*Article* **Fructooligosaccharide Supplementation Boosts Growth Performance, Antioxidant Status, and Cecal Microbiota Differently in Two Rabbit Breeds**

**Ayman H. Abd El-Aziz <sup>1</sup> , Mahmoud M. Abo Ghanima <sup>1</sup> , Walaa F. Alsanie 2,3, Ahmed Gaber 3,4 , Abd El-Wahab Alsenosy <sup>5</sup> , Ahmed A. Easa <sup>6</sup> , Sherif A. Moawed <sup>7</sup> , Sayed Haidar Abbas Raza <sup>8</sup> , Ahmed Elfadadny <sup>9</sup> , Hany Abo Yossef <sup>10</sup>, Wafaa M. Ghoneem <sup>11</sup> , Mustafa Shukry 12,\* , Amin Omar Hendawy <sup>6</sup> and Khalid Mahrose <sup>13</sup>**


**Simple Summary:** Rapidly rising incomes are dependent on animal protein production and the worldwide demand for livestock. It is expected that moving towards more intensive production systems to sustain this increased demand will depend on growth promoters. Some growth promoters, such as prebiotics, might be considered alternative non-antibiotic feed supplementation as they enhance performance without any side effects on the consumer's health. The present study inspected the influence of supplementation of β-fructan® (a commercial fructooligosaccharide; FOS) in the drinking water of growing rabbits on growth performance, carcass traits, hematological and biochemical indices, antioxidant status, and cecal microbiota of the NZW- and APRI-line rabbits (Animal Production Research Institute Line). FOS supplementation in rabbits enhanced growth carcass characteristics, significantly improving hematological parameters and antioxidant status, and minimized pathogenic Escherichia coli bacteria (from 3.45 in control groups to 2.89 and 2.24 (Log10 CFU g−<sup>1</sup> ) in 0.5 mL and 1 mL FOS-treated rabbits, respectively.

**Abstract:** The present study examined the effects of fructooligosaccharide (FOS) supplementation in drinking water on the growth performance, carcass characteristics, hematological and biochemical parameters, antioxidant status, and cecal microbiota of New Zealand White (NZW) and APRI rabbits. A total of 180 male NZW and APRI rabbits (aged five weeks; average live body weight

**Citation:** Abd El-Aziz, A.H.; Abo Ghanima, M.M.; Alsanie, W.F.; Gaber, A.; Alsenosy, A.E.-W.; Easa, A.A.; Moawed, S.A.; Raza, S.H.A.; Elfadadny, A.; Yossef, H.A.; et al. Fructooligosaccharide Supplementation Boosts Growth Performance, Antioxidant Status, and Cecal Microbiota Differently in Two Rabbit Breeds. *Animals* **2022**, *12*, 1528. https://doi.org/10.3390/ani12121528

Academic Editors: Rosalia Crupi and Raffaella Rossi

Received: 8 April 2022 Accepted: 9 June 2022 Published: 13 June 2022

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

**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/).

700 ± 39 g) were divided into six groups (30 rabbits/group; 5 replicates/group) in a two × three factorial arrangement. Rabbits of each breed were randomly assigned to one of three treatments of FOS (control; 0.00, FOS-0.5, and FOS-1.0). Results showed that rabbits' final body weight, FBWG, and carcass traits were considerably enhanced compared to those in the control group. The interaction effect of the supplement with the rabbit breed increased the growth, carcass traits, and hematobiochemical and antioxidant parameters with increasing FOS levels. In the cecum of both rabbit breeds, the total bacterial count and *Escherichia coli* population were considerably low, with a substantial increase in the number of *Lactobacilli* supplemented by FOS. In conclusion, FOS supplementation enhanced growth and carcass traits by improving the hematobiochemical parameters and antioxidant status and reducing cecal pathogenic bacteria in both breeds.

**Keywords:** antioxidant status; carcass; fructooligosaccharide; growth; haemato-biochemical parameters; cecal microbiota

#### **1. Introduction**

Improving animal productivity and boosting immunity using natural substances is a primary goal in animal breeding [1–7]. Recent studies have shown that immunostimulants, such as probiotics and prebiotics, have the potential to be used as protective and environment-friendly substitutions to antibiotics in mammals and poultry species [8–11]. These compounds are a possible method to enhance animal health and performance without antibiotics [12]. Prebiotics, such as inulin-type fructans and galacto-oligosaccharides, reveal immune-stimulating properties to the host through selective promoting of growth and/or encouraging the growth of some beneficial bacteria (i.e., probiotics) [13,14]. Fructooligosaccharides (FOSs) are considered the popular forms of prebiotics that consist of short-chain and undigested carbohydrates [9,15] because the β-linkages between fructose monomers cannot be hydrolyzed by the endogenous enzymes [16]. FOS is derived from the cell wall of the yeast, *Saccharomyces cerevisiae*, and has been reported to possess the ability to improve growth performance, decrease pathogenic bacterial count, and enhance immunity in two different rabbit breeds (New Zealand White and V-line rabbits) [1].

FOS may accelerate the gut fermentation of beneficial microorganisms, such as *Lactobacillus* and *Bifidobacterium*, and reduce the accumulation of pathogenic bacteria, such as *Clostridium perfringens* and *Escherichia coli* [15,17–19], thus enhancing the general health of animals [1,6,17]. Therefore, FOS is considered to be a prebiotic [20]. Dietary FOSs was reported to improve the growth traits (body weight, weight gain, and feed conversion ratio) and immune responses of broilers [21–23].

To our knowledge, there are no reports on adding FOSs to the drinking water of growing rabbits. Hence, this study was conducted to detect the possible effects of adding FOS (β-fructan®, a commercial FOS) in drinking water on the productive performance, carcass characteristics, hematobiochemical parameters, oxidative stress, and cecal microbiota of New Zealand White (NZW) and APRI rabbits. We hypothesized that oral FOS supplementation in combination with rabbit breed would enhance growth, improve blood biochemistry and antioxidant status, and improve microflora population diversity to alleviate the weaning stress of the rabbits.

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

#### *2.1. Ethical Declaration*

This research was performed after the approval of the Ethics of the Institutional Committee of Animal Husbandry and Animal Wealth Development Department, Faculty of Veterinary Medicine, Damanhour University, Egypt (DMU/VetMed-2019-/0145).

#### *2.2. Animal Rearing and Study Design*

APRI rabbit was produced by crossing Baladi Red bucks with a V line to create F1 ( <sup>1</sup> 2 B 1 <sup>2</sup>V) stock, and it was continued for two generations of intersex mating to attain performing constancy. A total of 180 weaned APRI and NZW rabbits (male, aged five weeks, weighing 700 ± 39 g) were collected and allocated to six groups (30 rabbits per group), and each group was divided into five replicates, each with six rabbits. The rabbits were assigned at random using a two × three completely factorial design (NZW and APRI-line with three treatments of a commercial FOS known as β-fructan®). The control group was not treated with FOS, and the first and second groups were supplemented with FOS-0.5 mL and FOS-0.1 mL, respectively. The experimental groups received 0.5 and 1.0 mL β-fructan (1,3 pharmaceutical grade 10%) per liter of drinking water for three sequential days per week (Glencore Company, Ann Arbor, MI, USA). Each rabbit in the 0.5 mL β-fructan-treated group was supplemented with 349.8 mg of β-fructan during the eight-weeks experimental period, while in the 1 mL β-fructan-treated group, each rabbit was supplemented with 699.75 mg of β-fructan. Rabbits were housed in galvanized wire batteries with standard dimensions (60 × 35 × 35 cm). All cages were supplied with galvanized steel feeding hoppers and automatic drinkers (nipples). Plastic ear tags identified rabbits. Freshwater was provided *ad libitum*, and a standard pelleted ration was provided *ad libitum* twice daily at 8 am and 2 pm. The pellets measured 1 cm in length and 0.4 cm in diameter. Rabbit cages were regularly cleaned and disinfected. Urine and feces dropped beneath the batteries were removed every morning.

#### *2.3. Experimental Feed Diet Preparation*

Diet was prepared following the NRC [24] and Lebas [25] recommendations (Table 1). The analysis of the ingredients was performed according to AOAC [26].


**Table 1.** Ingredients and chemical composition (%) of the basal diet.

<sup>1</sup> Dicalcium phosphate: 20% phosphorus and 25% calcium; <sup>2</sup> limestone: 34% calcium. <sup>3</sup> Amounts per kg: Vitamin A—12,000 and 900 IU of vitamin A and D3, respectively. While 2 mg of each vitamin K3, B1, and B6. 50 mg of vitamin E, 6 mg vitamin B2, 0.01 mg vitamin B12, 0.2 mg biotin, 20 mg pantothenic, 50 mg niacin, 5 mg folic acid, 8.5 mg manganese, 70 mg zinc, 75 mg iron, 5 mg copper, 0.75 mg iodine, 0.1 mg selenium. <sup>4</sup> Nitrogen free extract (NFE) was calculated by difference = 100 <sup>−</sup> (moisture % + CP% + EE% + CF% + Ash %). <sup>5</sup> Digestible energy (DE) was calculated according to values given in the feed composition tables of the NRC [24].

#### *2.4. Productive Performance and Carcass Characteristics*

At the start of the fifth week, the animals were weighed individually until the end of the experiment (13 weeks of age). The rabbit's daily feed consumption was calculated every week to evaluate the feed conversion ratio (FCR). Final body weight (FBW), body weight gain (BWG), and total feed consumption (TFC) were determined. Fifteen rabbits from each group were randomly selected to evaluate carcass characteristics at the end of the experiment (13th week). Rabbits were fasted for 12 h before being slaughtered. After removing the skin and bones, the carcasses were measured individually to evaluate the weight and percentage of the dressed animals. The offal weight includes blood, viscera, lungs, skin, arms, and tail. The obtained results were presented as the % of live weight [27]. The dressing % was calculated as hot carcass weight × 100/fasting weight. The carcass was divided into three cuts, viz., (1) the two forelegs (including the thoracic muscle inserting system), (2) the loin (the abdominal wall and the riveting after the seventh thoracic rib), and (3) the hind legs (including the sacral bone and the lumbar vertebra after the sixth lumbar vertebra).

#### *2.5. Hematology and Biochemical and Serum Oxidative Stress Evaluations*

Two blood samples were collected from the lateral ear vein (30 rabbits) during the slaughter. One sample contained an anticoagulant and was used to determine the count of white blood cells (WBCs), red blood cells (RBCs), lymphocytes, monocytes, and mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), platelet count, hematocrit %, and hemoglobin concentration [28]. The other blood sample was centrifuged (15 min, 3000× *g*) at 15–24 ◦C for plasma separation and stored at −20 ◦C until analysis. Total protein, albumin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels were measured in plasma using commercial kits. Moreover, the levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were evaluated using the colorimetric method (kits obtained from Bio-diagnostic, Cairo, Egypt).

#### *2.6. Bacterial Count*

Total bacteria, *E. coli*, and *lactobacilli* were all counted using the ring-plate method in the rabbit cecum sample [29,30].

#### *2.7. Data Analysis*

The attained results were statistically analyzed with the general linear model procedure of SAS® (Cary, NC, USA) [31]. Homogeneity of variances among studied groups was verified [32]. The analysis was performed using this model: Yijk = µ + S<sup>i</sup> + E<sup>j</sup> + SEij + eijK, where µ = observed mean for the concerned treatment, S<sup>i</sup> = breed effect, E<sup>j</sup> = treatment effect, SEij = interaction effect of breed and treatment, and eijk = the error related to individual observation using Duncan's multiple range test [32]. According to Ahrens et al. [33], the percentages were converted into arcsine values. Results were considered statistically significant at *p* ≤ 0.05.

#### **3. Results**

The FBW, FBWG, TFC, and FCR of rabbits supplemented with oral FOS were considerably enhanced compared to those in the control group (Table 2). The NZW rabbits treated with 1% FOS in drinking water showed the largest FBW, followed by APRI rabbits in which drunk water increased with the same level of FOS. BWG was more significant in NZW rabbits that consumed 1% and 0.5% FOS and APRI rabbits that consumed water supplemented with 1% FOS than their counterparts. Increasing FOS concentrations in drinking water decreased (*p* < 0.001) the amount of feed consumed in both breeds. The NZW rabbits that consumed water supplemented with 1% and 0.5% FOS and the APRI rabbits that consumed 1% FOS-supplemented water showed the lowest (*p* < 0.001) FCR compared with the other groups (Table 2).


**Table 2.** Growth performance of rabbits as affected by breed and supplementation of the diets with fructooligosaccharide (FOS).

Means within each column for each division with no common superscript letters are significantly different (*p* < 0.05). SEM = standard error of means.

Total giblets, gastrointestinal tract, liver, and dressing % in NZW and APRI rabbits that consumed FOS-supplemented water were significantly enhanced due to FOS and the interaction between FOS and breed (*p* < 0.001) compared with the control groups (Table 3). The difference between FOS levels was insignificant.

**Table 3.** Carcass traits of rabbits as affected by breed and supplementation of the diets with fructooligosaccharide (FOS) (%).


Means within each column for each division with no common superscript letters are significantly different (*p* < 0.05).

Substantial improvements (*p* < 0.001) were found in the two breeds in hematobiochemical and antioxidant parameters (Tables 4 and 5), which were enhanced with increasing FOS levels (FOS-1.0), with no detrimental effects on the kidney and liver. However, rabbits consuming 1% FOS had higher blood biochemicals and antioxidant parameters values than those receiving 0.5% FOS-supplemented water.

**Table 4.** Hematological parameters of rabbits as affected by breed and supplementation of the diets with fructooligosaccharide (FOS).


Means within each column for each division with no common superscript letters are significantly different (*p* < 0.05). SEM: standard error of the means; MCV: mean corpuscular volume; HCT; hematocrit; MCH: mean corpuscular hemoglobin; RDW: red cell distribution width.

In both rabbit breeds that consumed FOS-supplemented water, the cecum, total bacterial, and *E. coli* populations (Table 6) were considerably lower (*p* < 0.001), with a substantial increase in the Lactobacillus population compared with the control groups. Rabbits that consumed 1% FOS showed the most significant count of beneficial bacteria and a lower count of pathogenic ones compared with the other treatments.


**Table 5.** Blood biochemical parameters and some selected oxidative stress biomarkers of rabbits as affected by breed and supplementation with fructooligosaccharide (FOS).

Means within each column for each division with no common superscript letters are significantly different (*p* < 0.05). SEM: standard error of the means; ALT; alanine aminotransferase; AST; aspartate aminotransferase; GPX, glutathione peroxidases; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.

**Table 6.** Cecal bacterial counts of rabbits as affected by breed and supplementation with fructooligosaccharide (FOS) (Log10 CFU g−<sup>1</sup> ).


Means within each column for each division with no common superscript letters are significantly different (*p* < 0.05). SEM = standard error of means.

#### **4. Discussion**

This study investigated the possible effects of adding FOS in drinking water on the growth performance, carcass characteristics, hematobiochemical parameters, oxidative stress biomarkers, and cecal microbiota of NZW and APRI rabbits. Our results showed that supplementing water with FOS significantly enhanced the growth performance traits of the two rabbit breeds.

The beneficial effects of adding FOS to the drinking water of growing rabbits may be due to the augmentation of feed efficiency and absorption, which improves anabolic metabolism, enhances the intestinal response to pathogens, and increases serum protein levels, thereby encouraging rabbit growth [1,6,7]. Prebiotics provide suitable environments for the growth of helpful microflora and inhibit the growth of pathogenic bacteria, which may explain the improvement in growth performance [7].

Consistent with our findings, the rabbits' growth was enhanced with *Bacillus subtilis* and FOS with a more significant average daily BWG than the control [22]. In addition, Inmunair17.5® (*Propionibacterium acnes* and coli lipopolysaccharides) as a prebiotic in the drinking water of fattening NZW rabbits resulted in an enhancement of BW at marketing, BWG, and FCR [11]. Comparable findings reported that a diet supplemented with *S. cerevisiae* and probiotics accelerated the BWG and FCR of NZW rabbits [34,35]. By contrast, Rotolo et al. [36,37] found that the dietary supplementation of *S. cerevisiae* did not affect rabbits' BW, BWG, and FCR. Additionally, Zarei et al. [38] reported that dietary prebiotics did not modify FCR in laying hens. In broilers, Xu et al. [14] concluded that supplementation with 4 g of FOS/kg diet increased BWG and improved FCR.

Regarding carcass characteristics, our findings were consistent with those observed by Abd El-Aziz et al. [1], Mahrose et al. [6], and Abo Ghanima et al. [2]. Similarly, Mousa et al. [11] showed that dressing and giblet percentages were significantly higher in the carcasses of rabbits that drank water supplemented with 1 mL Inmunair17.5®/litter. However, Rotolo et al. [36] found nonsignificant changes in the carcass characteristics of growing rabbits treated with dietary prebiotics. Moreover, Ju´skiewicz et al. [39] concluded that increasing turkeys fed with a diet supplemented with FOS showed no differences from the control group. There were no significant changes between the two rabbit breeds regarding the breed impact on carcass traits in the present study. Such an absence of significant differences in carcass traits between genetic breeds has also been confirmed in previous studies [1,7].

Hematological measurements are valuable indicators for evaluating the animals' health statuses [4]. In our study, most hematological parameters were altered by the water supplemented with FOS in the two rabbit breeds. Our findings are consistent with those of Akrami et al. [13], who found that WBC counts were increased in fish fed with 1% FOS compared with the control group. They also found a nonsignificant elevation of RBCs, MCV, HCT, Hgb, and lymphocytes in the fish fed with a diet supplemented with 1% FOS.

In a study on birds, FOS supplementation resulted in low heterophil counts, indicating that FOS may reduce stress reactions and alleviate the possible damaging consequences on growth performance [9]. Moreover, broilers supplemented with FOS had more significant monocyte counts than broilers fed with the control diet. Monocytes comprise 5%–10% of peripheral blood leukocytes and can migrate rapidly in response to diseases, release cytokines, and differentiate into macrophages and dendritic cells to assist the innate immune response [40]. FOS supplementation increased monocyte %, suggesting that dietary FOS supplementation in broilers augments cytokine release and alleviates pathogenic infections rapidly [9]. This effect is probably due to the alteration in the gut microbiota, such as variations in the *Lactobacillus* profile, which shows diverse patterns for dendritic cell activation [41,42]. The findings concerning hematological indices revealed that these measurements were increased in rabbits that consumed water supplemented with FOS-0.5 and FOS-0.1. Hoseinifar et al. [43] mentioned that WBC count, primarily lymphocytes, was significantly increased in belugas fed with 1 and 2 g kg−<sup>1</sup> dietary oligofructose. The high leukocyte count may increase activity and improve defense mechanisms during feeding. Leukocytes are imperative cells that stimulate the immune responses of fish. They produce antibodies and may exhibit macrophage activities [44]. Saha et al. [19] obtained similar results, where the MCH in broilers receiving a water-soluble organic additive at different doses fluctuated from that in the control.

The total protein and globulin levels were increased in the experimental groups treated with varying levels of FOS in their diets, indicating a more robust innate immune response. Globulin is believed to be the main protein that plays a significant role in immune response [5]. Moreover, FOS was found to have the potential to control enteric pathogens and alter immunity [1]. This result was also previously supported by Abd El-Gawad et al. [17] who concluded that ALT and AST activities were diminished with dietary FOS than in the control fish group. Our results failed to show significant differences in AST and ALT activities with FOS supplementation in drinking water.

Interestingly, our findings showed an increase in SOD, GPX, and T-AOC values in the supplemented groups of the two rabbit breeds. These results suggest that the FOSsupplemented drinking water could alleviate oxidative stress in the two breeds of growing rabbits and maintain their healthy. The first line of antioxidant enzymatic defense is believed to involve GPX, SOD, and T-AOC [5], which act as biomarkers of oxidative stress due to the inequality between the production and elimination of reactive oxygen species. The enhancement of antioxidant enzymatic activities in the present study with FOS supplementation in the drinking water of growing rabbits was also previously reported by Guerreiro et al. [45] and Zhang et al. [46] as FOS supplementation may relieve oxidative stress [17].

In the present study, FOS supplementation in drinking water caused a stimulatory impact on the growth of health-supporting bacterial species (*Lactobacillus*). Moreover, FOS supplementation decreased the total bacterial count and harmful or potential pathogens (*E. coli*) in the two rabbit breeds. Our results are consistent with those reported by Xu et al. [16] who examined the effects of FOS at doses of 0, 2, 4, and 8 g/kg diet on intestinal microbiota. The inclusion of FOS at a 4 g/kg diet resulted in a beneficial effect on *Bifidobacterium* and *Lactobacillus*, with an immediate reduction of *E. coli* growth in the broilers' gastrointestinal tract. Saminathan et al. [47] evaluated the impact of applying various oligosaccharides by isolating 11 *Lactobacillus* species from the gastrointestinal tract of fowls. The in vitro data revealed that Lactobacillus species utilized FOS more competently than mannan oligosaccharides. The increased availability of FOS may be related to particular enzymatic actions and the oligosaccharide conveyance technique of *Lactobacillus* species. Nevertheless, broilers' intestinal microbiota is further complicated than in vitro examinations. Prebiotics may be fermented not only by *Lactobacillus* species but also by other microbes in the gastrointestinal tracts of animals [23].

#### **5. Conclusions**

FOS supplementation in the drinking water of rabbits improved most growth performance parameters, carcass characteristics, hematobiochemical parameters, antioxidant status, and cecal microbiota in NZW and APRI rabbits. Moreover, the response of NZW rabbits to FOS supplementation was more significant than that of APRI rabbits.

**Author Contributions:** Conceptualization, A.H.A.E.-A., W.M.G., A.E.-W.A. and M.M.A.G.; methodology, W.F.A., A.G., A.E.-W.A. and K.M.; software, W.M.G. and A.A.E.; validation, S.A.M., K.M. and A.A.E.; formal analysis, S.H.A.R.; investigation., A.H.A.E.-A.; resources, K.M., A.E.-W.A. and M.S.; data curation, A.O.H., A.E., H.A.Y., W.M.G. and K.M.; writing—original draft preparation, M.S., A.O.H., M.M.A.G., and K.M.; writing—review and editing, W.F.A., A.G., S.H.A.R. and M.S.; visualization, H.A.Y., W.F.A., W.M.G. and A.G.; supervision, W.F.A., A.O.H. and A.G.; project administration, M.S. and K.M.; funding acquisition, W.F.A., A.G., M.S., A.E.-W.A. and A.H.A.E.-A. All authors have read and agreed to the published version of the manuscript..

**Funding:** The research was partially funded by Taif University Researchers Supporting Project number (TURSP2020/53), Taif University, Taif, Saudi Arabia.

**Institutional Review Board Statement:** All investigations in this study were carried out following the Native Experimental Animal Care Committee and approved by the Ethics of the Institutional Committee of Animal Husbandry and Animal Wealth Development Department, Faculty of Veterinary Medicine, Damanhour University, Egypt (DMU/VetMed-2019-/0145).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

**Acknowledgments:** The authors appreciate Taif University Researchers Supporting Project number (TURSP2020/53), Taif University, Taif, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest. The funder had no role in the study's design or interpretation of data.

#### **References**


### *Review* **Potential Use of Tannin Extracts as Additives in Semen Destined for Cryopreservation: A Review**

**Mohammed S. Liman 1,2 , Abubeker Hassen <sup>3</sup> , Lyndy J. McGaw <sup>4</sup> , Peter Sutovsky <sup>5</sup> and Dietmar E. Holm 1,\***


**Simple Summary:** Freezing of semen used for artificial reproductive technologies (ART) affects the survival and vigour of sperm cells due to excessive production of reactive oxygen species (ROS) during the freezing and thawing processes. ROS plays a physiological role in sperm function but excessive ROS production from damaged sperm cells can hinder sperm's motility and their ability to fertilise an oocyte. Tannins, a class of water-soluble plant polyphenols, are known to have antioxidant and other health-promoting effects and may serve as binders/acceptors to reduce the deleterious effects of excessive ROS produced during the freezing and thawing process. This review is the first to analyse the available data supporting the use of tannins as additives to semen extenders to improve the survival of cryopreserved spermatozoa during storage and after thawing. It is concluded that tannins and their derivatives have naturally protective properties with the potential to ameliorate sperm cell survival after freezing.

**Abstract:** Cryopreservation and storage of semen for artificial insemination (AI) result in excessive accumulation of reactive oxygen species (ROS). This leads to a shortened life span and reduced motility of spermatozoa post-thawing, with consequent impairment of their function. However, certain levels of ROS are essential to facilitate the capacitation of spermatozoa required for successful fertilisation. Tannins, as well-known antioxidant compounds, may act as ROS binders/acceptors/scavengers to inhibit the damaging effects of ROS. This review comprises an analysis of the semen cryopreservation protocol and health functions of tannins, as well as the effects of ROS on fresh and cryopreserved semen's longevity and fertilisation. Additionally, we surveyed available evidence of the effects of tannin extract feed supplementation on male fertility. We furthermore interrogated existing theories on tannin use as a potential additive to semen extenders, its relationship with semen quality, and to what degree existing theories have been investigated to develop testable new hypotheses. Emphasis was placed on the effects of tannins on ROS, their involvement in regulating sperm structure and function during cryopreservation, and on post-thaw sperm motility, capacitation, and fertilising ability. The diverse effects of tannins on the reproductive system as a result of their potential metal ion chelation, protein precipitation, and biological antioxidant abilities have been identified. The current data are the first to support the further investigation of the incorporation of tannin-rich plant extracts into semen extenders to enhance the post-thaw survival, motility, and fertilising ability of cryopreserved spermatozoa.

**Keywords:** cryopreservation; spermatozoa; tannin; polyphenols; semen additives; antioxidant

**Citation:** Liman, M.S.; Hassen, A.; McGaw, L.J.; Sutovsky, P.; Holm, D.E. Potential Use of Tannin Extracts as Additives in Semen Destined for Cryopreservation: A Review. *Animals* **2022**, *12*, 1130. https://doi.org/ 10.3390/ani12091130

Academic Editor: Eva Bussalleu

Received: 28 February 2022 Accepted: 22 April 2022 Published: 28 April 2022

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

**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/).

#### **1. Introduction**

Cryopreservation reduces the functional and structural integrity of spermatozoa due to the development of reactive oxygen species (ROS) [1,2]. ROS are produced during numerous chemical reactions in different parts of the mammalian body [1]. In the testes, ROS are produced during spermatogenesis within the seminiferous tubules and steroidogenesis in the interstitium [3]. Cryopreservation and storage of semen lead to changes in the sperm mitochondrial membrane and the resident electron transport chain [3], which result in the excessive release of ROS, hydrogen peroxide (H2O2), nitric oxide (NO), or superoxide anion (O<sup>2</sup> −), with consequences on sperm capacitation and the acrosome reaction [2]. Cryoprotectants are important for the cryo-survival of spermatozoa [4], and these may include egg yolk, glycerol [4], dimethyl sulphoxide (DMSO) [5], ethylene glycol [6], Triladyl® (a commercially available semen extender) [7], and butylated hydroxytoluene (BHT) [8–11]. Combinations of cryoprotectants such as glycerol and ethylene glycol [7,12] and acetamide together with lactamide [13] may also be employed. Antioxidant substances may reduce the impact of oxidative stress and thereby improve the quality of semen post-thawing [14]. Cryoprotectants are important for the cryo-survival of spermatozoa [15].

Low levels of ROS are, however, associated with increased sperm motility, viability, increased capacity for successful fertilisation during sperm–oocyte interactions, and fertility in mammalian species [16]. Antioxidant additives in semen diluents for cryopreservation should therefore not aim to eliminate ROS [17]. When ROS occur in small concentrations, they act as mediators of normal sperm function, whereas when present in excess, they are toxic to spermatozoa [14].

Sperm capacitation normally occurs in the oviduct and involves biochemical and structural changes that make the spermatozoa competent to attach to the zona pellucida of the oocyte, penetrate it, and fuse with the oolemma [18]. The cellular changes that occur include the activation of soluble adenyl cyclase that produces cAMP, the influx of Ca2+ ions, Zn ions [19,20], efflux of cholesterol from the plasma membrane, leading to its fluidity/fuseability, and the generation of more ROS, with a consequent increase in intracellular pH [7]. Additionally, activation of protein kinase A and downstream protein tyrosine kinases results in the protein phosphorylation of numerous proteins on tyrosine residues [21]. This process results in the hyperactivation of sperm tail motility, which is necessary for sperm detachment from the oviductal sperm reservoir and the penetration of the egg vestment at fertilisation. It was reported that controlled and low ROS generation plays a physiological role during the capacitation and acquisition of sperm's fertilising ability, with ROS-specific scavengers inhibiting the process [14,22,23]. These processes of ROS affecting the spermatozoa have been reviewed previously [24].

Thus, ROS homeostasis appears to be equally important for timely sperm capacitation within the female oviduct, and for the prevention of premature capacitation during semen processing and cryopreservation for artificial insemination (AI).

Plants contain combinations of complex polymeric phenols, which are amongst the most studied phytochemicals because of their diverse array of useful biological functions and health-promoting effects [14]. Consequently, their antioxidation effects on the production of ROS, sperm longevity, and fertilising potential were reviewed using the available peer-reviewed data on tannin extract supplementation for male fertility. The aim was to document the utilisation of the biological and reproductive health benefits of tannins, with a view to exploiting their potential for use as additives to improve the cryopreservation of semen. To our knowledge, this review is the first to recommend further structured evaluation of the value of tannin extracts or compounds as additives into semen destined for cryopreservation [14,25].

#### **2. Methodology**

This theoretical literature review (TLR) focused firstly on the existing evidence of the biological and health benefits of tannins, specifically with regard to their antioxidant properties and resultant inhibitory effects on lipid peroxidation, as well as their antiviral,

antibacterial, and anti-inflammatory effects in terms of protecting spermatozoa against microbial infections during semen processing, cryopreservation, and distribution. This first section is divided into three subsections addressing the cryopreservation of semen using tannins, and their relevant biological and health functions, respectively. Secondly, we investigated the current evidence on the effect of ROS on sperm viability/semen longevity, and on the requirement for low levels of ROS in semen fertility. first section is divided into three subsections addressing the cryopreservation of semen using tannins, and their relevant biological and health functions, respectively. Secondly, we investigated the current evidence on the effect of ROS on sperm viability/semen longevity, and on the requirement for low levels of ROS in semen fertility. Articles used in this review had a concise hypothesis, with keywords searched on databases including Google Scholar, Scopus/ScienceDirect, and Web of Science/Pubmed

This theoretical literature review (TLR) focused firstly on the existing evidence of the biological and health benefits of tannins, specifically with regard to their antioxidant properties and resultant inhibitory effects on lipid peroxidation, as well as their antiviral, antibacterial, and anti-inflammatory effects in terms of protecting spermatozoa against microbial infections during semen processing, cryopreservation, and distribution. This

Articles used in this review had a concise hypothesis, with keywords searched on databases including Google Scholar, Scopus/ScienceDirect, and Web of Science/Pubmed (which includes CABAbstracts, Medline and Zoological Records). Inclusion words: "additives"; "cryopreservation", "tannin-extracts" or "fractions", or "compound" and "health" or "biological" with emphasis on specific functions, namely "antiviral", "antibiotic", "antioxidant", and "protection against lipid peroxidation", excluding anticancer and antidiabetes. Database results for Google Scholar (645, 76%); Scopus/ScienceDirect (94, 11%); and Web of Science/Pubmed (105, 13%) are all published reports, respectively (Figure 1). The relevant reports used are represented as cited in this study. (which includes CABAbstracts, Medline and Zoological Records). Inclusion words: "additives"; "cryopreservation", "tannin-extracts" or "fractions", or "compound" and "health" or "biological" with emphasis on specific functions, namely "antiviral", "antibiotic", "antioxidant", and "protection against lipid peroxidation", excluding anticancer and antidiabetes. Database results for Google Scholar (645, 76%); Scopus/ScienceDirect (94, 11%); and Web of Science/Pubmed (105, 13%) are all published reports, respectively (Figure 1). The relevant reports used are represented as cited in this study.

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**2. Methodology**

**Figure 1.** Database of the number of reports from Google Scholar, Scopus, and Web of Science on tannin additives for cryopreservation (*n* = 844). **Figure 1.** Database of the number of reports from Google Scholar, Scopus, and Web of Science on tannin additives for cryopreservation (*n* = 844).

#### **3. Effect of ROS on Cryopreserved Spermatozoa**

#### **3. Effect of ROS on Cryopreserved Spermatozoa** *3.1. ROS Effect on Sperm Cryopreservation and Longevity*

*3.1. ROS Effect on Sperm Cryopreservation and Longevity*  ROS are a group of molecules (free radicals, oxygen ions, peroxides, etc.) that are produced during aerobic metabolism in the mitochondria of cells, and are important components of physiological processes and cellular signalling events [1]. The liquid or frozen semen preservation and its effect on semen quality were reviewed previously [14]. Oxidative damage in semen impairs spermatozoal function, resulting in a loss of motility, loss of mitochondrial activity, increase in deoxyribonucleic acid (DNA) damage, and lack of activation of apoptotic pathways [26]. Consequently, unresolved issues affecting fertility are encountered in artificially collected semen samples, such as infections, inadequate constituents of semen extenders and protocols adopted during cryopreservation processes, and the overall need for highly skilled intra-uterine ROS are a group of molecules (free radicals, oxygen ions, peroxides, etc.) that are produced during aerobic metabolism in the mitochondria of cells, and are important components of physiological processes and cellular signalling events [1]. The liquid or frozen semen preservation and its effect on semen quality were reviewed previously [14]. Oxidative damage in semen impairs spermatozoal function, resulting in a loss of motility, loss of mitochondrial activity, increase in deoxyribonucleic acid (DNA) damage, and lack of activation of apoptotic pathways [26]. Consequently, unresolved issues affecting fertility are encountered in artificially collected semen samples, such as infections, inadequate constituents of semen extenders and protocols adopted during cryopreservation processes, and the overall need for highly skilled intra-uterine insemination. Mammalian spermatozoa naturally contain antioxidants and ROS scavenging enzymes, such as glutathione (GSH), superoxide dismutase, and catalase (CAT) [27]. These endogenous antioxidants often are not sufficient to prevent lipid peroxidation during cryopreservation [28]. Excess ROS that develop during the storage of spermatozoa are largely responsible for damage to spermato-

zoa. The damage of the sperm plasma membrane due to the effect of ROS consequently exposes semen to lipid peroxidation, resulting from the high content of polyunsaturated acids, and DNA damage [29]. Thus, the cryopreservation of semen is dependent on the reversible reduction of the survival and metabolic activity of spermatozoa [30]. This could be achieved by the provision of an effective environment for the steady cooling of semen, with a focus on the development of extenders that maintain membrane integrity, increase motility, maximise sperm's ability to capacitate, prevent oxidative stress, and minimise the generation of reactive oxygen species (ROS) during cryopreservation and storage [31–33]; see Figure 2. sperm plasma membrane due to the effect of ROS consequently exposes semen to lipid peroxidation, resulting from the high content of polyunsaturated acids, and DNA damage [29]. Thus, the cryopreservation of semen is dependent on the reversible reduction of the survival and metabolic activity of spermatozoa [30]. This could be achieved by the provision of an effective environment for the steady cooling of semen, with a focus on the development of extenders that maintain membrane integrity, increase motility, maximise sperm's ability to capacitate, prevent oxidative stress, and minimise the generation of reactive oxygen species (ROS) during cryopreservation and storage [31–33]; see Figure 2.

insemination. Mammalian spermatozoa naturally contain antioxidants and ROS scavenging enzymes, such as glutathione (GSH), superoxide dismutase, and catalase (CAT) [27]. These endogenous antioxidants often are not sufficient to prevent lipid peroxidation during cryopreservation [28]. Excess ROS that develop during the storage of spermatozoa are largely responsible for damage to spermatozoa. The damage of the

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**Figure 2.** Demonstration of the homeostatic effects of ROS on sperm's fertilisation ability, and the potential benefits of tannins to ameliorate its effects, particularly following cryopreservation. **Figure 2.** Demonstration of the homeostatic effects of ROS on sperm's fertilisation ability, and the potential benefits of tannins to ameliorate its effects, particularly following cryopreservation.

The cytotoxic action of ROS on spermatozoa is mediated by high concentrations of phospholipid-bound polyunsaturated fatty acids (PUFA) in the sperm plasma membrane, especially docosahexaenoic acid (DHA), with six double bonds per molecule, which makes them susceptible to free radical attack [34]. Additionally, spermatozoa lack an enzyme apurinic/apyrimidinic endonuclease (APEI), which plays a significant role in DNA repair and base excision repair pathways [24]. Furthermore, the sperm DNA is hyper-condensed; thus, it is not easily accessible to repair The cytotoxic action of ROS on spermatozoa is mediated by high concentrations of phospholipid-bound polyunsaturated fatty acids (PUFA) in the sperm plasma membrane, especially docosahexaenoic acid (DHA), with six double bonds per molecule, which makes them susceptible to free radical attack [34]. Additionally, spermatozoa lack an enzyme apurinic/apyrimidinic endonuclease (APEI), which plays a significant role in DNA repair and base excision repair pathways [24]. Furthermore, the sperm DNA is hyper-condensed; thus, it is not easily accessible to repair mechanisms.

#### *3.2. Effects of ROS on Sperm's Fertilising Potential*

mechanisms.

*3.2. Effects of ROS on Sperm's Fertilising Potential*  Low levels of ROS are associated with increased sperm motility, viability, increased capacity for fertilisation during sperm–oocyte fusion, and general male fertility in mammalian species [16]. When ROS are in low concentrations, they act as mediators of Low levels of ROS are associated with increased sperm motility, viability, increased capacity for fertilisation during sperm–oocyte fusion, and general male fertility in mammalian species [16]. When ROS are in low concentrations, they act as mediators of normal sperm function, whereas in excess, they are toxic to spermatozoa. Sperm capacitation is a complex process by which spermatozoa acquire the ability to fertilise the mature oocyte. This occurs within the oviductal sperm reservoir and involves the biochemical and morphological changes that make the spermatozoon competent to attach to the zona pellucida of the oocyte, penetrate it, and fuse with the oolemma [18]. Conception rates in livestock AI depend on the quality of semen, which is generally low post-thawing, with the capacitation

and fertilisation processes being dependent on the effect of the sub-lethal dysfunction of spermatozoa [35]. Premature sperm capacitation brought about by cryopreservation and thawing is referred to as cryocapacitation [36] and, similarly to physiological capacitation, is irreversible and terminal, leading to a shortened sperm lifespan and eventual death before spermatozoa can reach the oviductal fertilisation site following AI. The selection of animals with good-quality semen for cryopreservation and AI is a critical step in improving the fertility levels of frozen–thawed semen [37,38]. Despite having satisfactory fertility testing in terms of fresh-stored semen, the frozen–thawed semen of some animal species does not meet standards of acceptable fertilisation results suitable for commercial AI programmes [38,39]. Accumulated evidence indicates that inherent male progeny variability is one of the factors in semen cryopreservation responsible for the marked differences in sperm cryo-survival [37–40]. Individual differences in sperm quality and cryo-survival are addressed by ongoing efforts to identify gene variants and differentially expressed sperm proteins associated with either high or low sperm cryotolerance in livestock species [41,42].

#### **4. Tannins**

#### *4.1. Properties of Tannins*

Tannins are sourced from a multitude of trees and shrubs. Notable for industrial importance are black wattle or black mimosa (Mimosa tannin, *Acacia mearnsii*), quebracho wood (*Schinopsis lorentzhii*), oak bark (*Quercus robur*), chestnut wood (*Castanea sativa*), mangrove wood (*Algarobilla chilena*), gambir (*Uncaria gambir*), the bark of several species of pines and firs, such as *Pinus radiata* and *Pinus nigra*, as well as many other plants harbouring extractable tannins [43–46]. Tannins are a renewable resource used in several fields, ranging from the traditional application of tanning to producing heavy-duty leather and as wood adhesives up until the 1960s and 1970s, whereafter new applications were investigated [44], such as the proposed use of chestnut tannin as an antimicrobial and to reduce mycotoxins [47]. Tannins dissolve in water to form colloidal solutions, with their solubility dependent on the degree of polymerisation [48]. They are soluble in alcohol and acetone, and react with ferric chloride [49]. They have moderate stability in aqueous solutions, especially during extraction with boiling water (decoctions), in which they decompose in 30 min into gallic acid, ellagic acid, and corilagin [44]. At the centre of hydrolysable tannins is a polyol carbohydrate (D-glucose), which is partially or completely esterified with a phenolic group such as gallic acid (gallotannins) or ellagic acid (ellagitannins). Hydrolysable tannins are hydrolysed by weak acids or weak bases to produce carbohydrates and phenolic acids. Condensed tannins (proanthocyanidins) are polymers of 2–50 (or more) flavonoid units joined by carbon-to-carbon bonds, which are not easily cleaved by hydrolysis.

#### *4.2. Extraction of Tannins*

Tannins, both hydrolysable and condensed, are commonly extracted with a mixture of water and acetone. Optimal yield may be obtained from fresh, frozen, or lyophilised material. Some tannin-rich extracts are available from varied sources and are used as supplements to improve reproduction.

#### *4.3. Medicinal Properties and Biological Functions of Tannins*

The health benefits of tannins include antioxidant, anti-carcinogenic, cardioprotective, antimutagenic, antiviral, antibacterial, haemostatic, and anti-inflammatory properties, as well as inhibition of lipid perioxidation [45,46] Hydrolysable tannins are often cited for their antimicrobial activity [46] and chemopreventive properties against degenerative diseases [50]. These multi-functional properties of tannins are utilised in the treatment of human diseases [51]. Hydrolysable tannins are also inhibitors of α-glucosidase, which is an enzyme known to be involved in the modulation of the absorption of glucose in tissues [48].

Antioxidants have been used in semen extenders, including cysteamine, taurine, trehalose, and selenium, to improve the motility, viability, and membrane integrity of postthawed semen [52,53], with significant results. Some other antioxidants, such as Vitamin C and E and catalase, have been used to supplement human, cattle, boar, rabbit, and stallion semen [54,55] In a study of the α-glucosidase inhibition and antioxidant activity of an oenological commercial tannin (Tan'Activ® toasted oak wood *Quercus robur*), the extraction and fractionation process yielded four fractions, with one of the fractions generating a subfraction with enhanced α-glucosidase inhibitory activity with an inhibitory concentration (IC50) of 6.15 µg/mL [56]. The oak wood is used for barrel staves in the winemaking process and the polyphenols are not only used in the ageing of wine but in maintaining aroma/flavour, as well as contributing useful health properties [57,58].

Synthetic water-soluble polymers such as polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) are used as tannin-binding agents for quantification and to neutralise the negative effect of tannins in animal diets [49]. The PVP is used to bind hydrolysable tannins, while PEG is used for condensed tannins. These groups of tannins both contain sufficient oxygen molecules in their chains to form strong hydrogen bonds, with the phenolic and hydroxyl groups in tannins serving to precipitate them from solutions [49].

#### *4.4. Use of Tannins as Supplements to Improve Reproduction Outomces or as Semen-Protective Agents*

Tannin extracts or compounds are extracted using ethanol or water into powdered substances and stored at −20 ◦C [56,59,60] for later use as supplements (feed) (Table 1) or added into semen extenders (Table 2), etc., after optimisation.

**Table 1.** Reported effects of tannin extracts used as food/feed supplements on reproduction outcomes in humans or animals.


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#### **Table 1.** *Cont.*

MW = Molecular weight.

Certain tannin concentrations have exerted efficiency in fertilisation, but with no effect on sperm kinematic parameters, acrosome integrity, mitochondrial membrane integrity, lipid perioxidation, or capacitation status or its viability [73]. The ethanol extract of a commercial oenological tannin (*Quercus robur*, toasted oak wood Tan'Activ®) had a biological effect at a concentration of 10 µg/mL, stimulating an increase (*p* < 0.001) in in vitro swine sperm capacitation at the tail principal piece (B pattern) and increased (*p* < 0.001) oocyte fertilisation rate [60]. However, at 100 µg/mL, the opposite effect was recorded on both sperm capacitation (B pattern) and fertilising ability, associated with higher sperm viability [60]. Where 5% crude tannin was added to the semen of the Bali breed of cattle for 14 days, it increased (*p* > 0.001) motility and viability, with a decrease in abnormal semen [76]. Guava (*Psidium guajava*) leaf extract, comprising 3% crude tannin, was added to liquid semen (stored for 15 days at 4–5 ◦C) of Ettawa crossbred Boer goats and improved (*p* < 0.001) the motility and viability and maintained intact plasma membranes of the spermatozoa, while a concentration of 24% of the crude tannin reduced viable sperm content [71]. Altogether, it appears that tannins may benefit extended semen through ROS scavenging and microbial growth limitation. It is yet to be determined if tannins may also convey cryotolerance during semen preservation.


**Table 2.** Tannin-rich extracts used as semen-protective agents in humans and various domestic animals.

#### **5. Conclusions**

To our knowledge, this is the first review recommending the addition of tannin-rich extract or compounds into semen destined for cryopreservation, exploiting their diverse effects on biological systems due to their potential for metal ion chelation and biological antioxidation. The varied biological roles, however, together with the enormous structural variations of these compounds, make it difficult to develop a model that allows accurate prediction of the role of tannins in any biological system. Therefore, it becomes imperative for studies to be conducted on tannin biological activities by determining their chemical structure, biological activity, and structure–activity relationships so that potential applications can be explored. While the inquiry into the biological activities of tannins is still in its infancy, it holds a promise of utility in livestock-assisted reproductive technology and human reproductive therapy. The addition of plant tannin extracts, extract fractions, or purified/synthetic compounds derived therefrom to semen may elevate the quality and viability of semen intended for cryopreservation. Beyond sperm cryopreservation, protocols for semen collection, processing, and liquid semen distribution in relevant livestock species could benefit from judicious, experiment-validated tannin supplementation, taking advantage of the antioxidant properties of tannins.

**Author Contributions:** Conceptualisation, A.H., D.E.H. and M.S.L.; methodology, A.H., L.J.M., D.E.H. and M.S.L.; validation, M.S.L.; A.H., D.E.H., L.J.M. and P.S.; formal analysis, M.S.L.; investigation, M.S.L.; resources, L.J.M., D.E.H. and A.H.; data curation, D.E.H. and M.S.L.; writing—original draft preparation, M.S.L.; writing—review and editing, M.S.L., D.E.H., A.H., L.J.M. and P.S.; visualisation, L.J.M.; supervision, D.E.H., L.J.M. and A.H.; project administration, D.E.H.; funding acquisition, M.S.L. and D.E.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Red Meat Research and Development Trust of South Africa project committee (RMRD SA). P.S. was supported by grant number 2021-67015-33404 from the USDA National Institute of Food and Agriculture, grant number 1R01HD084353 from NIH/NICHD, and a travel grant from the University of Missouri South African Education Program. The project was also supported by the "Translational Medicine Research Theme" of the Faculty of Veterinary Sciences, University of Pretoria.

**Institutional Review Board Statement:** The study was approved by the University of Pretoria research committee and Animal Ethics Committee of the University of Pretoria (REC 193-19 of 8 October 2020).

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** We would like to thank Susan Marsh (susanmarsh@up.ac.za), Faculty Library Manager: Jotello F. Soga Veterinary Science, for guidance in the use of search tools.

**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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