg; niacin 15 mg; folic acid 0.30 mg; D-pantothenic acid 10 mg; choline 600 mg; Mn 60 mg; Fe 50 mg; Zn 15 mg; I 0.5 mg; Co 0.5 mg. <sup>2</sup> NDF: Neutral Detergent Fiber; ADF: Acid Detergent Fiber; ADL: Acid Detergent Lignin. Estimated by Maertens et al. [31].

Rabbits were bred in single cages and maintained at a temperature range between 18 and 21 ◦C, relative humidity of 60%, and with a photoperiod of 16 h of light. Throughout the entire trial, water and feed were provided ad libitum.

At the slaughterhouse, the gastrointestinal tract was immediately removed from each rabbit. The content of the different digestive tract sections from each animal (stomach, duodenum, jejunum, ileum, caecum, and colon) were collected separately in 15 mL sterile

tubes and then stored at −80 ◦C until examination. Each sample was analyzed individually. The average body weights (±standard error) at weaning were 875 ± 55 g and 893 ± 75, while at slaughter, they were 2310 ± 82 g and 2357 ± 82 g in C and G groups, respectively.

#### *2.2. Microbiota Evaluation—Genomic Sequencing* 2.2.1. DNA Extraction

Using the commercial QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany), the bacterial DNA was extracted from each sample of intestinal contents following the manufacturer's protocol. DNA quality and quantity were checked using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA, and the obtained DNA was stoked at −20 ◦C until use.

#### 2.2.2. 16S Ribosomal RNA (rRNA) Gene Sequencing

Bacterial DNA was amplified using primers described in the literature [32] which target the V3-V4 hypervariable regions of the 16S rRNA gene. All the PCR amplifications were performed in 25 μL volumes per sample. A total of 12.5 μL of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., Wilmington, MA, USA) and 0.2 μL of each primer (100 μM) were added to 2 μL of genomic DNA (5 ng/μL). Blank controls (no DNA template added to the reaction) were also performed. A first amplification step was performed in an Applied Biosystem 2700 thermal cycler (ThermoFisher Scientific, Waltham, MA, USA). The samples were denatured at 95 ◦C for 3 min, followed by 25 cycles with a denaturing step at 98 ◦C for 30 s, annealing at 56 ◦C for 1 min, and extension at 72 ◦C for 1 min, with a final extension at 72 ◦C for 7 min. The amplicons were then cleaned with Agencourt AMPure XP (Beckman, Coulter Brea, CA, USA), and libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified using Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., Wilmington, MA, USA), pooled in equimolar proportion, and then sequenced in one MiSeq (Illumina, San Diego, CA, USA) run with 2 × 250-base paired-end reads.

#### 2.2.3. Sequence Analysis

The reads obtained by the 16S rRNA sequencing were analyzed as previously described [18]. One rabbit from the Goji group and two samples, both from G diet (caecum intestinal tract), were removed because they had a total number of counts <100.

#### 2.2.4. Alpha and Beta Diversity Indices

To assess the microbial diversity of the different rabbit gastrointestinal tracts the alpha (within-) and beta (across-) diversities were used. These indices were estimated starting from the OTU table, after filtering with more than 50 total counts, distributed in at least five samples. Besides the number of observed OTUs directly, within-sample microbial richness, diversity, and evenness were estimated using Chao1 and ACE (abundance-based coverage estimator) for richness, Shannon, Simpson, and Fisher's alpha for diversity [33,34], and Simpson E and Pielou's J (Shannon's evenness) for evenness [35]. The Bray–Curtis dissimilarity [36] was used to quantify the across-sample microbiota diversity. Prior to the calculation of these metrics, the OTU counts were normalized for uneven sequencing depth by cumulative sum scaling (CSS) [37]. Details of these analyses can be found in Biscarini et al. [38].

#### 2.2.5. Software

The QIIME 1.9 pipeline [39] was utilized both to analyze the reads obtained from 16S rRNA gene sequencing and to estimate most diversity indices. Own Python (https: //github.com/filippob/Rare-OTUs-ACE.git, accessed on 15 November 2021) and R (https: //github.com/filippob/sampleBasedRarefaction, accessed on 15 November 2021) scripts were used to estimate the ACE index and sample-based rarefaction. The figures were

generated with the ggplot2 R package [40]. The R environment for statistical computing [41] was used to perform the additional data handling and statistical analysis.

#### *2.3. Lactic Acid and Ammonia Quantification*

For the analysis of bacterial metabolites (lactic acid and ammonia), 1 g of caecal content was diluted in 1 mL of 1 M perchloric acid and 8 mL of distilled water. After homogenization, tubes were centrifuged for 10 min at 5000 rpm, and the supernatant was transferred to 2 mL Eppendorf tube and frozen at −20 ◦C until metabolite quantification. The spectrophotometric method for biological fluids was used for lactic acid determination in accordance with Pryce et al. [42]. Ammonia concentration was detected in line with Patton et al. [43]. Spectrophotometer was set at 565 nm and 660 nm respectively (Shimadzu Corporation UV-2550, Kyoto, Japan). All chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA).

#### *2.4. Statistical Analysis*

Differences in alpha diversity indexes between treatments at various taxonomic levels along the rabbit's gastrointestinal tract were tested with a linear model that took into account the hierarchical structure of within-subject nested data (consecutive sections of the gastrointestinal tract belonging to individual rabbits). The model had the following form:

$$\mathbf{y}\_{i\text{jkt}} = \mu + \mathbf{z}\mathbf{b}\mathbf{b}\mathbf{i}\mathbf{i}\_{\parallel} + \text{treatment}\_{\mathbf{k}} + \text{anatornic region}\_{\mathbf{t}(\mathbf{j})} + \mathbf{e}\_{i\text{jkt}}\tag{1}$$

where yijkt is the alpha diversity index value for record i from rabbit j with treatment k and anatomic region t, μ is the intercept, rabbitj is the systematic effect of the individual rabbits, treatmentk is the treatment effect (Goji vs. control), anatomic regiontk(j) is the effect of the anatomic region of the gastrointestinal tract nested within rabbitj, and eijkt is the residual.

$$\text{Var}(\mathbf{y}) = \text{Sign}\mathbf{a}\mathbf{a} + \text{I}\sigma\_{\mathbf{e}}^2$$

where Sigma is a block diagonal matrix, with 1 s on the diagonal and the covariances σij between records within rabbits in the off-diagonal block elements, I is the identity matrix, and σ<sup>e</sup> <sup>2</sup> is the residual variance.

A simplified version of Model (1) was used to evaluate differences between Goji and control samples; in particular, where the anatomic region effect was dropped and data from all gastrointestinal sections were analyzed jointly to evaluate the effect of Goji supplementation on the overall rabbit gut microbiota.

For Bray–Curtis dissimilarities (beta diversity), differences along the digestive tract were tested non-parametrically using the permutational analysis of variance approach (999 permutations; [33]).

#### **3. Results**

#### *3.1. Sequencing Results*

The microbiota structure of the gastrointestinal tract of C and G groups was characterized by a total of 6,122,359 and 7,156,769 high quality reads (after filtering), respectively, with a mean of 75,584 ± 38,864 reads for C and 90,592 ± 33,296 reads for G group. The evaluation of the sample-based and sequence-based rarefaction curves suggested that the depth of coverage was sufficient to describe the biological diversity within the samples (Figure S1).

#### *3.2. Taxonomic Composition of Gut Microbiota along the Rabbit Gastrointestinal Tract of C and G Groups*

Phylum relative abundances distribution along the gastrointestinal tract of C and G groups are summarized in Figure 1. Significative differences were found in microbiota composition between the experimental groups for two phyla, two classes, five orders, fourteen families, and forty-five genera (Table S1). Firmicutes represented the main phylum

in all sections of the digestive tract, especially in the most distal portions of caecum and colon (77–79% of total bacteria) for both groups, and Bacteroidetes the second (14–16% of total bacteria). The caecum and colon of rabbits treated with Goji berries showed differences regarding the abundance of Bacteroidetes (16%) compared to the control group (14%) although these were not statistically significant. As regards other phyla, Actinobacteria was present in the upper part of the gastrointestinal tract. In the jejunum, its relative abundance was higher in C than G group (7.5% for C vs. 5.5% for G group), while in the ileum the percentages were opposite (4.8% for C vs. 5.2% for G group); as with the Bacteroidetes, the differences regarding Actinobacteria were not significant. On the other hand, at the phylum level, Cyanobacteria and Euryarchaeota, the latter belonging to kingdom Archaea, were statistically different (*p* = 0.034 and *p* = 0.004, respectively) between the experimental groups, with higher relative abundances in the upper part of the gastrointestinal tract in G group.

Moreover, Clostridia represented the major class in all anatomic regions, while Ruminococcaceae and Lachnospiraceae were the most abundant families in the Goji group (Figure 2).

Figure 3 shows the comparison of the relative abundances of significant OTUs between treatments and along the rabbit's gastrointestinal tract. As shown in Table S1 and Figure 3, there were significant differences between the groups; Bacillales were predominant (*p* = 0.0032) in the G group, and *Bacillus* was the major genus in the stomach (*p* = 0.0036). Ruminococcaceae UCG-005, Lachnospiraceae NK4B4 group, and Christensenellaceae R-7 group were genera detected in all the digestive tracts with statistically significant different results between the groups. As reported in Table S1, the Lactobacillaceae family was significantly different (*p* = 0.0018) between the groups with *Lactobacillus* as the predominant genus in G group compared to C group.

#### *3.3. F/B Ratio*

The Firmicutes: Bacteroidetes (F:B) ratio followed a clear pattern along the rabbit's digestive tract starting at around 10 in the stomach, increasing clearly in the duodenum and jejunum, and finally decreasing again in the caecum and colon. The F:B ratio appeared to be significantly lower in the G group (Figure 4), in the duodenum (*p* = 0.0176) and jejunum (*p* = 0.000049). This was confirmed by bootstrapping (1000 replicates resampled with replacement from the original data, Figure 5), which provided further statistical support of the significance of F:B differences between G and C groups in the duodenum, jejunum and, slightly less so, in the ileum.

#### *3.4. Alpha Diversity Index—Treatment by Region*

Table 2 reports the values for the alpha diversity indexes estimated in the rabbits' gastrointestinal tract, in the two groups. Alpha diversity indexes were significantly different between treatments in the last portion of the digestive tract (Figure S2): six indexes were significantly different in the jejunum (ACE, Fisher's alpha, observed n. of OTUs, Shannon and Simpson diversity), two in the ileum (Equitability and Simpson E), three in the caecum (Chao1, ACE, Fisher's alpha), and two in the colon (Equitability and Simpson E).

**Figure 1.** Pie-chart of phylum relative abundances in control and Goji-treated rabbits along the gastrointestinal tract. For the analyses, 14 and 13 samples were used for the control and Goji groups, respectively.

**Figure 2.** Bubble chart of relative abundances of all taxa (≥1%) in the microbiota of the digestive tract of rabbits, grouped by taxonomic level. Control (blue = 14 rabbits) and Goji (yellow = 13 rabbits) experimental groups. The size of the bubble is proportional to the relative abundance, with 0.2, 0.4 and 0.6 hallmarks, as shown in the legend.

**Figure 3.** Significantly different OTUs. OTU significantly different between treatments from analysis of variance based on normalized counts: *p*-values (**A**) and counts per group and anatomic region of the rabbit digestive tract (**B**). *p*-value < 0.05 was used as cut-off. Darker colours indicate lower *p*-values (**A**) or higher counts (**B**). *p*-values are in the range 10−15–0.049, from dark brown to light yellow. For the analyses, 14 and 13 samples were used for the control and Goji groups, respectively.

71

**Figure 4.** Distribution of the F:B ratio (Firmicutes to Bacteroidetes) in control and Goji-treated groups along the gastrointestinal tract. For the analyses, 14 and 13 samples were used for the control and Goji groups, respectively.

**Table 2.** Alpha diversity indices along the digestive tract of rabbits in the two experimental groups (14 controls and 13 Goji-treated; two more samples, both from caecum intestinal G diet were removed because they had a total number of read counts < 100). \* indicates significant difference (*p* < 0.05) between control and Goji groups.


#### *3.5. Beta Diversity Index (Clustering Treatment X Anatomic Portion)*

Figure 6a shows the clustering of samples (C and G groups) from Bray–Curtis dissimilarities (first three dimensions from non-metric multidimensional scaling). The distance between groups were significantly different (*p* < 0.01) from permutational multivariate analysis of variance (PERMANOVA, 999 permutations). This difference appeared to vary along the gastrointestinal tract, with jejunum, caecum, and colon showing the clearest differences, while the two groups mostly overlapped in the stomach, duodenum, and ileum (Figure 6b: first two NMDS dimensions only).

#### *3.6. Caecal Lactic Acid and Ammonia Quantification*

Regarding lactic acid quantification, G group showed a higher concentration than C group, suggesting higher bacterial activity (3.91 ± 1.59 and 1.01 ± 1.22 mmol/kg in C and G groups, respectively; *p* = 0.033). No significant differences in ammonia concentration were detected between the two groups (5.81 ± 2.22 and 5.89 ± 1.81 mmol/kg in C and G groups, respectively; *p* = 0.305).

**Figure 5.** Distribution of the F:B ratio (x-axis) along the digestive tract in Goji-treated (blue) and control (red) rabbits from 1000 bootstrapping replicates of the data. For the analyses, 14 and 13 samples were used for the control and Goji groups, respectively.

**Figure 6.** *Cont*.

**Figure 6.** (**a**): Non-metric multidimensional scaling plot of Bray-Curtis dissimilarities estimated from the OTU table. The plots show the first three NMDS dimensions (from left to right: dimensions one and two, one and three, two and three). Control samples in red circles, Goji-treated samples in blue triangles. (**b**): First two dimensions from the non-metric dimensional scaling of Bray-Curtis dissimilarities between control and Goji-treated samples along the digestive tract of rabbits. For the analyses, 14 and 11–13 samples were used for the control and Goji groups, respectively.

#### **4. Discussion**

Diet is one of the main factors affecting the composition of the microbiota in the digestive tract due to the relation between nutrients and microbial populations [44]. The bacterial populations inhabiting the different gastrointestinal compartments of the rabbit have been previously described [18]. For the first time, this study investigated the effect of Goji berry supplementation on microbiota composition in the different tracts of the digestive system and on caecal bacterial fermentations of adult rabbits.

The results of the present study showed a prevalence of Firmicutes in all the anatomic tracts in both experimental groups. This phylum is classified as the most efficient cellulose degrader [45] and it plays a fundamental role in rabbit digestion. Similar results were reported by both Cotozzolo et al. [18] and Arazzuria et al. [46]. This result was also supported by other studies investigating not only the caecal microbiota of rabbits [47,48] but also the gastrointestinal content and feces of both wild and domestic rabbits [49]. This is a common condition not only in hindgut fermenters, such as rabbits, but also in ruminants and monogastric animals [50].

Bacteroidetes was the second most abundant phylum, especially in the large intestine (caecum and colon tracts), and was slightly predominant in the G group. This phylum, not significantly different between the two groups and along the digestive tracts, is known for its role in the stimulation of gut-associated lymphoid tissue [46,48]. The abundance of Bacteroidetes is in accordance with what was already observed by Cotozzolo et al. [18] on the rabbit gastrointestinal microbiota and by Crowley et al. [49] on both domestic and wild rabbits. A further analysis of our samples with a shotgun metagenomic or metatranscriptomic approach, combined with immunological assays, could provide more information about the role of this relevant phylum in gut immunity.

Regarding other phyla, Verrucomicrobia were found in all sections, while Actinobacteria and Proteobacteria were found in the stomach and small intestine. Although with low levels in the core microbiome, the Euryarchaeota phylum, belonging to the kingdom

Archea, was statistically different between the two groups, with higher levels in the G group in all the digestive tracts. All species of this phylum were taxonomically assigned to the methanogenic genus *Methanobrevibacter* [51]. Though this phylum is not very common in the intestinal microflora of some species, such as horses and pigs [18], it is often found in the human gut with the role of increasing polysaccharide digestion by consuming the end products of bacterial fermentation [52].

Clostridia, anaerobic Gram-positive bacteria present in the intestinal microbiota of human, mouse, chicken, and pig, represented the major class in all anatomic regions, in accordance with Velasco-Galilea and co-workers [51]; they are prevalent, cellulosedegrading symbiotic microorganisms, helping the rabbit for plant material digestion [51].

The families of Ruminococcaceae and Lachnospiraceae were present in all anatomic parts, and both were higher in the G group. Ruminococcaceae are usually prevalent in healthy rabbits [53], while Lachnospiraceae is known to be associated with a decrease of mortality [54]. These two families appear to have an important role in fiber digestion, in particular of peptose and cellulose [55], and are significant producers of short-chain fatty acids [56]. Moreover, as previously reported [4], in mice a diet with Goji supplementation promotes butyrate-producing bacteria, including Lachnospiraceae and Ruminococcaceae families, preventing colitis; their high levels in the digestive apparatus also allow protective and beneficial effects towards different diseases, such as diabetes and heart disease [57].

Lactobacillaceae was another family that showed significant differences between the two groups, although present in small quantities. Within this family, *Lactobacillus* was the predominant genus. Lactobacilli are rare in the rabbit intestine, occupying less than 1% of the total intestinal bacteria [58], and their function in gut health is not fully understood. A recent study has shown that the total intestinal bacteria from rabbits tends to induce a higher inflammatory level than the total intestinal bacteria from chickens or pigs [59], probably because of the low abundance of Lactobacilli in the rabbit's intestine. Thus, the higher *Lactobacillus* abundance in rabbits supplemented with Goji could play a protective role against inflammatory diseases. Components of commensal bacteria can alleviate intestinal inflammation by regulating the expression of both pro-inflammatory and anti-inflammatory factors. Kawashima et al. [60] reported that bacterial double-stranded RNA, abundant in *Lactobacillus bacteria*, showed a regulatory function by triggering anti-inflammatory factor IFN-β production and inhibiting pro-inflammatory factors production.

The F:B ratio was at around 10 in the stomach, then increased in the duodenum and jejunum, and subsequently progressively decreased from the ileum to the caecum and colon. The F:B ratio appeared to be lower in the G group, significantly so in the duodenum and jejunum, less so in the ileum, caecum and colon, as also shown by the bootstrapping analysis. Studies in human microbiota and in animal models, have reported that the F:B ratio was directly related to body weight modifications and in particular to obesity [61]. In obese people, the population of Firmicutes shows an elevated proportion with a reduced Bacteroidetes population; this unbalance causes an altered F:B ratio [62]. Additionally, a strong correlation between the F:B ratio and milk fat yield has been observed in dairy cattle [63]. In previous studies, feed supplementation in livestock has been reported to alter the F:B ratio in the gut microbiota (e.g., grape pomace supplementation in cattle [38]). Further studies could investigate the link between Goji intake, F:B ratio and lipid metabolism in rabbits.

The alpha diversity results revealed higher microbial richness and diversity in bacterial composition independently from the treatments in the large intestine. That was an expected result because, as already demonstrated in other livestock species, the microbial densities (and also diversity) along the gastrointestinal tract is maximal in the fermenting compartments [64]. Indeed, Cotozzolo et al. found alpha diversity of the cecum and colon to be significantly higher than for the other compartments of the rabbit gastrointestinal tract [18]. As previously reported [18], this variability, typical of colon and caecum tracts, is due essentially to their physiological functions, such as fermentation of cellulose with production of volatile fatty acids (VFA) and their absorption for energy production. Goji berry

supplementation caused higher microbial richness, especially in the jejunum, ileum, and colon tracts, where six indexes were significantly different in the jejunum (ACE, Fisher's alpha, observed n. of OTUs, Shannon and Simpson diversity), two in the ileum (Equitability and Simpson E), three in the caecum (Chao1, ACE, Fisher's alpha), and two in the colon (Equitability and Simpson E). In particular, the principal families involved in the microbial richness were Ruminococcaceae and Lachnospiraceae, as well as *Lactobacillus* spp. These conditions could guarantee greater resilience toward dysbiosis in the gut microbiota, which is necessary to maintain homeostasis and, in turn, the healthy status of the gastrointestinal system [65]. The beta diversity was greatly influenced by Goji treatment, especially in caecum and colon tracts, which play fundamental roles in the digestion of fermenter animals, such as rabbits. Conversely, less influence of this treatment was found in the stomach, duodenum, and ileum tracts.

The differences in microbiota composition are due to the environmental conditions, such as pH modifications, along the gastrointestinal tract. In adult rabbits, the principal substrates for caecal microorganisms are polysaccharides and protein. Caecal microorganisms ferment available nutrients, converting them to metabolites (e.g., short-chain VFA, ammonia, H2, CH4, CO2) and compounds that are incorporated into microbial cells [66]. Our results for caecal bacterial fermentations indicate that Goji berry supplementation did not influence proteolytic activity and ammonia production. On the other hand, Goji supplementation stimulated lactic acid fermentation, indicating changes in the intestinal microbiota in favor of specific bacterial populations. The caecum represents the main site of fermentative activity in the rabbit due to the presence of an abundant microbial flora [1]. Rabbits produce large amounts of VFA and lactate by fermentation of dietary carbohydrates, such as xylan and pectin, in the hindgut [67–69]. Lactobacilli are strong producers of lactic acid and, for this reason, can compete against pathogenic bacteria [70]. Regarding Goji berry supplementation, several authors [71,72] have confirmed the beneficial effects of this integration on the physiology and health of the gut acting on the intestinal microbiota composition of human and mice. Castrica et al. [15] reported that the incorporation of 3% *w/w* of Goji berries in the rabbit diet was able to increase the Lactobacilli population in rabbit meat.

This is a preliminary study on the effect of Goji berry supplementation on gastrointestinal microbiota of the rabbit and, although its practical implications are currently limited, it may represent a starting point for future exploratory research. Further experimental trials could be addressed to evaluate whether caecal fermentative activities (VFA production) could be affected by changes in microbial community composition. Moreover, evaluation of digestive efficiency by performing an in vivo digestibility trial could integrate the study of the microbiota composition of the rabbit. Finally, it could be interesting to evaluate the impact of microbiota modification on the maturation and activity of the immune system, as well as on resistance to infectious diseases, animal welfare and the productive performance of the rabbit.

#### **5. Conclusions**

The present study demonstrated that Goji berry supplementation can modulate gastrointestinal microbiota composition and caecal fermentations of the rabbit. In particular, *Lycium barbarum* fruit increased the growth of the phylum Bacteroidetes as well as of Ruminococcaceae, Lachnospiraceae, and *Lactobacillus* in the caecum and colon, and as a consequence, lactic acid production. The mechanism of absorption and integration of the bioactive molecules contained in the fruit, and their influence on the microbiota population, should be investigated to appropriately use Goji berries' probiotic properties. The use of this natural compound needs to be further studied for its implications for both commercial performance and animal resistance to infection, as its supplementation could reduce the incidence of health problems in livestock and consequent antibiotic treatments.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/ani12010121/s1: Figure S1: Evaluation of the sample-based and sequence-based bacterial rarefaction curves to check the depth of coverage; Figure S2: *p*-values for the difference in alpha diversity indices between control and Goji-treated rabbits, along the gastrointestinal tract; Table S1: Significantly different OTUs between Goji-treated and control rabbits in the gut microbiota (*p* < 0.05).

**Author Contributions:** Conceptualization, L.M., D.V. and G.B.; data curation, P.C., F.B., L.M., B.C., O.B. and M.C.; formal analysis, G.C., F.B., L.M., M.L.M., O.B. and G.B.; funding acquisition, P.C., M.L.M., B.C., D.V. and G.B.; investigation, G.C., E.C., A.M., M.C., M.S. and S.A.; methodology, P.C., E.C., B.C., A.M. and S.A.; project administration, L.M., M.L.M., A.M. and G.B.; resources, F.R., M.L.M., D.V. and G.B.; software, F.B. and L.M.; supervision, P.C., O.B., D.V., M.S. and G.B.; validation, P.C., F.B., F.R. and G.B.; visualization, F.B., B.C. and A.Q.; writing—original draft, P.C., G.C., F.B., E.C., L.M., F.R., M.L.M., A.Q., S.A. and G.B.; writing—review and editing, G.C., L.M., A.M., A.Q., S.A. and G.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by FAR 2019 of the University of Sassari.

**Institutional Review Board Statement:** The Albanian Ministry of Agriculture and Rural Development, National Authority of Veterinary and Plants protection authorized the protocol of the experimental trial (prot. 824/2021).

**Data Availability Statement:** The data presented in this study are available in the article and Supplementary materials. Further information is available upon request from the corresponding author.

**Acknowledgments:** The authors gratefully acknowledge the collaboration and support of Giovanni Migni.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Effect of Sustained Administration of Thymol on Its Bioaccessibility and Bioavailability in Rabbits**

**Kristina Bacova 1,2, Karin Zitterl Eglseer 3, Gesine Karas Räuber 3, Lubica Chrastinova 4, Andrea Laukova 1, Margareta Takacsova 1, Monika Pogany Simonova <sup>1</sup> and Iveta Placha 1,\***


**Simple Summary:** The purpose of this study was to investigate the bioavailability and metabolic path of thymol, a major constituent of *Thymus vulgaris* L., in the rabbit organism. Oral bioavailability is a key parameter affecting the efficacy of substances, but it is not surprising that it does not correlate satisfactorily with efficacy. The main limitation factors are rate of absorption, metabolism, and excretion processes. In this work, the thymol metabolic path in the rabbit organism was determined for the first time after its sustained oral administration. We confirm intensive absorption of thymol from the gastrointestinal tract; our results point to metabolism and accumulation in kidney tissue and intensive metabolic and excretion processes in the liver. Some metabolic processes were present also after thymol withdrawal. Thymol as a lipophilic substance was found only in trace amounts in fat and muscle tissue as a consequence of its conversion into hydrophilic metabolite and greater elimination in the rabbit organism. This paper highlights the insufficient knowledge of modes of action of plant compounds in animal organisms.

**Abstract:** The objective of this study was the detection of thymol in rabbit plasma, tissues, large intestinal content, and faeces. Forty-eight rabbits were divided into control and experimental groups (thymol 250 mg/kg feed). Thymol was administered for 21 days and then withdrawn for 7 days. Concentration of thymol in the intestinal wall (IW) was significantly higher than in plasma (*p* < 0.05) and liver (*p* < 0.05); in the kidneys it was significantly higher than in plasma (*p* < 0.05) and liver (*p* < 0.05) during thymol addition. Thymol in IW was significantly higher than in plasma also after withdrawal (*p* < 0.01). Significant correlation (rs = −1.000, *p* < 0.01) between IW and plasma points to the intensive absorption of thymol from the intestine, while the correlation between plasma and liver (rs = 0.786, *p* < 0.05) indicates intensive biotransformation and excretion processes in liver. Significant correlation between liver and kidney (rs = 0.738, *p* < 0.05) confirms the intensive metabolism of thymol in the kidney. During the withdrawal period, thymol was detected above trace amounts only in faeces, and was significantly higher than in the colon during both periods (*p* < 0.01). Results show intensive biotransformation of thymol in the rabbit organism.

**Keywords:** rabbit; thymol; absorption; distribution; accumulation; excretion

#### **1. Introduction**

In recent years, natural products have assumed great importance as antibiotic replacement additives and as growth promoting agents in food animals. There is large pressure

**Citation:** Bacova, K.; Eglseer, K.Z.; Räuber, G.K.; Chrastinova, L.; Laukova, A.; Takacsova, M.; Simonova, M.P.; Placha, I. Effect of Sustained Administration of Thymol on Its Bioaccessibility and Bioavailability in Rabbits. *Animals* **2021**, *11*, 2595. https://doi.org/ 10.3390/ani11092595

Academic Editors: Alessandro Dal Bosco and Cesare Castellini

Received: 3 August 2021 Accepted: 1 September 2021 Published: 3 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on the animal production industry to improve animal treatment as well as production performance, and to ensure the safety of products for human consumption while minimizing economic losses [1]. Even though, the benefits of herbal additives depend on the biological activities of their compounds and their pharmacokinetics, their precise mode of action at the molecular level has not yet been fully elucidated [2,3].

To our knowledge, bioaccessibility, bioavailability, and metabolism of phenolic compounds have been studied in vitro in humans [4,5] and in chickens [6]. No information is available about absorption, distribution, and deposition of natural compounds at target sites in the rabbit organism. The rabbit gastrointestinal tract has characteristic features compared with other animal species, such as the relative importance of the well-developed caecum, and a separation mechanism within the proximal colon [7]. The efficiency of the rabbit's digestion depends in large part on the production and ingestion of caecotrophes, which must be considered as an integral part of the rabbit´s digestion system. The process of caecotrophy may be considered as "pseudorumination" which improves feed utilization [8,9].

One of the crucial aspects of the beneficial effect of natural compounds is the amount present in the gut as a result of their release from feed, and their consequent ability to pass through the intestinal barrier. Intestinal absorption of many compounds is limited by a range of biological and physiological barriers in the gastrointestinal tract. Biological barriers are represented mainly by the mucus layer and epithelial cell layer, which is composed of villus and crypt cells. Physiological factors include enzymatic activities in the intestinal lumen, specific transport mechanisms which are able to limit absorption, and intestinal transit time [10].

In terms of the potential role of thymol as feed additive for animals, the aim of our study was to try to produce a more detailed view and better understanding of the mechanism of its absorption, distribution, and accumulation in the rabbit organism after its sustained application into the rabbit's diet.

#### **2. Materials and Methods**

#### *2.1. Animals Care and Use*

The trial was carried out at the experimental rabbit facility of the National Agricultural and Food Centre, Research Institute for Animal Production, Nitra, Slovakia. The protocol was approved by the Institutional Ethical Committee, and the State Veterinary and Food Office of the Slovak Republic approved the experimental protocol (4047/16-221).

#### *2.2. Animals and Housing*

After weaning at 35 days of age, 48 rabbits of both sexes (meat line M9) were randomly divided into a control group (CG) fed a standard diet and an experimental group (EG) fed a standard diet into which 250 mg/kg of thymol was incorporated in powder form (≥99.9%, Sigma-Aldrich, St. Louis, USA). All experimental wire-net cages (61 cm × 34 cm × 33 cm) were kept in rooms with automatic temperature control (22 ± 4 ◦C) and photoperiod (16L:8D). The rabbits could feed ad libitum and had free access to drinking water. The experiment lasted 28 days. The rabbits received feed with thymol addition for 21 days (56 d of age) and for the next 7 days (63 d of age) the thymol was withdrawn. Initial live weight was 1006 ± 98 g in CG and 1035 ± 107 g in EG (2044 ± 24 g in CG, 1965 ± 58.7 g in EG at 56 d of age, and 2671 ± 72 g in CG, 2796 ± 60 g in EG at 63 d of age). Eight rabbits in each group were killed at 56 or 63 d of age using electronarcosis (50 Hz, 0.3A/rabbit for 5 s), immediately hung by the hind legs on the processing line and quickly bled by cutting the jugular veins and the carotid arteries.

#### *2.3. Diet and Chemical Analyses*

The standard diet consisted of a commercial diet for growing rabbits (KKZK, Liaharensky podnik Nitra a.s., Nitra, Slovakia) with ingredients and chemical composition as shown in Table 1. The diet was administered in the form of pellets with an average

size of 3.5 mm. The feed was stored in darkness to protect against degradation processes. The Association of Official Analytical Chemists (AOAC) methods [11] were used to determine the proportions of crude protein (no. 990.03, CP), ash (no. 942.05), and dry matter (no. 967.03, DM) in the diet, while DM amount was also determined for the tissues, gut content, and faeces. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analysed according to Van Soest et al. [12].

**Table 1.** Ingredients (%) and chemical composition (g/kg feed) of experimental diet.


<sup>1</sup> The vitamin-mineral premix provided per kg of complete diet: Retinyl acetate 5.16 mg, Cholecalciferol 0.03 mg, Tocopherol 0.03 mg, Thiamin 0.8 mg, Riboflavin 3.0 mg, Pyridoxin 2.0 mg, Cyanocobalamin 0.02 mg, Niacin 38 mg, Folic acid 0.6 mg, Calcium 1.8 mg, Iron 70 mg, Zinc 66 mg, Copper 15, Selenium 0.25 mg.

#### *2.4. Thymol Stability in Feed*

Thymol evaporation in feed was analysed every week during thymol application using high-performance liquid chromatography (HPLC) according to the modified method of Ocel'ová [6] and Pisarˇcíková et al. [13]. Samples were analysed in triplicate. Briefly, 2 mL of methanol was added into a glass tube containing 0.2 g of milled feed and thymol was extracted in an ultrasonic bath. The methanolic extract was then analysed using the HPLC method with an Ultimate 3000 HPLC-system liquid chromatograph (Dionex, Sunnyvale, CA, USA). The chromatographic analyses were evaluated by means of Chromeleon® Software Version 6.80 SR10 Build 2906 (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.5. Sampling*

To determine the thymol content in plasma, blood (1.5 mL) from eight rabbits was collected from the marginal ear vein (*vena auricularis*) into heparinized Eppendorf tubes and plasma was obtained after centrifugation at 1180× *g* for 15 min. The gastrointestinal tract was removed from the body cavity and was divided into small intestine, caecum, and colon (n = 8). Caecum and colon content were removed, and the small intestinal lumen was gently washed with 0.9% NaCl solution. Obtained samples of gut content and intestinal wall together with plasma, liver, kidney, muscle (*musculus longissimus dorsi*) and spleen tissue, fat, and faeces were immediately frozen in liquid nitrogen and stored at −70 ◦C until analysis. All samples were collected at both experimental days (56 or 63 d of age).

#### *2.6. Thymol Analyses in Plasma, Tissues, Large Intestinal Content and Faeces*

Detection of thymol in samples of plasma, tissues and faeces was performed using headspace solid-phase microextraction followed by gas chromatography coupled with the mass spectrometry method as described by Bacova et al. [14] and Placha et al. [15]. Briefly, detection and quantification were carried out using a GC/MS (type HP 6890 GC) system coupled with a 5972-quadrupole mass-selective detector (Agilent Technologies GmbH, Wilmington, DE, USA). Detection of thymol was confirmed by comparing its specific mass spectrum and retention time with those of the reference compound. Additionally, the Kovats index was calculated. Calibration curves were generated by plotting the peak-area

ratios of thymol to o-cresol used as an internal standard (Sigma-Aldrich, St Louis, MO, USA) against the known thymol concentrations. The selective ion mode was used for quantitative analysis of thymol. The mass fragments m/z 135 and m/z 150, as well as m/z 107 and m/z 108, were monitored as characteristic for thymol and o-cresol, respectively. Calibration curves were prepared from blank samples spiked directly with 50 μL thymol (Applichem, Darmstadt, Germany) in standard solutions with known concentrations as follows: for plasma 48, 100, 200, 400, and 800 ng of thymol per mL, for intestinal wall 100, 200, 400, 800, 1000 ng; for liver, kidney, muscle, caecum and colon content 100, 200, 400, 800, 1000, 2000 ng; spleen, fat 24, 50, 100, 200, 400 ng; faeces 200, 400, 800, 1600, 2000, 4000 ng of thymol per g of tissue. Each point on the calibration curve was analysed as a duplicate. The peak of thymol was detected around 19 min and the o-cresol peak occurred around 10 min in all samples. Samples for thymol detection were prepared using the method described by Ocel'ová et al. [16]. Enzyme β–Glucuronidase Helix pomatia Type HP-2 (aqueous solution, ≥100,000 units/mL, Sigma-Aldrich, St Louis, MO, USA) was added to samples to cleave thymol from its glucuronide and sulphate, since only free thymol should be detected in the GC system.

#### *2.7. Statistical Analysis*

Data collected were analysed using the Kolmogorov–Smirnov test for normal and non-normal distribution. All data were not accepted as parametric. The Kruskal–Wallis test with post hoc Dunn´s Multiple Comparison test was used to determine the differences between plasma and tissues or caecal, colon content and faeces. Results are presented as mean value ± standard error of mean (SEM). Differences were considered significant at *p* < 0.05. Correlations of thymol concentrations between plasma and intestinal wall, plasma and liver, and liver and kidney were analysed using nonparametric Spearman's Rank Correlation and expressed as Spearman's correlation coefficient (rs). Statistical analyses were performed using Graph Pad Prism (GraphPad Software, San Diego, CA, USA). The experimental unit was the animal's cage.

#### **3. Results**

#### *3.1. Thymol Stability in Feed*

Concentration of thymol in feed during the period of the experiment with its addition was relatively stable at 274 μg/g DM–0 d; 255 μg/g DM–7 d; 236 μg/g DM–14 d.

#### *3.2. Thymol in Plasma and Tissues*

Level of thymol in the intestinal wall was significantly higher than in plasma (*p* = 0.0211) and liver (*p* = 0.0305), and in the kidneys it was significantly higher than in plasma (*p* = 0.0259) and liver (*p* = 0.0415) during the period of thymol addition (Table 2). Thymol in fat (19.9 ± 7.36 ng/g DM, n = 2) and muscle (26.6 ng/g DM, n=1) during this period was found only in a small number of samples, and only in trace amounts in others. For this reason, those samples were not included in the statistical evaluation. Significant correlation was established between thymol concentration in plasma and intestinal wall (rs = −1.0, *p* < 0.01), plasma and liver (rs = 0.786, *p* < 0.05) and liver and kidney (rs = 0.738, *p* < 0.05, Figures 1–3). Even though thymol content was determined only in trace amounts during the period without thymol addition, the differences were statistically evaluated. Thymol in intestinal wall was significantly higher than in plasma in this period (*p* = 0.0035, Table 2).

#### *3.3. Thymol in Caecum, Colon and Feces*

Thymol in faeces was significantly higher than in the colon during both experimental periods, even if only in trace amounts without further thymol addition (*p* < 0.01, Table 3). During this period only thymol in faeces was detected above trace amount.


**Table 2.** Thymol content in plasma (ng/mL) and tissue (ng/g DM).

a,b Values within a column with different superscript letters differ significantly (*p* < 0.05). Data are presented as mean ± standard error of mean (SEM).

**Figure 1.** Correlation between plasma (ng/mL) and intestinal wall (ng/g DM).

**Figure 2.** Correlation between liver (ng/g DM) and plasma (ng/mL).

**Figure 3.** Correlation between liver and kidney (ng/g DM).

**Table 3.** Thymol content in caecum, colon, and faeces (ng/g DM).


a,b Values within a column with different superscript letters differ significantly (*p* < 0.05). Data are presented as mean ± standard error of mean (SEM).

#### **4. Discussion**

The epithelial cells in the small intestine wall contain various metabolic enzymes and transporters. The intestinal microflora possesses a wide range of metabolic processes including hydrolysis of glucuronides, sulphate esters, and amides. Enzymes in the intestinal microflora can hydrolyse drug metabolites, especially glucuronide conjugates, and convert them back to the parent compound [10]. The parent drugs excreted and/or released by the action of gut microflora are reabsorbed by intestinal cells. Metabolites are continuously excreted into the large intestine, where they are again hydrolysed and reabsorbed [17].

After being absorbed from the GIT, thymol becomes metabolized during processes of biotransformation and becomes more hydrophilic. The metabolites, mainly sulphates and glucuronides, are transported across the intestinal epithelium by active processes involving transmembrane proteins. Many transporters such as peptides, vitamins, amino acids, and sugars play important roles in the translocation of drugs and were identified in large amounts in the GIT [18].

Rubió et al. [19] and Pisarˇcíková et al. [13] have confirmed that thymol, which is a small lipophilic molecule, is not detected in unmetabolized form in plasma, as they detected only its conjugates (thymol sulphate and glucuronide). They also detected thymol conjugates in the duodenal wall, which points to active biotransformation of thymol in the organism.

Significant correlation (rs = −1.00, *p* < 0.01) between thymol content in the intestinal wall and plasma in our experiment indicates intensive absorption of thymol from the intestine. Placha et al. [15] and Ocel'ová et al. [20] also confirmed the intensive absorption of thymol from all intestinal segments in broiler chickens after four weeks of diet supplementation with thyme essential oil. They found significant correlation between thymol content in plasma and individual intestinal segments. Although we found six times lower concentration of thymol in plasma during its addition and seven times lower after its withdrawal (even if only in trace amounts) in comparison with the intestinal wall, we

can confirm that some metabolic processes were still active after thymol withdrawal from feed (Tables 2 and 3).

The rabbit caecum is the largest part of the large intestine and contains approximately 40% of the intestinal content. The primary mechanism by which nutrients are released from intestinal content is microbial fermentation, and its products are absorbed through the intestinal wall or are reingested as caecotrophes. The retained particles from the proximal part of the colon also provide substrate for caecal microbiota. The mucus which coats the caecotrophes protects them and allows the fermentation processes to continue in them until they reach the intestine. The composition of the intestinal flora depends to a large extent on the caecotrophic microbial population [21].

Intestinal microflora and epithelial cells play a crucial role in metabolic processes because they produce a wide range of metabolic enzymes [22]. The counts of bacterial flora are highly variable in different parts of the gastrointestinal tract [23]. We assume that the metabolic enzymes responsible for thymol biotransformation in the caecum and consequently in caecotrophes are expressed in large enough amounts that they can affect metabolic activities and consequently exert influence on the amount of thymol and its metabolites in the caecum.

There are some conditions affecting the absorption of compounds in the GIT. In addition to their rate of dissolution in the intestinal fluids, they must also be able to cross membranes in each part of the GIT. In case they are not able to cross these membranes by the time they reach the colon, the extent of intestinal absorption is not sufficient, and the compounds are excreted in faeces [24]. All these circumstances may explain the high concentrations of thymol in the caecum and faeces, not only during its addition but also after its withdrawal from feed (Table 3).

The first study which confirmed the presence of thymol metabolites (thymol sulphate and glucuronide) in the duodenal wall of broiler chickens after sustained consumption of thyme essential oil also confirmed the key role of the intestine in the metabolism of thymol [13]. Ocel'ová [6] detected thymol in the liver at a level of 8.9% of its concentration in the intestinal wall and observed significant correlation between thymol concentrations in liver and plasma, and liver and intestinal wall, which might indicate sufficient absorption of thymol from the intestinal wall to the liver through the vena portae. We found 15% (with thymol) and 29% (without thymol) of thymol in liver compared with its content in the intestinal wall, and significant correlation between thymol concentrations in plasma and liver (rs = 0.7857, *p* < 0.05). These results are in agreement with the findings of the above-mentioned authors and point to intensive biotransformation and excretion processes in the liver.

Between the enzyme systems and efflux transporters there exist mutual processes which can affect the efficiency of drugs in the intestine. These couplings can prolong the exposure of drugs in vivo and are crucial for enteric and enterohepatic recycling. Some parent compounds are absorbed and metabolized in the intestinal cells, while some portions are effluxed back into the intestinal lumen or transported to the mesenteric vein and are taken up by hepatocytes [17]. Excreted metabolites from enterocytes are converted back to the parent compound and again reabsorbed. Compounds in the liver are metabolized and together with parent compounds are excreted with bile into the duodenum and are then reabsorbed there [14,17]. These repeating processes together with processes of caecotrophy could explain the thymol content in plasma, tissues, and intestinal content after thymol withdrawal, although only in trace amounts. We must point out that we detected the sum of metabolized and non-metabolized thymol, as the thymol metabolites were cleaved by added enzyme, β-glucuronidase. However, the extent of absorption of compounds (drugs) and the contribution to their bioavailability by metabolic enzymes and transporters or receptors present on the intestinal membrane are still not clear.

In the present study, a significantly higher level of thymol in the kidney in comparison with liver tissue and plasma confirmed its metabolism and/or accumulation in this organ (Table 2). Our results are in agreement with our previous study [6], in which we detected a significantly higher thymol concentration in the kidney of chickens with thyme essential oil diet supplementation. Takada [25] detected thymol glucuronide and thymol sulphate in the urine of rabbits, and to our knowledge this is the only study concerned with the metabolic path of thymol in these animals. Data available so far indicate that thymol sulphate could be reabsorbed in the proximal tubule after glomerulary filtration, and that cleavage of this metabolite is achieved by enzymes located at the luminal brush border, so that subsequently thymol could be reabsorbed [26]. These authors demonstrated that although the liver is the most important organ for biotransformation, kidney microsomes demonstrate more effective metabolic processes than liver or intestinal microsomes. The significantly higher content of thymol in the kidney and the correlation between liver and kidney (rs = 0.7381, *p* < 0.05) in our study also confirm the intensive metabolism in kidney tissue. However, further study of renal metabolism mechanisms is necessary to confirm these findings.

Before drug compounds can reach systemic circulation and therapeutic targets, several barriers must be overcome [18]. Although lipophilic molecules evidently pass the barriers with ease by transcellular routes, various efflux transporters are preferentially bound with lipophilic molecules, seriously limiting their absorption. The first-pass metabolism which occurs in the intestinal wall decreases the chance of molecules which get into epithelial cells to reach the systemic circulation. Once compounds are absorbed in the GIT, the next major barrier awaits in the form of the first-pass metabolism in the liver. Drugs passing through the portal vein encounter hepatocytes and are metabolized there [18]. Based on these processes we can explain the trace amounts of thymol detected in rabbit muscle and fat in our experiment. Trace amounts of thymol in the muscle tissue of broiler chickens were also detected by Haselmeyer et al. [27] after thymol addition (55 mg/kg DM feed), and by Ocel'ová [6] after thyme essential oil application. Lipophilic substances which are not metabolized during the process of biotransformation are less readily eliminated and accumulate in fat tissue [22]. As we found only traces of thymol in this tissue, we can hypothesize that the majority of thymol was metabolized and converted into hydrophilic substances.

Finally, little is known about the bioactivity of thymol and thymol metabolites, further studies are needed to evaluate the distribution of thymol in different tissues at different levels and establish its suitable concentration for a beneficial effect on animal health.

#### **5. Conclusions**

Our results showed that, thymol was efficiently absorbed from the intestinal lumen and intensive metabolic processes in liver and kidney were observed, while accumulation in fat and muscle tissue was low, probably due to its intensive biotransformation into hydrophilic substances which were then excreted. We confirm some metabolic processes involving thymol even after its withdrawal from feed, as a consequence of caecotrophy. Oral bioavailability of plant compounds is a challenge for scientists because their metabolic processes in the animal organism should be understood at molecular level.

**Author Contributions:** Conceptualization: I.P.; methodology: I.P., K.B.; validation: I.P.; formal analysis: I.P., K.Z.E., K.B., G.K.R., M.T.; M.P.S.; investigation: I.P., K.B.; resources: I.P., K.Z.E.; data curation: I.P., L.C., A.L.; writing—original draft preparation: I.P., K.B.; writing—review and editing: K.Z.E.; visualization: I.P., K.B.; project administration: I.P.; funding acquisition: I.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Scientific Grant Agency of the Ministry for Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences (VEGA 2/0009/20), as well as the Austrian Federal Ministry for Science, Research and Economics, OeAD, Ernst Mach Grant Action Austria-Slovakia.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the State Veterinary and Food Office of the Slovak Republic on 1 December 2016 (approval number SK CH 17016).

**Data Availability Statement:** Data availability upon reasonable request to the corresponding author.

**Acknowledgments:** The authors gratefully acknowledge the technical support provided by M. Madarova at the University of Veterinary Medicine and Pharmacy, Kosice, Slovakia and L. Ondruska, V. Parkanyi and R. Jurcik at the National Agricultural and Food Centre, Research Institute for Animal Production, Nitra, Slovakia. The authors also thank Andrew Billingham for improving the written English of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


### *Article* **Effect of Goji Berry (***Lycium barbarum***) Supplementation on Reproductive Performance of Rabbit Does**

**Egon Andoni 1,†, Giulio Curone 2,†, Stella Agradi 2, Olimpia Barbato 3, Laura Menchetti 4,\*, Daniele Vigo 2, Riccardo Zelli 3, Elisa Cotozzolo 5, Maria Rachele Ceccarini 6, Massimo Faustini 2, Alda Quattrone 3, Marta Castrica <sup>7</sup> and Gabriele Brecchia <sup>2</sup>**


**Simple Summary:** Infectious diseases represent serious problems for the reproductive performance of livestock animals because they negatively affect not only the welfare of the animals, but also the profitability of the farm. Moreover, the European Community continues to promote the reduction of the use of antibiotics and hormones in animal breeding. In this context, it is necessary to find new nutritional approaches to reduce the negative energy balance, and at the same time, to reinforce the immune system of the animals. In this research, the effect of goji berry supplementation on the reproductive activity and productive performance of rabbits is evaluated. *Lycium barbarum* fruit is considered a nutraceutical natural product containing various biologically active substances that show health benefits for both humans and animals. In particular, the berry can modulate hormones and metabolites involved in energy balance and reproduction, stimulate and balance the immune system activity, contributing to the defense of the organism against pathogens. Our results suggest that the integration with goji berry in the rabbit diet at 1% affects the reproductive activity, influencing the pattern secretion of luteinizing hormone (LH) and estrogens, as well as the sexual receptivity. Moreover, the fruit induced a higher milk production, improving the productive performance of young rabbits.

**Abstract:** Goji berry shows a wide range of beneficial properties in human health, but only a few studies evaluated its effects in livestock animals. The objective of this research was to assess the effects of goji berry supplementation on the hormonal profile, productive, and reproductive performance of does. Two months before artificial insemination, 105 nulliparous does were randomly divided into three groups (*n* = 35) based on the dietary treatment: commercial diet (C), or a diet supplemented with either 1% (G1), or 3% (G3) of goji berry, respectively. The results showed that receptivity was higher in G1 than in the C group (*p* < 0.05). Trends toward significance for differences between the G1 and G3 groups in marginal means of LH concentrations (*p* = 0.059), and between G1 and C in LH AUC values (*p* = 0.078), were evidenced. Estrogen concentrations showed a more fluctuating trend but a significant interaction effect (*p* < 0.001). The G1 group showed higher litter weight than C at birth (*p* = 0.008) and weaning (*p* < 0.001), as well as higher litter size at weaning (*p* = 0.020). The G1 group also exhibited the highest mean milk production (*p* < 0.01). In conclusion, goji berry influenced

**Citation:** Andoni, E.; Curone, G.; Agradi, S.; Barbato, O.; Menchetti, L.; Vigo, D.; Zelli, R.; Cotozzolo, E.; Ceccarini, M.R.; Faustini, M.; et al. Effect of Goji Berry (*Lycium barbarum*) Supplementation on Reproductive Performance of Rabbit Does. *Animals* **2021**, *11*, 1672. https://doi.org/ 10.3390/ani11061672

Academic Editors: Iveta Plachá, Monika Pogány Simonová and Andrea Lauková

Received: 12 May 2021 Accepted: 1 June 2021 Published: 3 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

reproductive and productive performance, probably via modulating hormonal patterns and milk production in rabbits. However, further studies are needed to validate these preliminary results.

**Keywords:** receptivity; fertility; estrogen; LH; milk production

#### **1. Introduction**

*Lycium barbarum*, also known as wolfberry or Goji berry, is a functional food and plant medicine that has been used in China and Asian countries for 2300 years to restore well-being, improve eyesight, and nourish the kidneys and liver [1]. Recent studies have shown that goji berries possess various benefits for human health such as anti-aging [2], antioxidant [3], antidiabetic [4], hypolipidaemic [5], anticancer [6], cytoprotective [7], neuroprotective [8,9], immunomodulatory [10], gastroprotective [11], radiation protecting [12], and visual protecting effects [13]. As a consequence of all these beneficial properties and of the increasing public awareness of health and the quality of life, the plant and the fruit of *Lycium barbarum* have become extraordinarily popular in Western countries, where its cultivation and consumption have increased [1]. Moreover, the fruit seems to have a high safety profile at different dosages [14], although a few studies reported mild toxicity [15] and adverse effects such as urticarial-like reactions related to its use [16]. Goji berry contains a high quantity of carbohydrates, dietary fiber, protein, macro and micronutrients, and low levels of fat [17]. Besides the high nutritional value, it contains many biologically active compounds such as polysaccharides, carotenoids, phenylpropanoids, phenolics, and flavonoids [18]. Several studies showed that the wide range of effects of goji berries are principally due to the biological properties of polysaccharides [1]. They represent the most abundant constituents of the fresh and dried berry (46–51% and 5–8%, respectively), and are found principally branched and in the water-soluble form [14].

The research activity concerning goji berries was mainly carried out on humans, laboratory animals, and on specific cell lines in vitro [4,9,10,19], while only a few investigations have been carried out on livestock animals [20], including rabbits [21–23]. The rabbit is considered a livestock animal, and the productive efficiency of rabbit farms is strongly influenced by reproductive performance, especially those of the rabbit does. Generally, nulliparous does show higher fertility than primiparous and multiparous does [24,25]. The major causes of the reduced fertility rate of primiparous does are both the intensive reproductive rhythms to which they are subjected, and the negative energy balance due to the overlap between pregnancy and lactation [26,27]. Moreover, the profitability of the breeders could be reduced by the high culling and mortality rate of the animals, and the costs related to the purchase of medicines and veterinary services as a consequence of the sanitary status of the farm. Poor hygiene and incorrect artificial insemination practices are often linked to the onset of clinical or subclinical endometritis and metritis, which reduce the reproductive performance of the does [28,29]. Local and systemic diseases and/or inflammatory status induce the release of chemokines by the activation of TLR4 receptors that mobilize and activate immune cells [30]. At the systemic level, an alteration of the hormonal secretion of the gonadal axis could be induced; whereas at a local level, the inflammatory mediators and other signaling molecules can influence cellular, vascular, and endocrine functions impairing the reproductive activity of the animals [31,32].

Infectious diseases are generally fought with antibiotics, yet in the last decade, the abuse of these drugs led to the onset of antibiotic resistance with a dangerous and direct impact on human and animal health [33,34]. For this reason, the European Community guidelines tend to reduce the use of antibiotics and hormones in animal husbandry, including rabbit farming [35]. In this context, there has been a growing interest in the study and research of nutraceutical products that show health-promoting effects and with a potential in the prevention and treatment of several human and animal diseases, including those of the reproductive system [36,37]. Although there are numerous studies on the biological

activity of goji berries, their effects on the reproductive functions, gonadic axis activity, and fertility, are poorly known [38,39]. In particular, the research is strongly limited for the female of both humans and animals, including rabbit does [40,41]. The rabbit is, however, an excellent animal model for research, in particular with regards to the physiology of reproduction and several reproductive parameters [42–44].

Goji berry could be a natural strategy to improve the reproductive performance of rabbit farms. It is speculated that the fruit could reduce the incidence of reproductive infections/inflammation of the genital tract, acting principally on the immune system and the oxidative status of the organs as well as influencing the hormonal secretion of the ovarian axis. Therefore, the main objective of the present study is to evaluate the effect of goji berry dietary supplementation on the reproductive performance, fertility, LH, and estrogen secretion, as well as the milk production, of rabbit does.

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Design*

The experiment was conducted at the farm of the Agricultural University of Tirana, Faculty of Veterinary Medicine, Albania. The animals were maintained in accordance with Legislative Decree No. 146, implementing Directive 98/58/EC regarding the protection of animals that were kept for farming purposes. The experimental protocol was run with the permission of the Ministry of Agriculture and Rural Development, National Authority of Veterinary and Plants protection (prot. 824/2020), of Albania. All efforts were made to minimize animal distress and to use only the number of animals necessary to produce reliable results. Moreover, the responsible veterinarian for the farm checked the rabbits for health and welfare states daily.

Nulliparous New Zealand White rabbits (*n* = 105) of 4 months of age, weighing 3.5–3.8 kg, were individually housed in controlled environmental conditions where the temperature ranged from +18 to +21 ◦C, the relative humidity from 55% to 65%, the artificial ventilation was 0.3 m3/s, and the lighting was scheduled 16 L:8 D at 40 lux. Rabbits were provided 150 g/d of commercial food and water ad libitum. The composition of the diet supplied to the does is described in Table 1 and is in agreement with previous studies [21–23]. Goji berries in dried form were provided by a farm of central Italy (Impresa Agricola Gianluca Bazzica, Foligno, Italy). They were ground into smaller pieces, mixed with the rest of the diet ingredients, and, finally, pelleted [23]. All rabbits completely consumed their daily rations.


**Table 1.** Formulation and chemical composition (as fed) of control (C) and experimental diets supplemented with 1% (G1) and 3% (G3) Goji berries.


**Table 1.** *Cont*.

<sup>1</sup> as percentage (%). <sup>2</sup> Per kg diet: vitamin A 11,000 IU; vitamin D3 2000 IU; vitamin B1 2.5 mg; vitamin B2 4 mg; vitamin B6 1.25 mg; vitamin B12 0.01 mg; alpha-tocopherol acetate 50 mg; biotine 0.06 mg; vitamin K 2.5 mg; niacin 15 mg; folic acid 0.30 mg; D-pantothenic acid 10 mg; choline 600 mg; Mn 60 mg; Fe 50 mg; Zn 15 mg; I 0.5 mg; Co 0.5 mg. <sup>3</sup> as Kcal/kg. Estimated by Maertens et al. [45].

The does were randomly divided into three different groups (*n* = 35/group), according to the dietary treatment: commercial diet (Control, C), and diet supplemented with 1% or 3% of goji berry, G1 and G3 groups, respectively. After a period of adaptation to the new feed for two months, the does were submitted to artificial insemination (AI) at 6 months of age, performed with a heterospermic pool of fresh semen (0.5 mL) diluted 1:5 in a commercial extender. At the moment of the insemination, receptivity was established by controlling for the color of the vulva, and ovulation was induced by an intramuscular injection of 10 μg of synthetic gonadotropin-releasing hormone (GnRH; Receptal, Hoechst-Roussel Vet, Milan, Italy) [31]. Day 0 was designed as the day of the insemination. Pregnancy was diagnosed by abdominal palpation 12 days after AI, and then, 25 pregnant rabbit does per group were followed until the weaning of the young rabbits (day 35). Lactation was controlled by opening the door of the nest one time a day until 18 days after parturition.

On the day of the AI, blood samples were collected every 60 minutes, starting 120 minutes before and up to 240 minutes after the AI and GnRH injection to evaluate LH and 17-β estradiol concentrations. The samples were withdrawn from the marginal ear vein by a butterfly needle of 24G connected to a syringe of 2.5 mL. Blood samples were inserted into tubes containing EDTA, and immediately centrifuged at 3000× *g* for 15 min; furthermore, plasma was stored frozen until it was assayed for hormone levels.

The following reproductive and productive indices were calculated: receptivity (color of the vulva [46] categorized as white, pink, or red), fertility (number of parturitions/number of inseminations × 100), milk production, litter weight (from delivery until day 18 of the whole litter, and from day 18 to weaning of the single animal), litter size, and pre-weaning mortality (calculated as the percentage of weaned kits/litter) [47]. From parturition to day 35, the mortality, the litter weight, and the litter size were recorded daily. Milk production was evaluated daily by weighing the does before and after suckling, from parturition until day 18 of lactation [48].

#### *2.2. Hormone and Metabolite Assays*

Plasma LH concentrations were evaluated using a commercial rabbit LH ELISA kit (Wuhan Fine Biotech Co., Ltd., Wuhan, Hubei, China). The determination procedure is based on a sandwich enzyme-linked immune-sorbent assay technology. The kit shows high sensitivity, with a limit of detection of 0.281 ng/mL and excellent specificity for the detection of LH. The intra- and inter-assay coefficients of variation were <8 and <10%, respectively. Values below and above the limits of detection of the test were considered to be 0 and 30 ng / mL, respectively.

Plasma 17β-oestradiol concentrations in plasma samples were assayed using a commercial RIA kit (Immunotech sro, Prague, Czech Republic) following the procedure indicated by the producer [49]. The limit of detection was 13,11 pg/ml and the intra- and

inter-assay coefficients of variation were <14.4 and <14.5%, respectively. Values below the limit of detection of the test were considered to be 0 pg/mL.

#### *2.3. Statistical Analysis*

Kolmogorov-Smirnov and Levene tests were used to verify assumptions. Hormone concentrations and milk yield were analyzed by mixed-design ANOVA followed by multiple comparison tests corrected using the Bonferroni–Sidak method. Mixed design ANOVA evaluated the effect of dietary treatment (i.e., group effect; 3 levels: C, G1, and G3 groups), change over time (i.e., repeated-measures effect; 7 levels for hormone concentrations and 18 levels for milk yield), and their interaction. The LH AUC (area under the curve) was calculated for each animal by the trapezoid method using LH values at each sampling time point from 0 to 240 from the GnRH injection [48,50]. LH AUC, litter size, and weights were compared between groups by one-way ANOVA. Finally, receptivity and fertility were analyzed by Chi-square tests to evaluate if there was an association between these parameters and dietary treatment. The proportions of each group were then compared by pairwise z-tests. Statistical analyses were performed with SPSS Statistics version 25 (IBM, SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.01 software (Inc., San Diego, CA, USA). We defined *p* ≤ 0.05 as significant and *p* < 0.1 as a trend.

#### **3. Results**

#### *3.1. Hormone Concentrations*

Regardless of the group, plasma LH levels reached a peak at 60–120 minutes after GnRH injection (20.3 ± 1.1 ng/ml and 15.8 ± 2.2 ng/ml at 60 and 120 min, respectively; *p* for time effect <0.0001) and returned to baseline levels after 240 minutes (Figure 1). Regarding the group effect, trends toward significance were found for both LH concentrations (*p* = 0.056) and LH AUC (*p* = 0.067). In particular, multiple comparisons showed that marginal means of LH concentrations tended to be higher in G1 than in G3 (7.9 ± 3.2 and 6.5 ± 3.1 ng/ml for G1 and G3, respectively; *p* = 0.059), while LH AUC tended to be higher in G1 than in C (2510 ± 175 ng/ml x h and 3031 ± 149 ng/ml x h for C and G1, respectively; *p* = 0.078).

**Figure 1.** Plasma luteinizing hormone (LH) concentrations from minutes −120 to 240 after GnRH injection in the control group (C) and does supplemented with 1% (G1) or 3% (G3) of Goji. Values are means and standard errors.

Estrogen concentrations showed a more fluctuating trend, even if the highest mean values, regardless of group, were found at 180 minutes after GnRH injection (*p* for time effect <0.0001; Figure 2). Estrogen concentrations also showed a significant interaction effect (*p* < 0.001) and a trend toward significance for a group effect (*p* = 0.065). In particular, marginal means of the G1 group tended to be higher than in C (6.2 ± 1.9 pg/mL and 8.5 ± 3.0 pg/mL for C and G1, respectively; *p* = 0.088), and multiple comparisons showed significant differences between groups at time 0 (*p* = 0.034). Moreover, rabbits of the G3 group showed a delayed estrogen peak compared to the other two groups.

**Figure 2.** Plasma estrogen (E2) concentrations from minutes −120 to 240 after GnRH injection in the control group (C) and does supplemented with 1% (G1) or 3% (G3) of Goji. Values are means and standard errors.

#### *3.2. Reproductive and Productive Performance*

As an indicator of a rabbit doe's receptivity, the color of the vulva was affected by nutritional treatment (*p* = 0.044). In particular, the percentage of does showing a white color of the vulva was lower in G1 than in the C group, while the percentage of does showing a red color was higher in G1 than in the G3 group (*p* < 0.05; Figure 3). Fertility, however, did not differ between groups (77%, 82%, and 74% for C, G1, and G3, respectively; *p* = 0.678).

Milk yield increased from the 1st (32 ± 4 g/d) to the 18th day post-partum (164 ± 4 g/d; *p* < 0.001) in all groups (Figure 4), although, G1 showed the highest marginal means (112 ± 9 g/d, 122 ± 9 g/d, and 111 ± 8 g/d for the C, G1, and G3 groups, respectively; *p* < 0.05).

**Figure 3.** Relative frequency distribution of the color of the vulva used as an indicator of sexual receptivity in does of the control group (C) and does supplemented with 1% (G1) or 3% (G3) of Goji.

**Figure 4.** Milk production from day 1 to 18 post-partum of the control group (C) and does supplemented with 1% (G1) or 3% (G3) of Goji. Values are means and standard errors.

Pre-weaning mortality (*p* = 0.176) and litter size at birth (*p* = 0.249) did not differ between groups; however, rabbits of the G1 group showed higher litter size at weaning (*p* = 0.020), as well as higher litter weight at birth (*p* = 0.008) and at weaning (*p* < 0.001) compared to the C group. The G3 group showed intermediate values in litter weight at birth and litter size at weaning (*p* < 0.05), while their litter weight at weaning was higher than the C group (*p* < 0.01; Table 2).


**Table 2.** Productive performance of the control group (C) and does supplemented with 1% (G1) or 3% (G3) of Goji (*n* = 25 does/group).

RMSE: root-mean-square error. a: b: Means sharing the same superscript are not significantly different from each other (*p* < 0.05).

#### **4. Discussion**

To our knowledge, this is the first study that evaluates the effect of goji berry on the reproductive activity of rabbit does. The present work suggests that the diet integration with goji berry could affect the hormonal pattern of LH and 17β-oestradiol, increase receptivity, milk production, litter weight at birth, litter weight, and size at weaning. The encouraging findings of our research indicate that the berry could be used in rabbit nutrition, although further studies are needed to confirm the current outcomes, in order to understand the mechanism of action as well as to evaluate the economic convenience of its use.

It has long been assumed that rabbit does do not have a well-defined estrus cycle, nor do they show well-specified estrus manifestation, so they are quite often erroneously considered to be in permanent estrus. Actually, the does present a period during which they accept mating (oestrus) as well as a period during which they reject the male (dioestrus). Sexual receptivity could be measured by a behavioral test in the presence of a male [51], and by evaluating the color and turgidity of the vulva [46]. In particular, the mating acceptance behavior in the presence of a male (lordosis position) and the red color of the vulva could be considered as estral manifestations of the doe. The relationships between sexual behavior, vulva color, and circulating concentrations of reproductive hormones have been studied at different reproductive stages, but a firm conclusion has not yet been reached [52,53]. Our results showed that, based on the evaluation of the color of the vulva, the sexual receptivity is higher in supplemented does, namely in the G1 group compared to the C group. Moreover, animals of the same group also showed higher 17β-oestradiol plasma concentrations compared to the other groups at the moment of AI, suggesting a link with the receptivity. In agreement with our findings, the results of several studies highlight that the turgidity and the red color of the vulva, and more in general, the receptive behavior of the doe, can be related to high plasma levels of estradiol [54] as well as to the presence of ovulatory follicles in the ovaries [52]. In fact, the presence of tertiary follicles in the ovaries are responsible for the elevated 17β-oestradiol plasma concentrations, which in turn induce physiological changes in the reproductive organs, such as hyperemia at the vulvar level and act on the hypothalamus which induces the behavioral estrus signals that lead to the acceptance of the male [55]. Moreover, it is quite well-defined that the color of the vulva at the AI greatly influences fertility [56] and ovulation frequency [40]. In our study, although the receptivity and 17β-oestradiol plasma levels showed differences between the groups, they are not correlated with an increase in fertility. The lack of significance for the fertility of the rabbit does, however, could also be affected by the high sensitivity of the chi-square test comparative to the sample size. It was reported that *Lyceum barbarum* polysaccharides were able to restore the production of sex steroids in the ovaries of female senile rats [41]. Moreover, Liu et al. [40] found that *Lycium barbarum* polysaccharide improved the ratio between the different types of follicles, increased the litter size, weaning survival, and hormonal secretion, and reduced the damage of the ovary during repeated superovulation in mice. It was also reported that the administration of an extract of *Lycium chinense Miller* induced ovulation in adult female rabbits [57]. Based on our findings, goji berry seems to be able to enhance sexual receptivity and affects the estrogen secretion of rabbit does. It

would also be interesting to evaluate the effect of the supplementation of the fruit on the primiparous and lactating does, in which the receptivity and fertility are usually reduced for their higher negative energy balance and the inhibitory effect of prolactin secretion on the hypothalamus. This preliminary result can encourage the research to explore a possible role of goji berry in the preparation of female rabbits to AI, with the aim to reduce the use of exogenous hormones in rabbit farms. Nonetheless, further investigation is required to confirm these results and to establish the mechanism of action of the goji fruit, given the scarce literature on this topic.

To our knowledge, this is the first report that describes the effect of goji berry on the pattern of LH secretion in rabbits. In the present study, goji berry seems to affect the gonadal axis activity by modifying the LH secretion and the estrogenic activity of the ovary. The rabbit is an induced ovulatory species, in which ovulation is induced by the neuroendocrine reflex. The penile intromission, mounting, and pheromones induce the release of GnRH in a higher pulse-frequency from the medial basal hypothalamus that acts on the GnRH receptors on the gonadotropic cells in the anterior pituitary and determines the LH ovulatory peak within 15–75 minutes [58,59]. In our study, the exogenous administration of GnRH bypassed the neuro-endocrine ovulatory reflex, acting directly on the gonadotropic cells triggering LH release. In the present study, the LH ovulatory peak occurred within 60 minutes by the injection of synthetic gonadotropin in all the groups. These findings are similar to those reported by Brecchia et al. [49], that also found similar values of LH plasma concentrations after the induction of the ovulation. With respect to our results, other authors showed differences in the LH plasma levels detected and/or on the time of the peak of the gonadotropin after the injection of the GnRH analogs, probably linked to a different method of assay or to an individual variation among the animals [60,61].

In our study, the G1 group tended to differentiate from G3 and C in LH mean concentrations and LH AUC, respectively. The effect of goji berry on the LH concentration has been poorly investigated in humans and animals, in particular in the female. Several studies evaluated the effect of goji berry on the male reproductive tract and hormone concentrations in other species. The administration of goji berry polysaccharides reduced the testis spermatic injury induced by Bisphenol A and significantly increased the LH plasma levels of male mice [62]. Another study showed that *Lycium barbarum* polysaccharides exhibited a protective effect on fertility and reproductive hormone secretion impairments by activating the gonadal axis in streptozotocin-induced type-1 diabetic male mice [63]. Goji berry polysaccharides had protective effects against damage to the testicular tissue, annexed glands, and LH, FSH, and testosterone secretion of normal and hemicastrated male rats induced by heat exposure (43 ◦C), and on the DNA damage to mouse testicular cells induced by hydrogen peroxide [64]. Recent research showed that dietary integration with goji berry had positive effects on boar semen quality; in particular, it improved progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate compared to the control group [65]. Moreover, the wolfberry group showed a significant reduction of the head, tail, and total abnormality rates in both fresh semen and semen stored for 72 hr in comparison to the control group [65]. Finally, it was reported that goji polysaccharides improved sperm quality and fertility rate after cryopreservation in male Cashmere goats [39]. These findings support the idea that goji berry may affect the reproductive hormonal secretion, and as a consequence, the reproductive performance of the animals, including rabbits. It remains to be established which component could be responsible for the action on the gonadal axis of rabbit does as well as the site and the mechanism of action. An up-regulation of the pituitary receptors and/or increased secretion of GnRH secretory neurons, or the synthesis of LH by the gonadotropic cells of the pituitary, might be speculated.

In the present research, we found that a low dose of goji berry integration increases milk production and the weight of the litter at birth and weaning. These results are in agreement and confirm the data reported by Menchetti et al. [47], though they used a smaller sample size. Given the missing data on this topic, we can speculate that some

components of the *Lycium barbarum* berry may favor the production of milk in different ways, as reported for other nutraceutical substances in other animal species: increasing the concentration of the components and precursors of milk at the level of the mammary gland [66], stimulating the proliferation of the mammary epithelial cells enhancing the uptake of some precursors of milk such as propionate and butyrate [67], enhancing the secretion of some hormones such as prolactin [68], and acting as key regulators of the signal transduction pathways during the synthesis of the components of milk [69]. Goji berry is rich in free amino acids, particularly in L-arginine [70] which is able to increase milk production in cows [67] and sows [68].

The energy balance of the does in commercial rabbit farms is generally critical. In fact, females are quite often inseminated during lactation when a high energy output is present because of the milk secretion. The loss of energy is not completely compensated for by feed intake, and consequently, the does increase the mobilization of fat reserves and lose energy which negatively affects the reproductive activity [71]. Several metabolic hormones and metabolites are involved in maintaining energy homeostasis during both pregnancy and lactation in rabbit does [26,27]. These hormones and metabolites acting by complex interactions can influence the ovarian axis hormonal secretion, and as a consequence, they play a role in the relationship between energy balance and reproductive efficiency [27]. Moreover, the development of the mammary gland and milk secretion is under hormonal control in adult rabbits [72]. Milk production is influenced mostly by the reproductive (estrogens, progesterone, and prolactin) and metabolic (GH, corticosteroids, thyroid hormones, and insulin) hormones [73]. It was shown that goji berry affects the secretion pattern of metabolic signals such as leptin, insulin, NEFA, and glucose, improving energy homeostasis [22]. Moreover, *Lycium barbarum* has therapeutic effects in glucosides of Tripterygium wilfordii Hook f (GTW)-induced dyszoospermia rats, improving the semen quality and positively affecting the secretion of reproductive hormones, including prolactin [74]. Consequently, it might be suggested that the higher milk yield in the group supplemented with 1% of goji compared to control does in our study can be due to the hormonal framework of both reproductive and metabolic hormone levels.

Our findings showed that the does fed with goji berry had a higher litter size at weaning. Other authors found that a low percentage of goji berry induced lower preweaning mortality and, at the same time, higher litter size and weight at the opening of the nest (day 18) and at weaning (day 35) compared to animals fed without goji berry supplement [47]. In the same study, the rabbits that received a diet integrated with goji berry exhibited not only a higher mean body weight during the fattening period and at slaughter, but also a better feed conversion rate compared to control animals. The higher quantity of milk ingested by the litters belonging to the goji groups is probably responsible for the greater productive performance. In fact, a link between the quantity and quality of milk produced by the female and the growth performance of the litter, at least until the weaning was reported [68]. It should be noted that rabbits supplemented with the highest dose of goji berry (i.e., 3%) showed no relevant effects compared to the control group; however, they showed minor changes compared to those obtained with the lowest dose. Then, a non-linear dose-dependent effect was found confirming that the use of high percentages of goji requires further investigation. It has been shown, for example, that substances contained in the *Lycium barbarum,* such as polyphenols, may have negative effects when they reach high levels or are administered for long periods, altering the hormonal and/or energy balance in both male and female farm animals [47,75]. In particular, polyphenols could have estrogen-agonistic or antagonistic effects and could interfere with reproductive functions at different levels of the gonadal axis [75]. However, the effects of polyphenols could be species-specific and depend on the polyphenol profile of each plant [75].

The effects of goji berry dietary supplementation in livestock animals are limited, but there is evidence that the fruit can enhance growth performance in piglets [76], broiler chickens [20], and hybrid groupers [77]. It was reported that some components of the fruit can enhance the intestinal absorption of nutrients [78] and show antioxidant and immunomodulatory actions [79]. There is evidence that *Lycium barbarum* induces changes in the intestinal microbiota, which in turn, can favor the digestion and the absorption of the nutrients [20,80]. Moreover, goji berry polysaccharides can stimulate the gut immune system, increasing the defense of the animals against pathogen infection through direct and indirect actions such as competition for common nutrients and niches and the increase of host defense, respectively [20,75]. Polysaccharides, acting as a prebiotic, favor the growth of beneficial bacteria, which in turn stimulate the immune system and in particular, the innate immune response [81]. It is possible to suggest that the different compounds of goji berry included in the milk and in the feed may stimulate the growth of the animals, influencing the digestion and absorption of the feed by action on the intestinal microbial population. Moreover, the microbiota affecting the development, maturation, and response of the immune system, may contribute to reducing the infectious disease and the mortality of the rabbit. Taken together, these mechanisms can explain the positive effects of goji berry on the growth performance, and more in general, may contribute to the maintenance of good health status and animal welfare.

#### **5. Conclusions**

In conclusion, the present study provided experimental support for the effects of goji berry on the hormonal profile as well as the reproductive and productive performance of rabbit does. In particular, 1% goji supplementation influences the hormonal pattern and increases both receptivity and milk production of the does, as well as the growth of young rabbits, although the effects on fertility are limited.

Therefore, the use of this natural and nutraceutical product could represent a new strategy to reduce the use of drugs in animal farming. At the same time, it could represent an added value, not only for animal welfare as well as their productive and reproductive performance but also for the rabbit industry and breeding. Although these preliminary findings are encouraging and suggest a potential use of the berries on rabbit nutrition, further studies with a larger number of animals are required to definitely establish both the efficacy and the mechanism of action of the goji berry to improve the reproductive performance of rabbit does, as well as to make a cost-analysis to assess the sustainability of its use in rabbit breeding. In addition, the dose-effect of goji berries could be very interesting for further research.

**Author Contributions:** Conceptualization, E.A., G.C., and G.B.; Data curation, O.B. and E.C.; Formal analysis, L.M., R.Z., and E.C.; Funding acquisition, D.V., M.F., and G.B.; Investigation, S.A., O.B., R.Z., M.R.C., and M.C.; Methodology, E.A., G.C., L.M., and G.B.; Project administration, G.B.; Resources, E.A., D.V., M.F., and G.B.; Software, L.M. and M.F.; Supervision, G.C. and G.B.; Validation, G.B.; Visualization, L.M.; Writing—original draft, G.C., L.M., A.Q., and G.B.; Writing—review and editing, E.A., G.C., L.M., D.V., A.Q., and G.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The experimental protocol was run with the permission of the Ministry of Agriculture and Rural Development, National Authority of Veterinary and Plants protection (prot. 824/2020), of Albania. Every effort has been made to reduce animal discomfort and to use only the number of animals sufficient to produce valid results.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Goji Berries were gently provided by Impresa Agricola of Gianluca Bazzica, Foligno (Italy). The authors acknowledge support from the University of Milan through the APC initiative. The authors wish also to thank Giovanni Migni for his excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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